Separation Recovery and Purification in Biotechnology 1986 Recent Advances and Mathematical Modeling

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Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.fw001
Separation, Recovery, and

Purification in Biotechnology
In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1986.
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ACS
SYMPOSIUM
SERIES

Separation, Recovery, and

Recent Advances and
Mathematical Modeling
Juan A. Asenjo, EDITOR
Columbia University
Juan Hong, EDITOR

Illinois Institute of Technology
Developed from a symposium sponsored by

the Division of Microbial and Biochemical Technology
at the 190th Meeting
of the American Chemical Society,
Chicago, Illinois,
September 8-13, 1985
American Chemical Society, Washington, D C 1986

314
Library of Congress Cataloging-in-Publication Data

Separation, recovery, and purification in
biotechnology.
(ACS symposium series, ISSN 0097-6156; 314)

Includes bibliographies and index.

1. BiotechnologyTechniqueCongresses.
2. BiomoleculesPurificationCongresses.
3. Biological chemistryTechniqueCongresses.
I. Asenjo, Juan ., 1949- . II. Hong, Juan.
III. American Chemical Society. Meeting (190th: 1985:
Chicago, Ill.) IV. Series.
TP248.24.S47
1986
ISBN 0-8412-0978-2
660'.6'028
86-10833
Copyright 1986
American Chemical Society
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IBM Research Department

FOREWORD
The ACS SYMPOSIUM SERIES was founded in 1974 to provide a
medium for publishing symposia quickly in book form. The

format of the Series parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that, in order to save time, the
papers are not typeset but are reproduced as they are submitted
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the supervision of the Editors with the assistance of the Series
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research are acceptable, because symposia may embrace both
types of presentation.


Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.pr001
PREFACE
ONE OF T H E MOST DIFFICULT and challenging problems facing large-scale
biotechnology today is to find and develop appropriate recovery, separation,
and purification processes. The area of large-scale bioseparations is one to
which biologists, physical biochemists, and particularly biochemical engineers have important contributions to make. Some of the most recent
advances and developments that have already started to find practical
applications are
membrane separations, including the use of membrane bioreactors
and liquid emulsion membranes;
continuous or semicontinuous chromatographic separations, including the use of a number of affinity methods and monoclonal antibodies;
two-phase extraction processes such as aqueous systems and the use
of reverse micelles;
precipitation techniques;
electrically driven separation processes;
methods of product secretion, cell permeation, disruption, and
selective enzymatic lysis of microbial cells for intracellular product release;
product solubilization and renaturation of proteins or polysaccharides
present in inclusion bodies or granules.
This book covers several of the emerging areas of separations in
biotechnology and is not intended to be a comprehensive handbook. It
includes recent advances and latest developments in techniques and
operations used for bioproduct recovery in biotechnology and applied to
fermentation systems as well as mathematical analysis and modeling of such
operations. The topics have been arranged in three sections beginning with
product release from the cell and recovery from the bioreactor. This section
is followed by one on broader separation and concentration processes, and
the final section is on purification operations. The operations covered in
these last two sections can be used at a number of different stages in the
downstream process.
A crucial question remaining is how to design a flowsheet or product
recovery operation sequence. Three main points to keep in mind are
(1) integrating recovery with the fermentation system, (2) integrating the
different separation and purification stages to design the optimum sequence,
and (3) assessing the possibility of a continuous operation.
Revised versions of papers presented in the symposium upon which this
book is based as well as papers presented in other sessions that were relevant
IX

to the topic have been included in this volume. In addition, we have included
a few keynote chapters on areas we felt had not been well covered at the
meeting.
We gratefully acknowledge the assistance of many reviewers who helped
us with critical and constructive comments on the original manuscripts. We
would also like to acknowledge the support and well-organized help of the
staff at the ACS Books Department.

Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.pr001
J U A N A. A S E N J O
Columbia University
New York, NY 10027
JUAN HONG
Illinois Institute of Technology

Chicago, IL 20742

1
Protein Release from Chemically Permeabilized
Escherichia coli

Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.ch001
David J. Hettwer and Henry Y. Wang

Department of Chemical Engineering, The University of Michigan, Ann Arbor,
MI 48109-2136
An important
factor complicating the recovery of
recombinant proteins from Escherichia coli is their
intracellular location. An alternative to the commonly
used method of releasing these proteins by mechanical
disruption is to chemically permeabilize the cells. The
objective of this research was to characterize the protein release kinetics and mechanism of a permeabilization process using guanidine-HCl and Triton-X100. The
protein release kinetics were determined as a function
of the guanidine, Triton, and cell concentrations. Some
of the advantages over mechanical disruption include
avoidance of extensive fragmentation of the cells and
retention of
the nucleic acids
inside the cell
structure.
The
recent
development o f recombinant DNA t e c h n o l o g y has made i t
f e a s i b l e t o produce i n t e r f e r o n , human growth hormone, i n s u l i n , and
other
proteins
i n the bacterium E s c h e r i c h i a c o l i .
An important
factor complicating
t h e r e c o v e r y p r o c e s s i s t h e r e t e n t i o n o f the
p r o t e i n product i n s i d e the m i c r o b i a l c e l l .
T h i s has n e c e s s i t a t e d t h e
development o f p r o c e s s e s c a p a b l e o f r e l e a s i n g p r o t e i n from E. c o l i .
Protein release
on an i n d u s t r i a l
scale
i s commonly a c h i e v e d
by
mechanically
b r e a k i n g t h e c e l l i n a h i g h p r e s s u r e homogenizer o r a
ball mill.
D i s r u p t i o n i n a h i g h p r e s s u r e homogenizer i s caused by
p r e s s u r e g r a d i e n t s e s t a b l i s h e d when a p r e s s u r i z e d c e l l s u s p e n s i o n i s
f o r c e d t h r o u g h a narrow o r i f i c e , whereas w i t h a b a l l m i l l , d i s r u p t i o n
i s caused by shear f o r c e s g e n e r a t e d by g r i n d i n g t h e c e l l s
with
a b r a s i v e p a r t i c l e s (1.).
These m e c h a n i c a l l y
based p r o t e i n r e l e a s e methods have s e v e r a l
undesirable properties.
One problem i s t h a t e x t e n s i v e
fragmentation
of t h e c e l l s makes t h e subsequent c e n t r i f u g a t i o n d i f f i c u l t
(2,3).
Adding t o the problem o f c e l l fragment removal i s t h e h i g h v i s c o s i t y
imparted t o t h e s o l u t i o n by t h e r e l e a s e d n u c l e i c a c i d s
(4). A
n u c l e i c a c i d removal step
i s necessary t o decrease the s o l u t i o n
viscosity
and a v o i d
potential
interference
with
fractional
p r e c i p i t a t i o n and chromatography (5.).
Another u n d e s i r a b l e
property
i s t h a t t h e h a r s h a c t i o n o f m e c h a n i c a l d i s r u p t i o n causes t h e r e l e a s e
0097-6156/ 86/ 0314-0002$06.00/ 0

1986 American Chemical Society


1.
HETTWER AND WANG
Protein Release from Chemically Permeabilized E. coli
of n e a r l y a l l t h e s o l u b l e c e l l u l a r p r o t e i n .
Extensive p u r i f i c a t i o n
schemes a r e r e q u i r e d t o i s o l a t e t h e p r o d u c t from these e x t r a n e o u s
cellular proteins.
One a l t e r n a t i v e t o m e c h a n i c a l d i s r u p t i o n i s t o t r e a t t h e c e l l s
w i t h membrane a c t i v e compounds t h a t c a n p e r m e a b i l i z e the c e l l t o
protein
without
causing
e x t e n s i v e breakage
o f the c e l l .
The
o b j e c t i v e o f t h i s r e s e a r c h was t o study the p r o t e i n r e l e a s e k i n e t i c s
and mechanism o f a p e r m e a b i l i z a t i o n p r o c e s s u s i n g g u a n i d i n e - H C l and
Triton-XlOO.
Guanidine-HCl,
a
chaotropic
agent,
has been
demonstrated
t o be c a p a b l e o f s o l u b i l i z i n g
p r o t e i n from E. c o l i
membrane fragments
( 6 ) . Presumably,
t h i s occurs v i a guanidine's
i n t e r a c t i o n w i t h water w h i c h a l l o w s h y d r o p h o b i c
groups
t o become
t h e r m o d y n a m i c a l l y more s t a b l e i n an aqueous phase ( 7 ) . T r i t o n - X l O O ,
a n o n i o n i c detergent that has a h i g h b i n d i n g a f f i n i t y f o r hydrophobic
species,
i s very
effective
i n binding
t o and s o l u b i l i z i n g
p h o s p h o l i p i d s from E. c o l i i n n e r membrane and o u t e r w a l l
fragments
(8) .
Methods
C e l l p r e p a r a t i o n . E s c h e r i c h i a c o l i K12, s t r a i n W3110, was grown i n a
14 l i t e r f e r m e n t e r a t 37C, pH 7.0 u s i n g d e f i n e d media.
Additional
n i t r o g e n was s u p p l i e d by NHÔH which was a u t o m a t i c a l l y f e d t o c o n t r o l
the pH. The f e r m e n t a t i o n b r o t h was h a r v e s t e d i n t h e l a t e e x p o n e n t i a l
phase and c o o l e d t o 4C.
The c e l l s were immediately c e n t r i f u g e d a t
4C and washed w i t h b u f f e r (.1M T r i s , pH 7.0).
F o l l o w i n g a second
c e n t r i f u g a t i o n , t h e c e l l s were resuspended i n b u f f e r t o g i v e a dense
c e l l suspension (^50 g p r o t e i n / 1 ) .
Cell permeabilization.
The p e r m e a b i l i z a t i o n p r o c e s s was s t a r t e d by
adding 30 ml o f t h e c e l l s u s p e n s i o n t o 70 ml o f a b u f f e r e d s o l u t i o n
containing
guanidine-HCl
and/or
Triton
X100.
The r e p o r t e d
c o n c e n t r a t i o n s o f T r i t o n , g u a n i d i n e , and c e l l s always c o r r e s p o n d t o
the c o n c e n t r a t i o n s a f t e r m i x i n g t h e s e s o l u t i o n s .
The m i x t u r e was
shaken a t 200 rpm i n a 4C i n c u b a t o r .
Samples were withdrawn a t
v a r i o u s times and were immediately c e n t r i f u g e d .
The s u p e r n a t a n t was
a s s a y e d t o determined t h e r e l e a s e o f t h e v a r i o u s c e l l components.
A n a l y s i s o f t h e p e l l e t was done t o p e r f o r m a mass b a l a n c e .
Analysis
of c e l l
components.
Protein
was determined
w i t h the
B r a d f o r d dye b i n d i n g a s s a y u s i n g b o v i n e serum albumin as s t a n d a r d
(9) . I n t e r f e r e n c e by T r i t o n X100 was accounted f o r by e n s u r i n g t h a t
e v e r y sample had .2% T r i t o n .
I n o r d e r t o determine t h e amount o f
u n r e l e a s e d p r o t e i n from t h e sample p e l l e t s , a l l samples were t r e a t e d
f o r 5 minutes w i t h IN NaOH a t 100C.
DNA was determined by t h e d i p h e n y l a m i n e r e a c t i o n ( 1 0 ) .
Two 45
minute e x t r a c t i o n s a t 70C w i t h ,5N HCIO^ were used t o r e l e a s e
DNA
from the sample p e l l e t s .
I n t e r f e r e n c e from g u a n i d i n e was accounted
f o r by making each sample .4M g u a n i d i n e .
RNA was determined by t h e o r c i n o l p r o c e d u r e ( 1 1 ) . Two 15 minute
e x t r a c t i o n s a t 70C i n ,5N HC10 were used t o r e l e a s e RNA from t h e
sample p e l l e t s .
I n t e r f e r e n c e from T r i t o n X100 was accounted f o r by
making each sample 1% T r i t o n .
4


Results
SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

and
Discussion
F i g u r e 1 shows the p r o t e i n , DNA,

and RNA
release p r o f i l e s
obtained
when E. c o l i c e l l s are m e c h a n i c a l l y d i s r u p t e d w i t h .1 mm g l a s s beads.
The
cell
concentration
profile,
normalized
to
the
initial
cell
c o n c e n t r a t i o n , was o b t a i n e d w i t h a b a c t e r i a l c o u n t i n g chamber.
The
decrease
in
the
cell
concentration
indicates
that
extensive
f r a g m e n t a t i o n o f the c e l l s
i s occurring.
A nearly mirror
image
r e l e a s e of DNA, RNA,
and p r o t e i n r e s u l t s as c e l l u l a r components s p i l l
out
i n t o the
extracellular fluid.
The
maximum p r o t e i n
release,
70%,
i s p r o b a b l y i n d i c a t i v e of a s i g n i f i c a n t amount of
cellular
p r o t e i n b e i n g a s s o c i a t e d w i t h the membrane and w a l l fragments.
A s i m i l a r c h a r a c t e r i z a t i o n f o r c e l l s t r e a t e d w i t h 2M g u a n i d i n e
and 2% T r i t o n i s shown i n F i g u r e 2.
The p r o t e i n r e l e a s e , based on
t o t a l c e l l u l a r p r o t e i n , l e v e l s o f f at
35%.
RNA
i s released to a
l e s s e r e x t e n t (^15%) and v e r y l i t t l e DNA
(^5%)
i s r e l e a s e d from the
c e l l s . The c o n s t a n t c e l l c o n c e n t r a t i o n i n d i c a t e s t h a t the r e l e a s e i s
not the r e s u l t of c e l l f r a g m e n t a t i o n .
From these r e s u l t s , t h r e e major d i f f e r e n c e s between c h e m i c a l
p e r m e a b i l i z a t i o n and m e c h a n i c a l d i s r u p t i o n can be i d e n t i f i e d .
First,
the
release
o c c u r s by
f u n d a m e n t a l l y d i f f e r e n t mechanisms.
With
m e c h a n i c a l d i s r u p t i o n the c e l l s are e s s e n t i a l l y t o r n a p a r t , whereas
w i t h c h e m i c a l t r e a t m e n t the c e l l s t r u c t u r e i s s t i l l p r e s e n t but
has
been a l t e r e d t o a l l o w r e l e a s e o f i n t r a c e l l u l a r components.
Second,
t h e r e i s a n e a r l y complete p r e f e r e n t i a l r e l e a s e of p r o t e i n over
DNA.
T h i r d , t h e r e i s a p a r t i a l s e l e c t i v e r e l e a s e of p r o t e i n over
RNA.
T h i s s e l e c t i v i t y may r e s u l t from a m o l e c u l a r s i e v i n g mechanism.
The
average p r o t e i n m o l e c u l a r weight i s 40,000 whereas the c e l l u l a r
DNA
has
a m o l e c u l a r weight o f 2.5
10
(12).
The m o l e c u l a r weight
d i s t r i b u t i o n of RNA;
18%
i s 25,000, 27%
i s 500,000, and
55%
is
1,000,000 i s such t h a t most o f the RNA
i s also s i g n i f i c a n t l y larger
than p r o t e i n s ( 1 2 ) .
These d i f f e r e n c e s suggest s e v e r a l advantages o f the
chemical
permeabilization
method.
First,
avoiding
cell
breakage
should
s i m p l i f y the c e l l removal s t e p .
Second, r e t e n t i o n of the n u c l e i c
a c i d s i n s i d e the c e l l s h o u l d e l i m i n a t e the need f o r a n u c l e i c a c i d
p r e c i p i t a t i o n step.
Another advantage i s t h a t the
permeabilization
p r o c e s s a l s o k i l l s the c e l l s t h e r e b y e l i m i n a t i n g the need f o r the
f e d e r a l l y mandated c e l l k i l l i n g s t e p .
Figure
2 showed t h a t % 35%
of the
total cellular protein is
r e l e a s e d upon t r e a t i n g the c e l l s w i t h 2M g u a n i d i n e and 2% T r i t o n .
A
more complete d e s c r i p t i o n of ;the e f f e c t o f v a r y i n g the g u a n i d i n e and
T r i t o n concentrations
on the f i n a l
amount of p r o t e i n r e l e a s e d i s
shown i n F i g u r e 3.
Two
s e t s o f e x t r a c t i o n s were c o n d u c t e d :
one
consisted
of
using
2%
Triton
with
a
range
of
guanidine
concentrations,
the o t h e r c o n s i s t e d o f u s i n g 2M
guanidine with a
range o f T r i t o n c o n c e n t r a t i o n s .
These r e s u l t s i n d i c a t e t h a t
the
guanidine-HCl
concentration
is
the
more
sensitive
parameter.
Manipulation
o f the g u a n i d i n e c o n c e n t r a t i o n
i n the p r e s e n c e of
2%
T r i t o n l e a d t o r e l e a s e y i e l d s t h a t ranged from 6% t o 60% whereas
v a r y i n g the T r i t o n c o n c e n t r a t i o n from 0% t o 8% i n the p r e s e n c e of 2M
g u a n i d i n e o n l y changed the y i e l d from 25% t o 40%.
The time p r o f i l e s of the 2M/2%, 2M, and 2% t r e a t m e n t s , shown i n
F i g u r e 4, i n d i c a t e a s y n e r g i s t i c e f f e c t between g u a n i d i n e and T r i t o n .
The p r o t e i n r e l e a s e p r o f i l e o f the 2M/2%
t r e a t m e n t i s not simply the

HETTWER AND WANG
EXTENT OF RELEASE OF CELL COMPONENTS (*),

CONCENTRATION OF UNDISRUPTED CELLS (%)
DNA

" RNA
IJII
5
1
10
11
12
DISRUPTION TIME (min.)
F i g u r e 1.
Extent of c e l l
breakage and r e l e a s e o f c e l l u l a r
p r o t e i n , DNA, and RNA d u r i n g m e c h a n i c a l d i s r u p t i o n w i t h .1 mm
g l a s s beads.
EXTENT OF RELEASE OF CELL COMPONENTS (*),

CONCENTRATION OF UNOISRUPTFn fTLLS (*)
TIME (hours)
F i g u r e 2.
R e l e a s e o f c e l l u l a r p r o t e i n , DNA, and RNA d u r i n g
treatment w i t h 2M g u a n i d i n e HC1 and 2% T r i t o n X100.


FINAL PROTEIN RELEASE (*)
(all
2% Triton X100)

^^^^Xall
1
0
2M Guanidine HC1)
1
2
I I
3
10
TRITON X100 (*v/v), GUANIDINE HC1 (M)
F i g u r e 3. E f f e c t o f T r i t o n X100
release y i e l d .
and g u a n i d i n e HC1 on the p r o t e i n
PROTEIN RELEASE (*)
2/2
2t1 GUANIDINE HC1
2* TRITON X100
Q
0
0
1
5
TIME (hours)
F i g u r e 4.
S y n e r g i s t i c e f f e c t on t h e p r o t e i n
between g u a n i d i n e HC1 and T r i t o n X100.
release

profile

1.
HETTWER AND WANG
a d d i t i o n o f t h e p r o f i l e s o b t a i n e d when 2M g u a n i d i n e and 2% T r i t o n a r e
used i n d i v i d u a l l y . The a c c e l e r a t i o n o f t h e r a t e o f p r o t e i n r e l e a s e
by T r i t o n may be r e l a t e d t o t h e a b i l i t y o f T r i t o n t o s o l u b i l i z e l i p i d
membranes. One would a n t i c i p a t e t h a t t h e c o m b i n a t i o n o f 2M g u a n i d i n e
and 2% T r i t o n a l t e r s t h e E . c o l i i n n e r membrane and o u t e r w a l l t o a
g r e a t e r extent t h a n e i t h e r i n d i v i d u a l t r e a t m e n t , t h e r e b y p r o d u c i n g a
more permeable c e l l .
The
effect of varying
the c e l l
concentration
on t h e p r o t e i n
r e l e a s e p r o f i l e o f 2M/2% t r e a t m e n t s i s shown i n F i g u r e 5. The c e l l
c o n c e n t r a t i o n s a r e e x p r e s s e d i n terms o f t h e p r o t e i n c o n c e n t r a t i o n o f
the e x t r a c t i o n s o l u t i o n . A l t h o u g h no s i g n i f i c a n t e f f e c t was o b s e r v e d
on t h e r e l e a s e p r o f i l e , t h e r e l e a s e y i e l d d e c r e a s e d by a f a c t o r o f
two upon i n c r e a s i n g the c e l l c o n c e n t r a t i o n from 3.6 g/1 t o 43.3 g/1.
The exact n a t u r e o f t h e r e a s o n f o r t h e d e c r e a s e d y i e l d a t h i g h c e l l
concentrations
i s n o t known.
However, d e p l e t i o n o f the g u a n i d i n e
and/or T r i t o n d u r i n g t h e p r o c e s s i s n o t o c c u r r i n g , as e v i d e n c e d by
the f a c t t h a t t r e a t i n g c e l l s a second time w i t h f r e s h g u a n i d i n e and
T r i t o n does n o t induce a d d i t i o n a l r e l e a s e
( d a t a n o t shown). I f
d e p l e t i o n o f t h e g u a n i d i n e and/or T r i t o n caused t h e p r o t e i n r e l e a s e
t o cease, one would expect t h a t a second t r e a t m e n t would cause
f u r t h e r r e l e a s e o f p r o t e i n from t h e p a r t i a l l y a f f e c t e d o r as y e t
unaffected c e l l s .
hole Broth
Protein Cone.
PROTEIN RELEASE (*)
3.6 g/1
12.5
g/1
27.4 g/1
43.3 g/1
TIME (hours)
F i g u r e 5.
profile.
Effect
of c e l l
concentration
on t h e p r o t e i n
release


Conclusions
Exposure o f E. c o l i
t o guanidine-HCl
and T r i t o n - X l O O
induces t h e
r e l e a s e o f c e l l u l a r p r o t e i n s . The r e l e a s e r a t e and y i e l d were found
to be dependent on t h e g u a n i d i n e , T r i t o n , and c e l l c o n c e n t r a t i o n s .
Higher
concentrations
of guanidine
and T r i t o n
and lower
cell
c o n c e n t r a t i o n s gave g r e a t e r r e l e a s e r a t e s and y i e l d s . G u a n i d i n e a l o n e
i s c a p a b l e o f r e l e a s i n g a s i g n i f i c a n t amount o f p r o t e i n .
Triton
r e l e a s e s a v e r y low l e v e l o f p r o t e i n but s u b s t a n t i a l l y i n c r e a s e s t h e
r a t e o f r e l e a s e when used i n c o n j u n c t i o n w i t h g u a n i d i n e .
The mechanism o f t h e r e l e a s e , a p e r m e a b i l i z a t i o n o f t h e c e l l , i s
fundamentally
different
from m e c h a n i c a l d i s r u p t i o n which i n v o l v e s
extensive fragmentation o f the c e l l s .
The a v o i d a n c e o f e x t e n s i v e
c e l l breakage s h o u l d s i m p l i f y t h e c e l l removal s t e p and r e t e n t i o n o f
the n u c l e i c a c i d s i n s i d e t h e c e l l should e l i m i n a t e t h e need f o r a
n u c l e i c acid p r e c i p i t a t i o n step.
F u r t h e r m o r e , s i n c e t h e treatment
k i l l s t h e c e l l s , a s e p a r a t e c e l l k i l l i n g s t e p may be u n n e c e s s a r y .
Acknowledgment
We would l i k e
t o acknowledge
Science Foundation.
partial
support
from
the National
Literature Cited
1.
Edebo, L. In 'Fermentation Advances'; Perlman, D., Ed.;

Academic Press: New York, 1969; p. 249.
2. Schutte, H; Kroner, K. H.; Hustedt, H.; Kula, M. R. Enzyme
Microb. Technol. 1983, 5, 143.
3. Bucke, C. In 'Principles of Biotechnology'; Wiseman, ., Ed.;
Surrey University Press: New York, 1983; p. 151.
4. Higgins, J. J.; Lewis, D. J.; Daly, W. H.; Mosqueira, F. G.;
Dunnill, P.; Lilly, M. D. Biotech. Bioeng. 1978, 20, 159.
5. Wang, D. I. C.; Cooney, C. L.; Demain, .; Dunnill, P.;
Humphrey, .; L i l l y , M. In 'Fermentation and Enzyme
Technology'; John Wiley Sons: New York, 1979; Chap. 12.
6. Moldow, C. J. Membrane Biol. 1972, 10, 137.
7. Hatefi, Y.; Hanstein, W. In 'Methods in Enzymology'; Fleischer,
S.; Packer, L.; Eds.; Academic Press: New York, 1974; p. 770.
8. Schnaitman, C. J. Bact. 1971, 108(1), 545.
9. Bradford, M. Anal. Bioohem. 1976, 72, 248.
10. Burton, K. Biochem. J. 1956, 62, 315.
11. Herbert, D.; Phipps, P. J.; Strange, R. E. In 'Methods in
Microbiology'; Norris, J. R.; Ribbons, D. W.; Eds.; Academic
Press: London, 1971; p. 210.
12.
Brock, T. In 'Biology of Microorganisms'; Prentice-Hall:
Englewood Clifts, New Jersey, 1979; p. 131.
RECEIVED March 26, 1986

2
Structured and Simple Models of Enzymatic Lysis and
Disruption of Yeast Cells

J. B. Hunter and J. A. Asenjo

Biochemical Engineering Laboratory, Department of Chemical Engineering and Applied
Chemistry, Columbia University, New York, NY 10027
Microbial cell-wall-lytic enzymes are widely

used in the laboratory for cell breakage, proto-plasting of yeasts and bacteria, and for studies
of the structure and composition of microbial
cell walls (1). Recently lytic systems have come
under consideration as a specific and chemically
mild way to rupture microbial cells on an industrial scale (2,3). There appear to be attractive commercial applications of lytic systems
for the recovery of enzymes, antigens and
other recombinant products accumulated within
cells, for upgrading of microbial biomass for food
and feed uses (4,5) and for the manufacture of
functional biopolymers from cell wall carbohydrates (6).
This paper presents two models of enzymatic
lysis of yeast cells; a simplified two-step model,
accounting for protein release at cell lysis
followed by proteolysis, and a more complex mechanistic model which describes the removal of the
two layers of the yeast wall and the extrusion
and rupture of the protoplast and organelles.
The use of these models in predicting the release
and breakdown of microbial proteins, and the application of the structured model to enzyme recovery will also be discussed.
One p r o b l e m i n p r o d u c t i o n o f r e c o m b i n a n t p r o t e i n s i s r e c o v e r y o f
the f i n i s h e d product from the c e l l s which accumulate i t . T h i s
problem i s p a r t i c u l a r l y acute i n the case o f y e a s t s and f u n g i , which
have tough, t h i c k c e l l w a l l s which are d i f f i c u l t t o r u p t u r e mechanic a l l y o r by s o n i c a t i o n .
Product s e c r e t i o n i s not always f e a s i b l e ,
even f o r l o w - m o l e c u l a r - w e i g h t p r o d u c t s , a l t h o u g h a newly developed
s e c r e t i o n p r o c e s s f o r y e a s t (7) a p p e a r s p r o m i s i n g .
0097-6156/ 86/ 0314-0009$06.50/ 0


10
There a r e numerous examples of overproduced r e c o m b i n a n t p r o t e i n s

w h i c h p r e c i p i t a t e i n t r a c e l l u l a r l y i n E_. c o l i , f o r m i n g d e n s e i n c l u s i o n
b o d i e s ( 8 ) ; t h e s e p r o d u c t s i n c l u d e i n s u l i n and s o m a t o s t a t i n , b o t h
very small p r o t e i n s . In yeast, recombinant v i r a l surface antigen
p r o t e i n s a r e n o t s e c r e t e d , b u t a s s e m b l e i n t o p a r t i c l e s ( 9 ) . Subc e l l u l a r s t r u c t u r e s such as m i t o c h o n d r i a , l y s o s o m e s o r t h e v a c u o l e
must a l s o be r e c o v e r e d by c e l l b r e a k a g e , f o r use e i t h e r as b i o c a t a l y s t s (10) o r as an i n i t i a l s t e p i n t h e p u r i f i c a t i o n o f enzymes
a s s o c i a t e d w i t h s u c h s t r u c t u r e s . U n t i l now, t h e s e p r o d u c t s h a v e g e n e r a l l y b e e n h a r v e s t e d by m e c h a n i c a l l y r u p t u r i n g t h e c e l l s i n a
homogenizer, bead m i l l or French p r e s s .
The h i g h s h e a r f i e l d s , e l e v a t e d t e m p e r a t u r e s and g a s - l i q u i d i n t e r f a c e s g e n e r a t e d i n t h e s e
d e v i c e s can denature p r o t e i n s , e s p e c i a l l y multi-enzyme complexes
and m e m b r a n e - l i n k e d p r o t e i n s (11) . M o r e o v e r , t h e s e p a r a t i o n
of c e l l d e b r i s from the products i s e s p e c i a l l y complicated i f the
product i s p a r t i c u l a t e , f r a g i l e or membrane-associated.
L y t i c enzyme s y s t e m s p r o v i d e a c h e m i c a l l y m i l d , l o w - s h e a r
and c a t a l y t i c a l l y s p e c i f i c a l t e r n a t i v e t o m e c h a n i c a l c e l l d i s ruption.
D e p e n d i n g on t h e p a r t i c u l a r l y t i c s y s t e m e m p l o y e d and
i t s p u r i t y , t h e e n z y m e s may be e n g i n e e r e d t o a t t a c k c e l l w a l l comp o n e n t s a l o n e , w i t h o u t p r o d u c t damage.
The e n z y m e l y s o z y m e ,
active
a g a i n s t some b a c t e r i a l c e l l w a l l s , h a s b e e n u s e d t o h a r v e s t b o v i n e
g r o w t h h o r m o n e g r a n u l e s f r o m _E. c o l i ( 8 ) , a n d a m e m b r a n e - a s s o c i a t e d
h y d r o x y l a s e c o m p l e x f r o m P. p u t i d a ( 1 1 ) ; u s e o f o t h e r b a c t e r i o l y t i c enzymes f r o m a v a r i e t y o f m i c r o b i a l s o u r c e s h a v e a l s o been
reported (3).
I n v e s t i g a t i o n s i n t o t h e s u b c e l l u l a r l o c a t i o n o f enzyme a c t i v i t i e s i n m i c r o b i a l c e l l s s u g g e s t t h a t one o r more enzyme p r o d u c t s
c o u l d be s p e c i f i c a l l y f r a c t i o n a t e d f r o m a s i n g l e b a t c h o f c e l l s
by p r o p e r l y c o n t r o l l i n g c e l l d i s r u p t i o n .
Invertase i n yeast i s
p o s s i b l y the best example of t h i s p r i n c i p l e .
The s t u d i e s l e a d i n g
to d i s c o v e r y of i t s l o c a t i o n ( i n the p e r i p l a s m i c space) have
b e e n s u m m a r i z e d by P h a f f ( 1 2 ; p . 1 7 1 - 1 7 3 ) , and a s a m p l e p r o c e s s f o r
i t s r e c o v e r y h a s b e e n p r o p o s e d ( 4 ) . The r e c o v e r y o f s e v e r a l d i f f e r e n t enzymes i n h i g h y i e l d and h i g h r e l a t i v e p u r i t y s h o u l d be
p o s s i b l e u s i n g a c o m b i n a t i o n o f l y t i c enzymes, s u r f a c t a n t s and o s m o t i c s u p p o r t b u f f e r s t o s e l e c t i v e l y and s e q u e n t i a l l y r e l e a s e p r o t e i n s from p a r t i c u l a r s t r u c t u r e s .
C e l l f r a c t i o n a t i o n b y m e c h a n i c a l r u p t u r e h a s a l r e a d y come u n d e r
investigation.
Two s e p a r a t e s t u d i e s o f m e c h a n i c a l r u p t u r e o f y e a s t
showed d i f f e r e n t r a t e s o f r e l e a s e f o r enzymes i n d i f f e r e n t c e l l
l o c a t i o n s (13,14).
W a l l - l i n k e d and p e r i p l a s m i c enzymes w e r e r e leased r e l a t i v e l y f a s t e r than t o t a l p r o t e i n , s o l u b l e cytoplasmic
e n z y m e s a t a b o u t t h e same r a t e , a n d t h e m i t o c h o n d r i a l e n z y m e f u m a r a s e
l a t e r t h a n t o t a l p r o t e i n (13) . P r o t e o l y s i s by t h e y e a s t s
own
e n z y m e s was n o t f o u n d t o b e a p r o b l e m .
A c t i v i t i e s of the r e l e a s e d
e n z y m e s d e c l i n e d s l o w l y o r n o t a t a l l w h e n d i s r u p t i o n was
cont i n u e d a f t e r t h e end o f p r o t e i n r e l e a s e , and t h e e f f e c t o f s h e a r
was
not separated from the e f f e c t of p r o t e o l y s i s .
S h e t t y and K i n s e l l a
(15) a l s o f o u n d a l o w r a t e o f p r o t e o l y s i s a f t e r m e c h a n i c a l d i s r u p t i o n ,
though t h i o l r e a g e n t s added t o weaken the c e l l w a l l s b e f o r e d i s r u p t i o n c a u s e d an i m p o r t a n t i n c r e a s e i n t h e e x t e n t of p r o t e i n b r e a k down .
1

2.
HUNTER AND ASENJO
Model
Enzymatic Lysis and Disruption of Yeast Cells
11
Background
Yeast c e l l structure.
The e x t e n s i v e body o f l i t e r a t u r e on c e l l
c o m p o s i t i o n and s t r u c t u r e has r e c e n t l y been reviewed b y B a l l o u
and e a r l i e r by Phaff (12).
A s a n e n g i n e e r i n g a p p r o x i m a t i o n , t h e c e l l w a l l o f y e a s t may b e
c o n s i d e r e d a s a t w o - l a y e r s t r u c t u r e . ( F i g u r e 1) T h e i n n e r w a l l i s
composed o f a m i x t u r e o f b r a n c h e d 3(1-3) a n d 3(1-6) l i n k e d g l u c a n s ,
glucose polymers s i m i l a r t o c e l l u l o s e (12).
The o u t s i d e o f t h e
g l u c a n l a y e r i s covered w i t h a mannan-protein complex c o n s i s t i n g
of a c r o s s - l i n k e d n e t w o r k o f p r o t e i n m o l e c u l e s , t o w h i c h a r e a t t a c h e d two t y p e s o f mannan: a n a c i d i c o l i g o s a c c h a r i d e , a n d a h i g h e r
m o l e c u l a r w e i g h t p h o s p h o m a n n a n h a v i n g a d.p. o f a b o u t 1 0 0 ( 1 7 ) .
From t h e p e r s p e c t i v e o f c e l l l y s i s , t h i s m a n n o p r o t e i n l a y e r s e r v e s
to p r o t e c t t h e g l u c a n s from h y d r o l y t i c enzymes (18,19,20).
Within
t h e t w o w a l l l a y e r s i s t h e p r o t o p l a s t , c o m p r i s e d o f a p l a s m a membrane e n c l o s i n g the c y t o s o l and the s u b c e l l u l a r s t r u c t u r e s .

wall
(16)
Enzymes o f t h e l y t i c system.
M i c r o b i a l y e a s t - l y t i c enzyme
systems are w i d e l y d i s t r i b u t e d i n n a t u r e , and have been i s o l a t e d from
R h i z o c t o n i a sp., ( 4 ) , B a c i l u s c i r c u l a n s (21), Coprinus macrorhizus
(22),
a n d C y t o p h a g a s p . ( 2 3 ) , among o t h e r s o u r c e s .
C r u d e y e a s t l y t i c enzyme s y s t e m s c o m p r i s e s e v e r a l h y d r o l y t i c
a c t i v i t i e s , o f t e n i n c l u d i n g c h i t i n a s e , mannanase, and a v a r i e t y o f
p r o t e a s e s a n d g l u c a n a s e s ( 1 ) . Only two o f t h e s e a c t i v i t i e s , a l y t i c
protease anda l y t i c glucanase, are e s s e n t i a l f o r l y s i s
(19,24,20).
L y t i c glucanases u s u a l l y b i n d p r e f e r e n t i a l l y t o l o n g c h a i n s o f 3(1-3)
g l y c o s i d i c l i n k a g e s , such a s those found i n m i c r o f i b r i l l a r y e a s t w a l l
glucan.
I n g e n e r a l , the l y t i c g l u c a n a s e s have a n endo- a c t i o n
p a t t e r n b u t some a t t a c k e x o - w i s e , r e l e a s i n g o l i g o s a c c h a r i d e s o f
5 glucose u n i t s from the s t r u c t u r a l yeast glucan.
Other glucanases,
with d i f f e r e n t substrate s p e c i f i c i t y anda c t i o n p a t t e r n s , are u s u a l l y
p r e s e n t i n t h e l y t i c s y s t e m and a c t s y n e r g i s t i c a l l y t o d e g r a d e
i n s o l u b l e y e a s t g l u c a n t o g l u c o s e a n d d i s a c c h a r i d e s (25) .
Lytic
p r o t e a s e s have a c h a r a c t e r i s t i c h i g h a f f i n i t y f o r the y e a s t w a l l s u r f a c e , and o f t e n have anomalously low a c t i v i t i e s a g a i n s t c o n v e n t i o n a l
protein substrates. Their role i n l y s i s of viable yeast c e l l s
cannot be s u b s t i t u t e d by o r d i n a r y p r o t e a s e s . (20,26).
We u s e d a l y t i c s y s t e m f r o m O e r s k o v i a x a n t h i n e o l y t i c a L L - G 1 0 9
f r o m t h e c o l l e c t i o n o f M. L e c h e v a l i e r , a t R u t g e r s U n i v e r s i t y .
F i l t e r e d c u l t u r e b r o t h was u s e d a s t h e enzyme s o u r c e .
Details of
t h e enzyme p r o d u c t i o n a r e g i v e n e l s e w h e r e ( 2 7 , 2 8 ) .
The l y t i c a c t i v i t y o f the O e r s k o v i a system i s due t o a l y t i c p r o t e a s e and an
endo 3 ( 1 , 3 ) g l u c a n a s e ( 2 0 ) , p o s s i b l y s u p p l e m e n t e d w i t h a n exo 3 ( 1 - 3 )
glucanase removing a 5-sugar u n i t from the c h a i n (29).
Sequence o f c e l l l y s i s .
Enzymatic c e l l l y s i s begins w i t h b i n d ing o f the l y t i c protease t o the outer mannoprotein l a y e r o f t h e
wall.
The p r o t e a s e opens up t h e p r o t e i n s t r u c t u r e , r e l e a s i n g w a l l
p r o t e i n s and mannans, a n d e x p o s i n g t h e g l u c a n s u r f a c e b e l o w ( F i g u r e
2).
Next, the glucanase a t t a c k s the i n n e r w a l l and s o l u b i l i z e s the
g l u c a n ( 1 9 ) . When t h e c o m b i n e d a c t i o n o f p r o t e a s e a n d g l u c a n a s e
has opened a s u f f i c i e n t l y l a r g e h o l e i n the c e l l w a l l , the plasma
membrane a n d i t s c o n t e n t s a r e e x t r u d e d a s a p r o t o p l a s t ( 1 ) . I n
o s m o t i c a l l y s u p p o r t e d b u f f e r s c o n t a i n i n g .55 t o 1.2M s u c r o s e o r

12

Mannoprotein Units
Cell Membrane
Figure 1
Structural Glucan Units
Double-layered structure o f the yeast w a l l ,

t h e c e l l membrane
Figure
Schematic o f l y s i n g
yeast
enclosing
cell

2.
HUNTER AND
ASENJO
13
m a n n i t o l , the p r o t o p l a s t remains i n t a c t but i n d i l u t e b u f f e r s i t l y ses i m m e d i a t e l y , r e l e a s i n g c y t o p l a s m i c p r o t e i n s and the

organelles
w h i c h may t h e m s e l v e s l y s e .
Meanwhile, p r o t e i n s r e l e a s e d from the w a l l and the cytoplasm are
subject t o a t t a c k by product-degrading p r o t e a s e contaminants i n the
l y t i c system (28,30).

Models
Mathematical models w i t h d i f f e r e n t l e v e l s o f s t r u c t u r e are usef u l f o r the d e s i g n o f r e a c t o r s , t o c a r r y out s i m u l a t i o n s t u d i e s , f o r
p r o c e s s o p t i m i z a t i o n and f o r i n c r e a s i n g our u n d e r s t a n d i n g o f t h e
mechanistic, b i o l o g i c a l behavior o f biochemical
systems.
H i s t o r i c a l l y t h e r e has been l i t t l e p u b l i s h e d work on models o f
microbial cell lysis.
The models proposed f o r o v e r a l l c e l l l y s i s
have been e l e m e n t a r y and t h e i r a p p l i c a t i o n has been l i m i t e d .
Firsto r d e r and M i c h a e l i s - M e n t e n m o d e l s h a v e b e e n u s e d t o e s t i m a t e t h e
performance o f a sample l y s i s p r o c e s s (2 3) , L y s i s o f f r e e z e - d r i e d
M i c r o c o c c u s l y s o d e i k t i c u s c e l l s b y l y s o z y m e was modeled w i t h a
second-order rate expression
(31) . A t t h e o t h e r e n d o f t h e s p e c t r u m
of mathematical c o m p l e x i t y i s a model o f l y s o z y m e - c a t a l y z e d degradat i o n o f s o l u b l e b a c t e r i a l c e l l - w a l l o l i g o s a c c h a r i d e s , f o c u s i n g on the
d e g r e e o f p o l y m e r i z a t i o n o f t h e s u b s t r a t e a n d t h e b i n d i n g modes o f
enzyme t o s u b s t r a t e s ( 3 2 ) .
A c c o u n t i n g f o r one enzyme a n d c a r b o h y d r a t e o l i g o m e r s u p t o d.p. 9, i t h a s n i n e d i f f e r e n t i a l e q u a t i o n s a n d
t e n p a r a m e t e r s , and was t e s t e d o n p u r i f i e d r a d i o l a b e l e d
oligosaccharides.
A l t h o u g h u s e f u l f o r e l u c i d a t i n g enzyme a c t i o n p a t t e r n s , s u c h
models are too d e t a i l e d t o be r e a d i l y a p p l i e d t o a multi-enzyme,
m u l t i - s u b s t r a t e system.
The t w o m o d e l s o f y e a s t l y s i s p r e s e n t e d h e r e
have been d e v e l o p ed t o s e r v e t w o d i f f e r e n t p u r p o s e s .
The s i m p l e model i s a lumped,
t w o - s t e p m o d e l w h i c h f o l l o w s t h e m a j o r f e a t u r e s o f t h e d a t a a n d may
prove u s e f u l f o r design o f l y s i s reactors.
The s t r u c t u r e d model,
w h i c h can a c c o u n t f o r the s o u r c e o f p r o t e i n w i t h i n the c e l l , was
developed t o g a i n a m e c h a n i s t i c b a s i s f o r p r e d i c t i n g the e f f e c t s o f
u n t e s t e d p r o c e s s c o n d i t i o n s , and t o a i d i n s i g h t i n t o the p h y s i c a l
p r o c e s s e s a t work d u r i n g l y s i s .
S
Simple model.
The s i m p l e model was b u i l t f o r compact d e s c r i p t i o n o f t h e d a t a i n a p r e - d e t e r m i n e d r a n g e o f y e a s t a n d enzyme c o n c e n trations.
I t t r e a t s c e l l l y s i s and p r o t e o l y s i s as s i n g l e - s t e p
r e a c t i o n s i n sequence.
Both r e a c t i o n s are modeled w i t h M i c h a e l i s Menten k i n e t i c s , even though y e a s t , the s u b s t r a t e o f the f i r s t
r e a c t i o n , i s p a r t i c u l a t e and the p r o t e i n s are s o l u b l e .
The d i f f e r e n t
enzymes o f t h e l y t i c s y s t e m a r e g r o u p e d t o g e t h e r i n t o a n a l l - i n c l u s i v e s i n g l e enzyme, E , b e a r i n g b o t h t h e p r o t e o l y t i c and y e a s t - l y t i c
activities.
A l l o f the c e l l s t r u c t u r e s are a l s o considered
together
a s a u n i f i e d y e a s t c e l l m a s s , Y.
When a c e l l i s a t t a c k e d b y e n z y m e s i t i s p r e s u m e d t o d i s s o l v e
i n s t a n t a n e o u s l y , r e l e a s i n g i t s e n t i r e mass a s s o l u b l e p r o t e i n s , p e p t i d e s andcarbohydrates.
The assumption o f i n s t a n t a n e o u s s o l u t i o n
o f t h e c e l l mass c o n s t r a i n s t h e m o d e l f o r use w h e r e t h e l y s i s medium
i s hypo-osmotic andp r o t o p l a s t s cannot s u r v i v e i n t a c t .

14
SEPARATION, RECOVERY, A N D PURIFICATION IN BIOTECHNOLOGY
O n l y two i n d e p e n d e n t v a r i a b l e s a r e u s e d : y e a s t ( Y ) a n d e n z y m e
(E);
t h e measured v a r i a b l e s a r e y e a s t , T C A - i n s o l u b l e p r o t e i n (),
T C A - s o l u b l e p r o t e i n ( p e p t i d e s , S ) , and c a r b o h y d r a t e s
(C); a l l are
e x p r e s s e d a s g/1 d r y b a s i s .
E n z y m e c o n c e n t r a t i o n was e x p r e s s e d a s
t h e v o l u m e p e r c e n t o f c r u d e l y t i c enzyme p r e p a r a t i o n added t o t h e
reaction mixture.
P r o t e o l y t i c and o t h e r c a u s e s f o r l y t i c enzyme
d e a c t i v a t i o n ( e . g . , t h e r m a l ) h a v e been assumed t o be n e g l i g i b l e ( 2 8 ) ,
k E'(Y
dY

dt
-k (Y-Y
a
(Y -
dP _
dY
>
" py
dt
dS _
-f
sy
dt
dC
Yco)
Yco)
k E-P
(2)
dt,
+ s +
dY/
k
_J2
+ S + K
mp 1 + -
dt,
(1)
1+
Y *
(3)
dY'
"~fcy
dt
(4)
.dt,
V a r i a b l e names and p a r a m e t e r v a l u e s
are given
i n Table
I.
On t h e r i g h t - h a n d s i d e o f e q u a t i o n 1, t h e i n i t i a l t e r m r e p r e
s e n t s a u t o l y s i s and t h e second t e r m , e n z y m a t i c l y s i s .
Equation 2
d e s c r i b e s p r o t e i n breakdown by p r o d u c t - d e g r a d i n g
proteases.
The
f i r s t t e r m on t h e r i g h t s i d e s t a n d s f o r t h e p r o t e i n r e l e a s e d f r o m
l y s i n g c e l l s , and t h e second term, breakdown o f t h e p r o t e i n a l r e a d y
in solution.
E q u a t i o n 3 shows t h a t p e p t i d e s a r e r e l e a s e d f r o m l y s i n g
y e a s t , b u t a l s o a r i s e f r o m b r e a k d o w n o f l o n g e r p r o t e i n s , P.
Since the protease a c t i v i t y against s o l u b l e p r o t e i n s i s con
s i d e r e d n o n - s p e c i f i c , b o t h l o n g - and s h o r t - c h a i n p r o t e i n s w i l l be
a t t a c k e d b y t h e e n z y m e w i t h e s s e n t i a l l y t h e same a f f i n i t y p e r g r a m
of s u b s t r a t e .
Hence, S w i l l a c t as a c o m p e t i t i v e i n h i b i t o r o f t h e
e n z y m e a c t i v i t y a g a i n s t P, w h e r e t h e i n h i b i t i o n c o n s t a n t i s e q u a l
to t h e M i c h a e l i s c o n s t a n t K ^ .
C a r b o h y d r a t e r e l e a s e i s shown i n
equation
4.
Parameters f o r t h e s i m p l e model were d e t e r m i n e d g r a p h i c a l l y by
Eadie-Hofstee p l o t t i n g of i n i t i a l r e a c t i o n rates
and s u b s t r a t e c o n
centrations.
D e t a i l s a r e g i v e n e l s e w h e r e (30).
As has been ob
served i n h y d r o l y s i s of other s o l i d s u b s t r a t e s , a r e s i d u e of nonl y s e d s u b s t r a t e was f o u n d a t e x t e n d e d r e a c t i o n t i m e s , w h e n d Y / d t
tended toward zero.
The e x t e n t o f r e a c t i o n was s t r o n g l y d e p e n d e n t
on i n i t i a l s u b s t r a t e and enzyme c o n c e n t r a t i o n s ( 3 3 , 3 4 ) .
An
e m p i r i c a l f u n c i t o n f o r Y^ was f i t t e d t o t h e u l t i m a t e t u r b i d i t y d a t a
f o r l y s i s r u n s a t a v a r i e t y o f i n i t i a l y e a s t and enzyme c o n c e n
t r a t i o n s u s i n g a l e a s t squares method.
The c a l c u l a t e d v a l u e s f o r
Yoo w e r e u s e d i n t h e s i m u l a t i o n s (30) . F i g u r e 3 s h o w s r e s u l t s o f
the simple model.
S t r u c t u r e d model.
T h i s model c o n s i d e r s l y s i s of t h e c e l l from
the v i e w p o i n t of p r o g r e s s i v e breakdown of the c e l l s t r u c t u r e s ,
s t a r t i n g f r o m t h e o u t e r w a l l l a y e r and p r o g r e s s i n g t o t h e s u b c e l l u l a r
s t r u c t u r e s i n s i d e t h e p r o t o p l a s t (35).
Here the c e l l i s d i v i d e d i n t o

HUNTER AND ASENJO
2.
Table
L.
15
Lumped m o d e l v a r i a b l e s a n d p a r a m e t e r s
V a r i a b l e s - Simple Model
Y
Y e a s t , mg/1
Y
Original yeast concentration
Yoo
U l t i m a t e y e a s t c o n c e n t r a t i o n , mg/1; p r o p o r t i o n a l
0

to
residual
turbidity.
^= a 4 b E + c Y
S
C
P r o t e i n ( T C A - i n s o l u b l e ) , mg/1
P e p t i d e s ( T C A - s o l u b l e ) , mg/1
C a r b o h y d r a t e s , mg/1
Enzyme, % ( v / v ) o f r e a c t i o n m i x t u r e
Parameters-Simple
Yoo c o n s t a n t s :
a:
b:
c:
d:
Model
3.6342 1 0 "
- 2 . 6 5 8 4 10 ~
6
6.0442 ""
-9.9603 1 0
Rate constant
for autolysis
Rate constant
for lysis,
M i c h a e l i s constant
3.987 1 0 - ^ m i n "
s i m p l e m o d e l 15.51 mg/L-min-%ez
for lysis,
for proteolysis,
1902 mg/L
kp
Rate constant
Michaelis constant, proteolysis,
4 5 9 8 mg/L
Inhibition
2 6 0 7 7 mg y e a s t / L
constant, proteolysis,
Fraction ofprotein
i n yeast
4.441
Fraction of peptides i n yeast
0.0777
Fraction of carbohydrates
0.3687
i n yeast
mg/L-min-%ez
0.4048
f y
g

16
SEPARATION, RECOVERY, AND
PURIFICATION IN BIOTECHNOLOGY

f o u r r e g i o n s ; t h e o u t e r w a l l o r w a l l p r o t e i n (WP); i n n e r w a l l o r w a l l
g l u c a n (WG) ; t h e c y t o s o l (CS) a n d t h e o r g a n e l l e s , h e r e g r o u p e d t o g e t h e r
as m i t o c h o n d r i a ( M I ) .
The l y t i c s y s t e m i s a p p r o x i m a t e d a s t h r e e
enzymes, a l y t i c g l u c a n a s e , Eg, w h i c h h y d r o l y z e s the i n n e r c e l l w a l l
g l u c a n , a l y t i c p r o t e a s e , Ep, w h i c h a t t a c k s o n l y t h e o u t e r w a l l l a y e r
and a d e s t r u c t i v e
p r o t e a s e , E^, a c t i v e a g a i n s t s o l u b l e p r o t e i n s .
P r o d u c t i n h i b i t i o n i s i n c l u d e d i n a l l enzyme r e a c t i o n s .
Adsorption
and d e s o r p t i o n o f t h e e n z y m e s t o t h e y e a s t w a l l i s n e g l e c t e d ,
since
a d s o r p t i o n k i n e t i c s a p p e a r e d i n s t a n t a n e o u s on t h e t i m e s c a l e o f o u r
measurements (35).
A s c h e m a t i c o f t h e r e a c t i o n p a t h w a y s i s shown i n
Figure
4.
Special
EGA
variables.
=
(WG
r-WP)
The g l u c a n h y d r o l y s i s r a t e i s n o t r e l a t e d d i r e c t l y t o t o t a l
g l u c a n c o n c e n t r a t i o n WG, b u t r a t h e r t o t h e a m o u n t o f g l u c a n made
a c c e s s i b l e to a t t a c k through removal of w a l l p r o t e i n from the
outside
of the c e l l .
EGA,
" e x p o s e d g l u c a n , a c c e s s i b l e " r e p r e s e n t s t h e amount
o f g l u c a n u n c o v e r e d by r e m o v a l o f t h e o u t e r w a l l .
The p r o p o r t i o n a l i t y
constant r i s the weight r a t i o of w a l l glucan to w a l l p r o t e i n .
PBR
The o v e r a l l r a t e o f s o l u b l e p r o t e i n h y d r o l y s i s , PBR,
protein
breakdown r a t e , a c c o u n t s f o r d e s t r u c t i o n o f s o l u b l e p r o t e i n by
the
destructive protease.
The r e l e a s e o f c y t o s o l i n t o t h e m e d i u m d e p e n d s o n t h e o s m o t i c
breakage of the p r o t o p l a s t s , which occurs at a r a t e approximately
p r o p o r t i o n a l t o t h e o s m o t i c g r a d i e n t a c r o s s t h e p l a s m a membrane ( 3 6 ) .
The i n t e r n a l o s m o l a l i t y o f t h e c e l l s was e s t i m a t e d t o b e 0.617
Os/L
( 3 5 ) , w h e r e 1 Os/L i s e q u i v a l e n t t o 1 M o l / L o f a n i d e a l s o l u t e .
The
e x t e r n a l o s m o l a l i t y i s t h e sum o f t h e c o n t r i b u t i o n f r o m t h e b u f f e r
s y s t e m i n t h e m e d i u m ( a b o u t 0.02M i n o u r e x p e r i m e n t s ) a n d
the
s u b s t a n c e s r e l e a s e d by l y s i n g p r o t o p l a s t s .
The s t a b i l i z a t i o n o f t h e
r e m a i n i n g c e l l s by t h e s e s u b s t a n c e s i s f a r s t r o n g e r t h a n c o u l d be
e x p e c t e d s o l e l y on t h e b a s i s o f o s m o t i c e f f e c t s , a n d c o u l d r e s u l t
from the r e l e a s e of c a t i o n s which i n t e r a c t w i t h s p e c i f i c
receptors
o n t h e p l a s m a membrane ( 3 7 ) .
The r e l e a s e o f s o l u b l e p r o d u c t s o f
g l u c a n a n d p r o t e i n h y d r o l y s i s a r e a l s o e x p e c t e d t o add t o t h e s t a b i l i
z i n g e f f e c t of the l y s a t e .
The e f f e c t i v e o s m o l a l i t y o f c e l l l y s a t e was f i t t o a L a n g m u i r
e x p r e s s i o n , w h e r e 0 S M i s t h e maximum s t a b i l i z i n g e f f e c t and
is
the e q u i l i b r i u m constant f o r i n t e r a c t i o n of the s t a b i l i z e r s w i t n
the
protoplasts.
The r e s u l t i n g e q u a t i o n ,
L
C S
C S
V
o s J
* +
o ~
>
1 +
( C S * + CS - CS)
osm
e x p r e s s e s t o t a l e f f e c t i v e o s m o l a l i t y i n the l y s i s medium. B
i s the
o r i g i n a l o s m o l a l i t y o f t h e l y s i s b u f f e r a n d CS* i s t h e sum
of p r o t e i n ,
p e p t i d e s and c a r b o h y d r a t e s p r e s e n t a t t h e s t a r t o f r e a c t i o n .
0SM
= B

HUNTER AND ASENJO
en
s
TIME - MINUTES
Figure
Simple model s i m u l a t i o n o f yeast l y s i s

Y e a s t c e l l m a s s , mg/1
mg/1
P e p t i d e s , mg/1
h y d r a t e s , mg/1
0.78 g/1 y e a s t c o n c e n t r a t i o n ; 1 0 % enzyme
Oligopeptides
Amino Acids
Soluble Proteins
Cytoplasmic Enzymes
- Carbo-
1. Protease attack
2. Glucanase attack
3. Release of
cell contents
4. Lysis of organelles
5. Glucan hydrolysis
6
Wall Protein
Wall Enzymes
Wall Mannan
Protein,
7. > Product proteolysis
/-\
Proteins
Organelles-<>- Organellar Enzymes
Carbohydrates
fl(l-3) Oligosaccharides
Glucose
Figure
Reaction
pathways f o r s t r u c t u r e d model

18
Based on t h e p r o d u c t OSM[/K
the s t a b i l i z i n g e f f e c t o f c e l l l y
s a t e a t l o w c o n c e n t r a t i o n s i s e q u i v a l e n t t o 4.4 10
Os/mg c y t o s o l
released (35).
c
Wall
hydrolysis
equations.
d(WP)
dt
1

WP
(Km
wjy_
WP
- WP
Ki
wp
wp
WP
Km
wp
(1)
EGA
wg g (Km
wg
WG
- WG
l + # ^
+
Km
Km
sg
wg
k
d(WG)
dt
(2)
R e l e a s e o f c y t o s o l and m i t o c h o n d r i a .
The o s m o t i c g r a d i e n t b e
tween p r o t o p l a s t s and b u f f e r o r m i t o c h o n d r i a and b u f f e r d r i v e s t h e
r e l e a s e o f p r o t e i n i n t o t h e medium. I f t h e o s m o l a l i t y o f t h e e x t e r n a l
medium e x c e e d s t h e i n t e r n a l o s m o l a l i t y o f t h e p r o t o p l a s t o r o r g a n e l l e ,
no r u p t u r e o c c u r s .
The o s m o l a l i t y d e c r e a s e s i n t e r n a l l y , and i n c r e a s e s
e x t e r n a l l y , as m a t e r i a l i s r e l e a s e d from t h e p r o t o p l a s t .
In addition,
the r e l e a s e o f c y t o s o l i s p r o p o r t i o n a l t o t h e s i z e o f t h e opening i n
t h e w a l l g l u c a n , up t o a maximum h o l e s i z e o f 1/3 o f t h e c e l l ^
sur
face area.
d(CS)
dt
(CS).k
(CS)
d(MI)
dt
f l
k^maxÔSM^
CS,
d(CS)
' dt
CS
CS
r m
- 0SM )] max(.33, 1
x
WG
)
WG
[ m a x ( 0 , 0.3 - OSK^.) ]
(3)
(4)
Soluble products.
V a l u e s f o r T C A - i n s o l u b l e p r o t e i n , p e p t i d e s and
c a r b o h y d r a t e s r e l e a s e d w e r e e s t i m a t e d b y summing t h e c o n t r i b u t i o n t o
each p o o l from t h e breakdown o f each c e l l u l a r s t r u c t u r e .
^
dt
- - f
pwp
f
pm
d(WP)
[ dt
- f
pes
'd(CS)'
dt
k
[ m a x ( 0 , 0.3 - OSM ) ] - PBR
rm

(5)
2.
HUNTER AND ASENJO
'd(WP)'
- - f
[dt
swp
^
dt
f
sm

d
dt
19
- f
d(CS)l
ses
dt
-k [ m a x ( 0 , 0.3 - OSM ) ] - M I + P B R
rm
_ d(WG)
dt
_ f
(6)
-d(CS)
' dt
T o t a l y e a s t c e l l m a s s w a s e s t i m a t e a s t h e s u m o f WG, WP, C S , a n d M I
( s t r u c t u r e s r e m a i n i n g w i t h t h e c e l l ) , w i t h a n added f a c t o r a c c o u n t i n g
for non-protein, non-carbohydrate
substances i n the c e l l .
T h e s e sums
generate v a l u e s f o r y e a s t , p r o t e i n , p e p t i d e s and carbohydrates f o r
comparison t o e x p e r i m e n t a l measurements.
The v a r i a b l e s f o r t h e s t r u c t u r e d m o d e l a r e l i s t e d
Parameter values are given i n Table I I I(35).
Table
II.
i n Table I I .
Structured Model V a r i a b l e s
EGA
Exposed glucan, a c c e s s i b l e f o r h y d r o l y s i s by glucanase
Initial
WG
Wall
WG
Original
CS
CS
q u a n t i t y o f y e a s t , mg/1 d r y b a s i s
g l u c a n , mg/1
a m o u n t o f g l u c a n ; = Y f W G , mg/1
0
CS*
Original
quantity of cytosol
Long-chain
Oligopeptides
M i t o c h o n d r i a l m a s s , mg/1
Q
Eg
material i n
W a l l p r o t e i n , mg/1
= Y f C S , mg/1
I n i t i a l amount o f o s m o t i c a l l y s t a b i l i z i n g
r e a c t i o n medium: P + S + C
Q
WP
C y t o s o l , mg/1
p r o t e i n (TCA - i n s o l u b l e ) , mg/1
(TCA - s o l u b l e ) , mg/1
O r i g i n a l mass o f m i t o c h o n d r i a
Osmotic s t r e n g t h o f b u f f e r ,
G l u c a n a s e enzyme o f l y t i c
protease
enzyme,
i n cell
= Y f M , mg/1
0
Os/kg
system, %(V/V) o f m i x t u r e
Ep
Lytic
E(j
Destructive o r product-degrading
%(V/V) o f m i x t u r e
WE
Yeast
e n z y m e i n w a l l s , mg/1
CE
Yeast
e n z y m e i n c y t o s o l , mg/1
ME
Yeast
e n z y m e i n m i t o c h o n d r i a , mg/1
protease,
%(V/V)

20
Table I I I ,
S t r u c t u r e d model parameters and t h e i r
Ratio of wall
to wall
R a t e c o n s t a n t , g l u c a n a s e on
Kg
Michaelis
Km,
sg

glucan
wp
Kin
wp
Ki
wp
mg/L-min-%ez
4424
mg/L
800
Rate constant, p r o t e o l y s i s
4.441 m g / L - m i n ~ % e z
Michaelis
o f WP
constant
Inhibition
o f WP
constant,
mg/L
459.8
mg/1
919.6
mg/1
proteolysis
of
constant
Rate constant
"rm
WG
M i c h a e l i s c o n s t a n t , g l u c a n a s e on
soluble glucan
Michaelis
^ p
1.326 mg WG/mg WP
10.58
WG
c o n s t a n t , g l u c a n a s e on
Rate constant, p r o t e o l y s i s
protein
1.441 m g / L - m i n - % e z
4598
f o r CS
leakage
mg/L
3.987 1 0 - " m i n "
Rate constant
f o r CS
release
1.6667 m i n "
Rate constant
f o r MI
breakage
0.6 m i n "
protein
i n wall
pes
Fraction
protein
i n cytosol
0.3753
Fraction
protein
i n mitochondria
0.75
pm
Fraction
peptides i n wall
^swp
Fraction
peptides
Fraction
peptides i n mitochondria
0.05
Fraction
of carbohydrates
0.3145
CCS
protein
protein
i n cytosol
0.1170
i n cytosol
Initial
fraction
fWG
Initial
fraction of wall
glucan
0.1922
fWP
Initial
fraction
protein
0.1450
Initial
fraction of mitochondria
fM
of cytosol
of wall
OSMi
Internal osmolality
0SM
Maximum e f f e c t i v e o s m o l a l i t y
released l y s i s products
ewp
of yeast
0.0566
fCS
0.9434
Fraction
pwp
values
i n yeast
cell
0.5612
0.7652
0.617
Os/L
0.539
Os
of
Equilibrium constant f o r osmotic

s t a b i l i z a t i o n of protoplasts
8.135 l O - ^ L / m g CS
P r o p o r t i o n o f enzyme i n w a l l
0.01
protein
P r o p o r t i o n o f enzyme i n c y t o s o l
0.01
P r o p o r t i o n o f enzyme i n m i t o c h o n d r i a
0.01

2.
HUNTER AND ASENJO
21

The s t r u c t u r e d m o d e l s i m u l a t e s t h e p r o g r e s s o f l y s i s i n t e r m s
of t h e c e l l ' s s t r u c t u r a l components d u r i n g l y s i s .
The decrease
i n WP s t a r t s i m m e d i a t e l y , a s i t i s t h e f i r s t c o m p o n e n t a t t a c k e d
by t h e l y t i c enzymes.
WG b r e a k d o w n l a g s WP r e m o v a l , a n d c y t o s o l
r e l e a s e l a g s g l u c a n breakdown, as suggested by the s e q u e n t i a l i t y
b u i l t i n t o the model.
The m i t o c h o n d r i a a r e r e l e a s e d l a s t , and
t e n d t o a c c u m u l a t e b e c a u s e t h e y a r e more r e s i s t a n t t o o s m o t i c r u p t u r e
than the p r o t o p l a s t s .
A t y p i c a l g r a p h i s shown i n F i g u r e 5 a . I n
F i g u r e 5b, s t r u c t u r e d m o d e l e s t i m a t e s o f y e a s t c e l l m a s s , p r o t e i n ,
p e p t i d e s a n d c a r b o h y d r a t e s a r e p r e s e n t e d f o r t h e same e n z y m e a n d
yeast concentrations.
Results
The s i m p l e a n d s t r u c t u r e d m o d e l s i m u l a t i o n s f o r y e a s t m a s s a n d
s o l u b l e p r o t e i n , p e p t i d e s and c a r b o h y d r a t e s a r e compared i n F i g u r e 6
f o r t h e y e a s t a n d e n z y m e c o n c e n t r a t i o n s h o w n i n F i g u r e s 3 a n d 4, a n d
in Figure 7 for a concentrated yeast c e l l slurry.
The simple model
f i t s the data f a i r l y w e l l a t both yeast c o n c e n t r a t i o n s , i n every
v a r i a b l e except the p e p t i d e s .
The f i t f o r a l l v a r i a b l e s a t l o n g e r
r e a c t i o n times i s d i r e c t l y r e l a t e d t o use o f the e x t e n t - o f - r e a c t i o n
t e r m Yoo i n t h e y e a s t l y s i s e q u a t i o n .
The s t r u c t u r e d m o d e l p r o v i d e s a d i s t i n c t i m p r o v e m e n t o v e r t h e
simple model, i n the i n i t i a l stages o f the r e a c t i o n .
The i n i t i a l
l a g s i n t h e h y d r o l y s i s o f t o t a l y e a s t mass a n d c a r b o h y d r a t e i n f i g ures 6 and 7 are very w e l l represented.
The p o s s i b i l i t y remains
t h a t the i n i t i a l l a g s r e l a t e p a r t l y t o a d s o r p t i o n o f l y t i c enzymes
t o t h e c e l l w a l l . On t h e t i m e s c a l e o f o u r e x p e r i m e n t s , h o w e v e r ,
a d s o r p t i o n appeared t obe instantaneous (35).
A t h i g h y e a s t c o n c e n t r a t i o n ( f i g u r e 7) a t t h e l a t e r s t a g e s o f
r e a c t i o n , the carbohydrates c o n t i n u e t o r i s e though t u r b i d i t y i s
l e v e l l i n g o f f . A p p a r e n t l y some w a l l h y d r o l y s i s i s o c c u r r i n g e v e n
though the t o t a l yeast s o l i d s c o n c e n t r a t i o n i s not v i s i b l y d e c l i n i n g .
T h i s r e s u l t i s i n c o n t r a s t t o f i g u r e 6, w h e r e t h e s t r u c t u r e d m o d e l
f o l l o w s carbohydrate data c l o s l e y , and the h y d r o l y s i s o f w a l l glucan
i s e s t i m a t e d t o go e s s e n t i a l l y t o c o m p l e t i o n .
Presumably the glucanase a t t a c k s t h e more s u s c e p t i b l e amorphous g l u c a n a t a h i g h e r r a t e
than the f i b r i l l a r glucan f r a c t i o n o f the w a l l .
Such dependence on
p h y s i c a l s t r u c t u r e i s w e l l known t o o c c u r i n e n z y m a t i c h y d r o l y s i s o f
c e l l u l o s e (38,34).
I f t h e a n a l o g y i s c o r r e c t , t h e amorphous c a r b o hydrateds c o u l d be s o l u b i l i z e d without s u b s t a n t i a l l y changing
the m i c r o f i b r i l network s t r u c t u r e i n the w a l l , o r r e l e a s i n g p r o t o plasts.
C e l l u l o s e / c e l l u l a s e system r e s u l t s a l s o suggest t h a t t h i s
e f f e c t o u g h t t o b e more p r o n o u n c e d a t t h e h i g h e r y e a s t - t o - e n z y m e
r a t i o shown i n F i g u r e 7, t h a n a t t h e l o w e r r a t i o o f F i g u r e 6.
A l i m i t a t i o n o f b o t h m o d e l s i s o v e r e s t i m a t i o n o f t h e amount o f
p r o t e i n r e l e a s e d . P e p t i d e p r e d i c t i o n s b y the s i m p l e model are too
high as w e l l .
The e f f e c t resembles a gap i n the m a t e r i a l b a l a n c e ,
as i f the models p r e d i c t a l a r g e r q u a n t i t y o f p r o t e i n a c i o u s m a t e r i a l
than i s a c t u a l l y present i n t h e c e l l s .
P o s s i b l y some c y t o p l a s m i c
p r o t e i n s are not r e l e a s e d d u r i n g p r o t o p l a s t breakage.
I n f i g u r e 6,
i n s o l u b l e p r o t e i n s could account f o r a s u b s t a n t i a l p a r t o f t h e
r e s i d u a l y e a s t a t 90 m i n u t e s d i g e s t i o n .


45
TIME - MINUTES
ure
Structured
5a
Cell
model s i m u l a t i o n
structures
CY
of yeast
cytosol
WP w a l l p r o t e i n
lysis
WG w a l l
- -MI
glucan
mitochondria
mg/1
45
TIME - MINUTES
5b
C e l l mass and r e l e a s e d compounds

"Y e a s t c e l l m a s s , mg/1
Peptides,
mg/1
- P r o t e i n , mg/1
Carbohydrates,
mg/1

HUNTER A N D ASENJO

Figure
Figure
TIME- MINUTES
TIME-MINUTES
TIME-MINUTES
TIME-MINUTES
Comparison o f simple and s t r u c t u r e d models - I n t i t i a l

y e a s t c o n c e n t r a t i o n 0.78 g/1 ( d . b ) , 1 0 % e n z y m e
S t r u c t u r e d model
Simple model
Comparison o f simple and s t r u c t u r e d models - I n i t i a l

y e a s t c o n c e n t r a t i o n 3 6 . 3 g/1 ( d . b ) 4 0 % e n z y m e
Symbols as i n f i g u r e 6

24
W o r k c o n t i n u e s i n two a r e a s :
p u r i f i c a t i o n of the l y t i c
system
to a l l o w p r o t e a s e and g l u c a n a s e l e v e l s t o be c o n t r o l l e d i n d e p e n d e n t l y
and i n v e s t i g a t i o n o f t h e r e l e a s e o f s i t e - s p e c i f i c y e a s t enzymes and
s u b c e l l u l a r f r a c t i o n s by e n z y m a t i c
lysis.
Applications

The s t r u c t u r e d model's d e t a i l e d a c c o u n t i n g o f t h e f a t e o f c e l l
s t r u c t u r e s c a n b e u s e d t o make p r e d i c t i o n s a b o u t t h e e f f e c t s o f a
number o f i m p o r t a n t p r o c e s s v a r i a b l e s , f o r e x a m p l e :
The r a t i o o f l y t i c p r o t e a s e t o g l u c a n a s e i n t h e l y t i c
system
T h e e f f e c t o f pH o r t e m p e r a t u r e o n s y n e r g i s m b e t w e e n t h e
l y t i c enzymes
Elimination of "destructive" protease (for bioactive protein
recovery) or supplementation with a d d i t i o n a l proteases
( f o r f o o d and f e e d a p p l i c a i t o n s )
The a d d i t i o n o f p r o t e a s e i n h i b i t o r s a t a p o i n t o r p o i n t s d u r i n g
l y s i s , e f f e c t i v e l y l o w e r i n g k p o r i n c r e a s i n g K^p.
Osmotic b u f f e r i n g s t r a t e g i e s f o r recovery of b i o a c t i v e p r o t e i n
from d i f f e r e n t s i t e s i n the c e l l .
C e l l F r a c t i o n a t i o n S i m u l a t i o n . The w a l l p r o t e i n , c y t o s o l and
o r g a n e l l e s o f y e a s t e a c h c o n t a i n enzymes w h i c h a r e found nowhere
else i n the c e l l .
Some e x a m p l e s o f t h e s e e n z y m e s i n c l u d e i n v e r t a s e
i n t h e w a l l s , g l y c o l y t i c pathway enzymes i n t h e c y t o s o l and f u m a r a s e
i n t h e m i t o c h o n d r i a (13) . A m o d e l o f r e c o v e r y o f t h e s e enzymes i s
offered here.
Enzyme a c c u m u l a t i o n .
F o r the purpose of s i m u l a t i o n , w a l l - l i n k e d
a n d p e r i p l a s m i c e n z y m e s (WE) a r e c o n s i d e r e d t o b e a p a r t o f t h e
outer wall protein.
C y t o p l a s m i c a n d m i t o c h o n d r i a l e n z y m e s (CE,ME)
a r e a s s u m e d t o b e some f r a c t i o n o f t h e c y t o p l a s m i c a n d m i t o c h o n d r i a l
mass, r e s p e c t i v e l y .
The e q u a t i o n s d e s c r i b i n g t h e i r r e l e a s e and
h y d r o l y s i s are e x a c t l y analogous t o equation 6 f o r t o t a l l o n g - c h a i n
protein.
d(WE)
dt
ewp
d(CE)
dt
d (ME)
dt
Variables
W ^ r m
and parameters
d(WP)
dt
(WE)
Lies)'
dt
CE
PBR
(8)
'PBR
[max(0,0.3-0SM )]'MI - M l
(9)
.PBR
(10)
are included i n Table I I I .
F i g u r e 8 shows a s i m u l a t i o n o f enzyme r e c o v e r y f r o m t h e w a l l ,
c y t o s o l and m i t o c h o n d r i a .
The c o n c e n t r a t i o n s o f r e c o v e r a b l e enzyme
a r e n o r m a l i z e d t o t h e i n i t i a l amount o f enzyme p r e s e n t i n t h e c e l l
site.
The c u r v e s r i s e a s enzyme i s r e l e a s e d f r o m a s i t e , t h e n f a l l
as i t i s h y d r o l y z e d .
I t may b e s e e n t h a t t h e l y t i c s y s t e m i s
u s a b l e even as a crude p r e p a r a t i o n t o r e c o v e r w a l l l i n k e d yeast enz
ymes i n 60 t o 8 0 % y i e l d .
The y i e l d o f y e a s t w a l l enzyme d e p e n d s on

HUNTER AND ASENJO

100
Figure
Release o f s i t e
8a
8b
Initial
Initial
- specific
enzymes - S i m u l a t i o n
y e a s t c o n c e n t r a t i o n , 0.78 g/1
y e a s t c o n c e n t r a t i o n , 36.33 g/1
W a l l enzyme
Cytoplasmic
M i t o c h o n d r i a l enzyme

enzyme

26
p r o t e a s e a c t i v i t y and i s t h e r e f o r e d i r e c t l y r e l a t e d t o t h e q u a n t i t y
of " d e s t r u c t i v e " protease present i n t h e l y t i c system.
The p r o d u c t
p u r i t y depends on t h e r e l e a s e o f p r o t e i n s f r o m o t h e r c e l l s i t e s , and
hence on o s m o t i c f a c t o r s . A t t h e h i g h e r y e a s t c o n c e n t r a t i o n ( F i g .
8 b ) , few o f t h e p r o t o p l a s t s and none o f t h e m i t o c h o n d r i a r e l e a s e
t h e i r enzymes i n t o s o l u t i o n .
W h i l e t h e y i e l d o f w a l l enzyme i s n o t
as good a s i n F i g u r e 8 a , t h e p u r i t y i s f a r h i g h e r . W i t h p r o p e r o s m o t i c s u p p o r t d u r i n g l y s i s , and b r e a k a g e o f o s m o t i c a l l y s t a b l e p r o t o p l a s t s by m e c h a n i c a l means, t h e w a l l , c y t o p l a s m i c and m i t o c h o n d r i a l
f r a c t i o n s c a n be o b t a i n e d s e p a r a t e l y .
A s i m u l a t i o n of s i t e - l i n k e d product recovery i s presented
i n F i g u r e 9 a n d T a b l e s I V a n d V.
The c a l c u l a t i o n s assume t h a t s i t e l i n k e d e n z y m e s WE, C E , a n d ME c o n s t i t u t e 1% o f t h e w a l l p r o t e i n ,
c y t o s o l and m i t o c h o n d r i a r e s p e c t i v e l y . I n t h e f i r s t l y s i s s t e p ,
u s i n g 2 0 % l y t i c enzyme b r o t h and o s m o t i c s u p p o r t , 9 3 % o f t h e w a l l
p r o t e i n ( a n d w a l l enzyme) i s r e l e a s e d f r o m t h e c e l l w a l l .
Some i s
h y d r o l y z e d by t h e " d e s t r u c t i v e " p r o t e a s e , b u t 73.8% o f i t s u r v i v e s
t o be r e c o v e r e d a t t h e end o f t h e f i r s t h o u r .
S i n c e o n l y 3% o f t h e
protoplasts burst during t h i s step, l i t t l e cytoplasm i s released.
T h e p r o t e i n c o n c e n t r a t i o n i n s o l u t i o n a t t h e e n d o f t h e h o u r i s 3.83
g/1 o f w h i c h a b o u t 1% i s WE.
I f t h e c e l l s were broken mechanically,
t h e w a l l enzyme w o u l d c o n s t i t u t e o n l y 0.35% o f t h e t o t a l p r o t e i n ,
even assuming t h a t i t c o u l d be c o m p l e t e l y s o l u b i l i z e d .
The s i m u l a t i o n a l s o s h o w s a r a t i o o f WE t o CE o f 7.8 i n t h e m e d i u m a t t h e e n d
o f t h e f i r s t s t e p , w h i c h c o m p a r e s t o a r a t i o o f 0.258 o n a t o t a l - c e l l
basis.
Table IV.
First
Process
lysis
c o n d i t i o n s f o r enzyme r e l e a s e s i m u l a t i o n
step:
Yeast
Enzyme
Buffer
T o t a l volume
36.33g
40%
0.3 O s / L
1L
R e a c t i o n m i x t u r e c e n t r i f u g e d ; '5% o f s u p e r n a t a n t a n d 1 0 0 % o f
p e l l e t r e t a i n e d and resuspended i n t w i c e t h e i n i t i a l volume
of enzyme/buffer s o l u t i o n .
Second l y s i s
step:
Digested yeast:
Enzyme
Buffer
T o t a l volume
Breakage:
by s t i r r i n g
26.90g
20%
0.3 O s / L
2-L
o r p a s s a g e t h r o u g h a pump
Protoplast rupture
Mitochondrial rupture
95%
0%

HUNTER AND ASENJO
25
A)
AFTER
PROTOPLAST
RUPTURE f
CS
CS

60
120
TIME-MINUTES
B)
\WP
\ LYSIS STEP 2
\
\
AFTER 1
PROTOPLAST
RUPTURE
Ml
\WG
\
LYSIS
STEP 1
s.
Ml
Ml
< i
60
120
T I M E - M INUTES
C)
100
CE
Lu
>
/WE
.*
50
Lu
/
WE
f c
!
0
60
120
T I M E - MINUTES
Figure 9
9a
Enzyme r e c o v e r y
Cell
from s u b c e l l u l a r
s t r u c t u r e breakdown
Wall protein
Cytosol
9b
9c
structures
- - Wall glucan
Mitochondria
C e l l s t r u c t u r e : Close-up, showing m i t o c h o n d r i a
Enzyme r e l e a s e , p e r c e n t o f o r i g i n a l enzyme i n c e l l
W a l l enzyme
C y t o p l a s m i c enzyme

28

Table
Initial
End o f
first
step
Start of
second
step
End o f
second
step
After
protoplast
rupture
36332
8.1
760.7
26902
3831.5
2135.0
26902
191.6
106.7
21660
966.9
684.9
4766
7307.3
2661.5
655.0
4449.8
222.5
3321.4
8634.7
5268.2
6983.0
20389
371.0
3380.2
19779
371.0
908.9
19779
0.028
908.9
17783
0.028
908.9
889.2
II
2780.1
2696.9
2696.9
2424.7
121.0
II
83.2
83.2
355.4
2659.1
mg/1
38.6
.97
4.18
4.18
II
4.95
.12
15.24
184.2
II
Yeast s o l i d s
mg/1
S o l u b l e p r o t e i n It
Soluble peptides "
Soluble
II
carbohydrates
Wall protein
Wall glucan
Cytosol
Mitochondria
(internal)
Mitochondria
(released)
W a l l enzyme
Cytoplasmic
enzyme
Mitochondrial
enzyme
V. Y i e l d s
II
II
It
T h e s e c o n d d i g e s t i o n was i n c l u d e d t o d e c r e a s e t h e amount o f
s t r u c t u r a l g l u c a n from about 50% t o about 13% o f i t s o r i g i n a l mass,
i n o r d e r t o make t h e c e l l s m o r e f r a g i l e , t h u s e a s i e r t o r u p t u r e
mechanically.
O n l y a s m a l l amount o f c y t o p l a s m i c p r o t e i n i s r e leased from the p r o t o p l a s t s during t h i s time - a d e s i r a b l e r e s u l t ,
s i n c e p r o t e i n s e q u e s t e r e d i n s i d e t h e p r o t o p l a s t s i s n o t a t t a c k e d by
protease.
A t t h e end o f t h e second h o u r t h e r e m a i n i n g p r o t o p l a s t s c a n
be b r o k e n m e c h a n i c a l l y by s t i r r i n g o r c e n t r i f u g a t i o n . P r o t e a s e
a c t i v i t y c a n be m i n i m i z e d by k e e p i n g t h e t e m p e r a t u r e l o w . Assuming
t h a t 95% o f t h e p r o t o p l a s t s (and none o f t h e s t u r d i e r
mitochondria)
a r e b r o k e n b y s t i r r i n g , t h e f i n a l p r o t e i n c o n c e n t r a t i o n i s 7.3 g / 1 ,
o f w h i c h 2.5% i s c y t o p l a s m i c enzyme.
Almost a l l of the mitochondria
(95.6%) a r e r e l e a s e d d u r i n g t h e second l y s i s and p r o t o p l a s t r u p t u r e ,
but they remain whole because t h e b u f f e r o s m o l a l i t y i s kept above
0.3 O s / L .
C e n t r i f u g a t i o n o f t h e f i n a l m i x t u r e p r o d u c e s 4.77 g/1 o f
a p e l l e t , o f w h i c h 2.66 g o r 5 6 % i s m i t o c h o n d r i a a n d 0.89 g o r
19%, p r o t o p l a s t s .
These s i m u l a t i o n s suggest an a d d i t i o n a l t e s t f o r t h e s t r u c t u r e d
model: t h a t i s , t o compare i t s p r e d i c t i o n s t o d a t a on r e l e a s e o f
s i t e - l i n k e d enzymes i n y e a s t .
T e s t s f o r c y t o p l a s m i c and m i t o c h o n d r i a l enzyme r e l e a s e w i l l b e a i d e d by p r e p a r a t i o n o f a l o w - p r o t e a s e
l y t i c system.

2.
HUNTER AND
ASENJO

Conclusions
The s i m p l e m o d e l i s c o n c e p t u a l l y s t r a i g h t f o r w a r d a n d g i v e s a n
approximate f i t t o the data over the e n t i r e range o f v a r i a b l e s
s t u d i e d : y e a s t c o n c e n t r a t i o n , enzyme c o n c e n t r a t i o n a n d t i m e .
The
p r o d u c t d i s t r i b u t i o n depends on the r e l a t i v e r a t e s o f l y s i s and
proteolysis.
U s i n g a s i n g l e enzyme p r e p a r a t i o n , a s was done h e r e ,
the r e l a t i v e r a t e s change o n l y w i t h y e a s t c o n c e n t r a t i o n .
I n prac
t i c e , however, i n h i b i t i o n o f the p r o t e a s e a c t i v i t y , supplementation
of the l y t i c a c t i v i t y w i t h p u r i f i e d glucanases o r m i x i n g o f l y t i c
systems from d i f f e r e n t s o u r c e s can b r i n g about l a r g e changes i n t h e
a c t i v i t y r a t i o , w h i c h may b e i n c o r p o r a t e d i n t o t h e s i m p l e m o d e l
by a d j u s t i n g
and k .
The s t r u c t u r e d m o d e l i s c o n s i s t e n t w i t h f e a t u r e s o f l y t i c e n
zyme a c t i o n a n d y e a s t s t r u c t u r e r e p o r t e d i n t h e l i t e r a t u r e . T h e
s e q u e n t i a l r e m o v a l o f the two w a l l l a y e r s , f o l l o w e d b y p r o t o p l a s t
r u p t u r e , a c c u r a t e l y d e s c r i b e s the e a r l y l a g i n p r o t e i n and carbo
hydrate release.
The p r e s e n c e o f r e s i d u a l s o l i d s a t l o n g r e a c t i o n
t i m e s was a c c o u n t e d f o r s t a b i l i z a t i o n o f p r o t o p l a s t s b y s u b s t a n c e s
released from l y s e d c e l l s .
The s t r u c t u r e d model can be used t o
e s t i m a t e t h e e f f e c t s o f s e v e r a l p r o c e s s a l t e r n a t i v e s , a s shown i n
a s i m u l a t i o n o f a p r o c e s s f o r r e c o v e r y o f s i t e - l i n k e d enzymes f r o m
yeast.
r
Acknowledgments
T h i s work was s u p p o r t e d b y a g r a n t f r o m the N a t i o n a l S c i e n c e
F o u n d a t i o n , (NSF) t o whom t h a n k s a r e d u e .
One o f t h e a u t h o r s
(JBH) was s u p p o r t e d b y g r a d u a t e f e l l o w s h i p s f r o m NSF a n d t h e
J o s e p h i n e de Karman F o u n d a t i o n d u r i n g p a r t o f t h i s work.
This
support i s a l s o g r a t e f u l l y acknowledged.
Literature Cited
1.
2.
3.
4.
5.
6.
Phaff, H.J., "Enzymatic yeast cell wall degradation", in Food

Proteins: Improvement through Chemical and Enzymatic
Modification, eds. R.J. Feeney and J.R. Whitaker, Adv. Chem.
Series, Vol.60, American Chemical Society, 1977.
Asenjo, J . . , "Process for the production of yeast-lytic
enzymes and the disruption of whole yeast cells", in
Advances in Biotechnology, Vol III: Fermentation Products,
C. Vezina & K. Singh, ed's., Pergamon, 1981; p. 295.
Le Corre, S., B.A. Andrews, and J.A. Asenjo, Enzyme Microb.
Technol., 1985, 7, 73-77.
Kobayashi, R., T. Miwa, S. Yamamoto, S. Nagasaki, Eur. J. Appl.
Microbiol. Biotechnol., 1982, 15, 14-19.
Okagbue, R.N. and M.J. Lewis, Biotech. Lett., 1983,
5, 731-736.
Jamas, S., C.-K. Rha, A.J. Sinskey, "Directed biosynthesis
of yeast glucans with known structure-function properties",
paper presented at the ACS 190th Annual Meeting, Chicago, IL,
September 9-13, 1985.

29
30
7.
8.
9.

10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Brake, A.J., J.P. Merryweather, D. Coit, U. Heberlein, F.R.

Masiarz, G.T. Mullenbach, .Valenzuela, and P.J. Barr,
Proc. Natl. Acad. Sci. USA, 1984, 81, 4642-46.
Schemer, R.G., L.F. Ellis, B.E. Schoner, Bio/Technology,
1985, 3, 151-54.
Valenzuela, P., D. Coit, M.A. Medina-Selby, C.H. Kuo, G.
Van Nest, R.L. Burke, P.Bull, M.S. Urdea, P.V. Graves,
Bio/Technology, 1985, 3, 323-326.
D'Souza, S.F., Biotechnol. Bioeng., 1983, 25, 1661-1664.
Fish, N.M. and M.D. Lilly, Bio/Technology, 1984, 2, 623-627.
Phaff, H.J., "Structure and biosynthesis of the yeast cell
envelope", in The Yeasts, Vol II, ed. R. Harrison and A.
Rose, Academic Press, 1971.
Follows, M., P.J. Hetherington, P.Dunnill, M.D. Lilly,
Biotechnol. Bioeng, 1971, 13, 549-560.
Marffy, F. and M.-R. Kula, Biotechnol. Bioeng., 1974, 16,
623-634.
Shetty, J.and J.Kinsella, Biotechnol. Bioeng., 1978, 20, 755-766
Ballou, C.E., "Yeast cell wall and cell surface", in
The molecular biology of the yeast Saccharomyces: Metabolism
and gene expression, ed. J.N. Stratton, E.W. Jones, J.R.
Broach, Cold Spring Harbor, 1982 ; pp. 335-361.
Ballou, C.E., Adv. Microb. Physiol, 1976, 14, 93.
Kitamura, ., Agric. Biol. Chem., 1982, 46, 963-969.
Kitamura, ., Agric. Biol. Chem., 1982, 46, 2093-99.
Scott, J.H. and R. Schekman, J. Bacteriol., 1980, 142, 414-423.
Rombouts, F.M. and H. Phaff, Eur. J. Biochem., 1976, 63,
121-130.
Ishida, K., M. Kawai, N. Mukai, U.S. Patent 3, 809,780,
May 7, 1974.
Asenjo, J.., and P.Dunnill, Biotechnol. Bioeng., 1981, 23,
1045.
Vrsanska, ., P. Biely and Z. Kratky, Z. Allg. Mikrobiol.,
1977, 17, 465-480.
Jeffries, T.W., and Macmillan, J.D., Carbohydr.Res.,
1981, 95, 87-100.
Obata, T., H. Iwata, and Y. Namba, Agric. Bio. Chem., 1977,
41, 2387-2394.
Andrews, B.A. and J.A. Asenjo, "Continuous Culture Studies
of the Synthesis and Regulation of Extracellular (1-3)
Glucanase and Protease Enzymes in Oerskovia xanthineolytica",
Biotechnol. Bioeng., 1986. (submitted for publication).
Hunter, J.B. and Asenjo, J.., "Kinetics of enzymatic lysis
and disruption of yeast cells: 1. Evaluation of two lytic
systems with different properties." Biotechnol. Bioeng., 1986
(submitted for publication).
Jeffries, T.W., Ph.D. Thesis, Rutgers University, 1976.
Hunter, J.B. and Asenjo, J . . , "Kinetics of enzymatic lysis
and disruption of yeast cells: 2. A simple model of lysis
kinetics" Biotechnol. Bioeng. 1986 (submitted for publication)
Bernath, V.R., and W.R. Vieth, Biotechnol. Bioeng., 1972, 14,
737-745.
Chipman, D. Biochemistry, 1971, 10, 1714-22.
Asenjo, J . . , Biotechnol. Bioeng., 1983, 25, 90.

2.
HUNTER AND ASENJO
34.
31
Lee. Y.-H., and L.T. Fan, Biotechnol. Bioeng. , 1983, 25,

939-966.
35. Hunter, J.B. and Asenjo, J.A., "A structured, mechanistic model
of the kinetics of enzymatic lysis and disruption of yeast
cells", Biotechnol. Bioeng., 1986 (being submitted).
36. Indge, K.J., J. Gen. Microbiology, 1968, 51, 425-432.
37. Indge, K.J., J. Gen. Microbiology, 1968, 51, 433-440.
38. Fan, L.T., Y.-H. Lee, D.H. Beardmore, Biotechnol. Bioeng.,1980,
22, 177-199.
Received March 26, 1986

3
Dual Hollow-Fiber Bioreactor for Aerobic Whole-Cell
Immobilization
1

Ho Nam Chang , Bong Hyun Chung , and In Ho Kim
Department of Chemical Engineering, Korea Advanced Institute of Science and

Technology, Dongdaemun, Seoul, Korea
Lucky Central Research Institute, Daeduk, Korea
Aerobically growing Escherichia coli, Aspergillus niger

and Norcardia mediterranei were immobilized in the
interstitial space of a dual hollow-fiber bioreactor
formed by a parallel arrangement of three microporous
polypropylene hollow fibers contained within a silicone
tubule. All three types of cells grew well and attain
ed high densities to reach 550-600 g dry cell weight
per liter of the cell containing volume. In the culti
vation of E. coli, cell growth among the fibers was not
uniform and leakage of cells through the fiber walls
was observed. The unlimited growth of A. niger expand
ed the silicone fiber and compressed the inner fibers
to reduce the substrate flow rates gradually to zero.
Only Nocardia mediterranei was grown successfully to
make possible long term operation of 50 days or more,
producing antibiotics rifamycin with a volumetric
productivity of 125 g/mL/h based on the volume occupi
ed by the immobilized cells. This corresponds to a 30fold increase over the productivity of a comparable
batch system.
For the past f i f t e e n years hollow-fiber membrane bioreactors have
been extensively used for immobilizing enzymes (1,2.), animal c e l l s
(3), microbial c e l l s (4,5) and plant c e l l s (6). Immobilization of
microbial c e l l s i n a hollow-fiber reactor offers some d i s t i n c t advan
tages over other methods: c e l l s can be e a s i l y immobilized without
much preparation; primary separation of products i s carried out;
very l i t t l e energy w i l l be consumed i n the scale-up operation. How
ever, there are some disadvantages as well. Insoluble substrates
can not be used and usually substrate pretreatment i s required to
prevent the f i b e r s from being blocked.
In general, polymer-based
hollow f i b e r s can not be repeatedly h e a t - s t e r i l i z e d . Inherently the
transport of gas i s d i f f i c u l t and thus the c u l t i v a t i o n of aerobic
c e l l s with high oxygen demand becomes d i f f i c u l t .
Robertson and h i s colleagues at Stanford University have exam
ined hollow-fiber membrane bioreactors as a means for continuous
0097-6156/ 86/ 0314-0032506.00/ 0


3.
CHANG ET AL.
Dual Hollow-Fiber Bioreactor
33
production of ^-lactamase and ethanol using IS. c o l i and S_. c e r e v i siae immobilized i n the sponge layer of asymmetric hollow f i b e r s
(7,8). In the _E. c o l i culture c e l l s leaked through the f i b e r walls,
and the production of carbon dioxide was a problem i n the ethanol
production. Recently Robertson and Kim (9) developed a dual hollowf i b e r bioreactor consisting of s i l i c o n e tubules for oxygen transport
and microporous polypropylene hollow f i b e r s for substrate transport
to study the production of tetracycline using Streptomyces aureofaciens. Higher productivity as compared to that of the batch fermentation was achieved. The production remained at high l e v e l s for
three days and declined sharply after that for unknown reasons.
The present study reports the f i r s t successful long-term c u l t i v a t i o n
of rifamycin-B producing Nocardia mediterranei and the problems
encountered i n growing IS. c o l i and A_. niger c e l l s i n the dual hollowf i b e r bioreactor.
Materials and Methods
Materials and s t r a i n s . Yeast extract, bactopeptone, malt extract
and trypton were products of Difco Laboratories and glucose was
from Hayashi Pure Ind. (Tokyo, Japan). E. c o l i (Sigma EC-1,
a l k a l i n e phosphatase-rich mutant) was from Sigma Chemical Co.
(St. Louis, MO) and Nocardia mediterranei (ATCC 21789) was from
American Type Culture C o l l e c t i o n . Aspergillus niger B-60 was obtained from Kubicek at Technical University Wien (Austria) who used
t h i s s t r a i n for c i t r i c acid production studies (10,11).
Bioreactor system.
The reactor used i n t h i s study was constructed
d i f f e r e n t l y from that of Robertson and Kim (9). The reactor was a
glass tubing of 30cm length (0.8 cm i.d.) i n which ten dual hollowf i b e r units were bundled together i n a p a r a l l e l assemblage. Each
unit had one s i l i c o n e tubule (Dow Corning, 0.147 cm i . d . , 0.196 cm
o.d.) that contained three microporous polypropylene hollow f i b e r s
(Enka, West Germany, 0.03 cm i . d . , 0.065 cm o.d.) inside.
Robertson
and Kim used one polypropylene hollow f i b e r i n which three s i l i c o n e
tubules were placed to make one polypropylene/silicone f i b e r assemblage. Thus the order i n s i l i c o n e and polypropylene f i b e r s was oppos i t e to the o r i g i n a l l y developed bioreactor. The cross section of
a dual hollow f i b e r unit i s shown i n Figure 1 wherein microbial c e l l s
are supposed to grow i n the r e s t r i c t e d i n t e r s t i t i a l space between
the two f i b e r walls. The detailed dimensions of the reactor are
shown i n Figure 2. The t o t a l volume of the glass tube based on the
16-cm e f f e c t i v e length was 8.04 cm^ and that of the i n t e r s t i c e for
c e l l growth was 1.12 cm^. The c e l l inoculum port was covered with
rubber through which inoculation could be made with a syringe needle.
Reactor operation.
The polypropylene hollow f i b e r s i n the reactor
were prewetted prior to inoculation with r e c i r c u l a t i o n of 50% ethanol
and s t e r i l i z e d chemically with 5% formalin solution. Then the reactor was washed by u l t r a f i l t r a t i o n of one l i t e r of autoclaved d i s t i l l e d
water. The reactor was placed i n a water bath maintained at a d e s i r ed temperature.
C e l l s were inoculated through the inoculation port
using a syringe needle. The detailed experimental setup i s shown i n
Figure 3.


NUTRIENT
CELL
NUTRIENT
INOCULUM OUT
EPOXY SEAL
\SILICONE RUBBER SEAL

235mm
300mm
F i g u r e 2. D e t a i l e d
bioreactor.
specification
o fa dual
hollow-fiber


3.
CHANG ET AL.
Dual Hollow-Fiber
Bioreactor
35
IS. c o l i seed culture from the l y o p h i l i z e d c e l l s was grown i n a

250 mL flask with a LB medium (yeast extract, 10 g/L, trypton, 10 g/L
, pH 7 adjusted with 1 NaOH) placed on a rotary shaker (250 rpm).
When the flask culture reached exponential growth phase, i t was d i l u
ted 20 times and inoculated into the reactor. The medium and a i r
flow rates were maintained at 2 mL/h and 100 mL/min, respectively.
In order to see nutrient consumption during the c e l l growth a LB
medium with 5% glucose was used. The temperature for the seed c u l t
ure and the reactor operation was 37C.
For the A_. niger culture, spores grown i n sugar agar slant were
diluted with s t e r i l i z e d d i s t i l l e d water to a concentration of 10-10
spores/L and inoculated into the reactor. The medium for the reac
tor operation consisted of sucrose, 60 g/L; ^
1 g/L; MgSO,
7H 0, 0.25 g/L; NH^NO^, 2.5 g/L and the pH was adjusted to 3.1 with
2N HC1. The flow rates for the medium and a i r were the same as i n
the IS. c o l i case. The temperature was maintained at 30C.
The medium for If. mediterranei seed culture were: glucose, 20
g/L; yeast extract, 5 g/L; bactopeptone, 5 g/L; malt extract, 5 g/L
(pH 7.3 adjusted with 1 NaOH). The seed culture was carried out
as i n the IS. c o l i culture except the temperature (30C). When the
glucose l e v e l dropped to 9 - 11 g/L, the seed culture was diluted
10 times and used i n the inoculation. For comparison, a batch c u l
ture was performed with the following medium composition: glucose,
110 g/L; yeast extract, 10 g/L; bactopeptone, 10 g/L; sodium barbi
t a l , 0.7 g/L. The batch fermentation was carried out i n a 500 mL
flask at 30C and at 250 rpm on a rotary shaker. For the hollowf i b e r reactor the medium flow rate was 1.7 mL/h and the a i r flow rate
was 100 mL/min. The medium composition for the reactor operation
was: glucose, 20 g/L; yeast extract, 5 g/L; malt extract, 5 g/L;
sodium b a r b i t a l , 0.5 g/L.
2
A n a l y t i c a l methods.
After the reactor operation the f i b e r s were cut
into 10 cm segments and dried i n an oven at 90C for 72 hours. The
dry mass density was obtained by taking the difference between the
dry mass of the cut f i b e r and that of an empty one of equivalent
length. This difference corresponds to the biomass accumulated i n
the i n t e r s t i t i a l space between the inner and outer f i b e r s .
Glucose
was determined by glucose analyzer (YSI model 23 A, Yellow Springs,
OH) and rifamycin was measured spectrophotometrically at 425 nm.
Microscopic techniques.
Morphological examination of JE. c o l i and
N_. mediterranei contained i n the reactor was done i n a Jeol trans
mission electron microscope (model 100CX). The sample was prepared
according to the method by Robertson and Kim (9). For the picture
of _A. niger reactor the f i b e r was cut into 1 mm pieces, which were
photographed with a l i g h t microscope.
Results and Discussion
Cultivation of E. c o l i .
Figure 4 shows glucose concentration and
pH h i s t o r i e s during the course of IS. c o l i c u l t i v a t i o n i n the reactor.
After 4 days IS. c o l i c e l l s began to appear i n the effluent, which
means that the c e l l s i n the reactor leaked through the pores of the
polypropylene membrane which were supposed to be smaller than the


36
Figure 3 Schematic diagram of experimental setup: 1. medium

reservoir, 2. p e r i s t a l t i c pump, 3. dual hollow-fiber bioreactor,
4. water bath, 5, a i r or pure oxygen bombe, 6. rotameter, 7.
humidifier, 8. inoculum syringe, 9. sampling bottle, 10. effluent
reservoir.
DAYS
Figure 4.
course of
; glucose
triangles
Glucose and pH h i s t o r i e s i n the effluent during the

the dual hollow-fiber reactor operation. - -, -
cone,
pH. The f i l l e d c i r c l e s and
represent the effluents containing E. c o l i c e l l s .

3.
CHANG ET AL.
37
size of IS. c o l i c e l l s . Figures 5(a) and 5(b) show the electron micr
ographs of the JE. c o l i c e l l s at the boundary of the polypropylene
f i b e r and i n the middle region between the two f i b e r s . The c e l l s
were packed l i k e tissue and some of the c e l l s penetrated into the
i s o t r o p i c membrane structure. The dry c e l l mass was 550 g/L, which
i s the highest c e l l mass ever reported i n the l i t e r a t u r e as shown
i n Table 1. This high c e l l mass compares well with 1 0 ^ E. c o l i
cells/mL achieved by Inloes et a l . (7) i n the sponge region of a
hollow-fiber reactor i f we assume that the mass of a single IS. c o l i
i s roughly 10~12g
Leakage of c e l l s and high c e l l densities seem
common i n t h i s type of reactors i n the case of IS. c o l i .
After the experiment the reactor was dismantled and each of ten
dual hollow-fiber units was v i s u a l l y examined. Only i n 4 out of the
ten f i b e r s c e l l s were densely packed, which suggests that the med
ium was not adequately supplied to many of these f i b e r s . Probably
the medium was not equally d i s t r i b u t e d among the f i b e r s . In other
words, i n some of f i b e r s the medium flow was not adequate to support
the c e l l growth i n the f i b e r . The nonuniform flow d i s t r i b u t i o n
among the f i b e r s of a hollow f i b e r device i s an i n t r i n s i c problem,
which was studied i n depth i n the authors* laboratory (16).
The
work of E, c o l i immobilization i n the dual hollow f i b e r reactor was
reported previously from the authors laboratory (17).

Culivation of A_. niger.

In the culture of A_. niger B-60, the c e l l s
did not leak through the f i b e r s . In other words, no mycelia were
detected i n the e f f l u e n t . In a l l the f i b e r s the c e l l s grew well and
appeared uniform along the f i b e r s , but the s i l i c o n e tubes were expan
ded and the polypropylene tubes were contracted. Figure 6(a) i s the
photograph of an empty s i l i c o n e tube and Figure 6(b) shows the cross
section of the f i b e r after 15 days of the A_. niger growth. It was
observed that the flow rate decreased gradually to zero meaning that
the pumping head of a p e r i t a l t i c pump was not s u f f i c i e n t to overcome
the flow resistance exerted by the growing fungi. Thus the continu
ous operation of the reactor was not f e a s i b l e . It i s suggested that
the control of the c e l l growth be needed after a growth period by
switching the culture to a nitrogen d e f i c i e n t medium or by some other
means.
Production of rifamycin by N. mediterranei.
Figure 7 shows the
results of shake flask culture for rifamycin production.
After
8 days of fermentation 60 g/L of glucose was consumed and 820 pg/mL
of rifamycin was produced. This gives a volumetric productivity
of 4.3 pg of rifamycin/mL/h. The continuous production of rifamycin
i n the dual hollow-fiber bioreactor i s shown i n Figure 8. The succ
essful production of the a n t i b i o t i c s continued more than 50 days
without showing signs of decreased production.
This i s i n contrast
to the t e t r a c y c l i n e production by Robertson and Kim that lasted for
a few days. The cause of t h i s decline i n Robertson and Kim s work
has not been understood. S t a b i l i t i e s i n the a n t i b i o t i c s production
i n the dual hollow- f i b e r bioreactor are speculated to be associated
with reactor design or producing organism or both.
Two d i s t i n c t differences are noted between the f l a s k culture and
the present reactor system. F i r s t , the effluent concentration of
rifamycin was ca. l/10th of that obtained i n the f l a s k culture.
1


38
Figure 5. Electron micrographs of densely packed E. c o l i K-12

cells,
(a). The c e l l s at the boundary of the polypropylene f i b e r
(pp).
Magnification, ,. (b). The c e l l s i n the middle space
between the s i l i c o n e tube and the polypropylene f i b e r .
Maginification, 20,000X.

3.
CHANG ET AL.
Dual Hollow-Fiber
Table 1.
Bioreactor
39
Comparison of c e l l mass of IS. c o l i i n various

fermentations.
System
Shake-flask culture
C e l l density
Reference
1 - 2 g/L

0 Submerged-culture under
controlled conditions
10 g/L
(12,13)
Submerged-culture with
pure oxygen supply and
semi-continuous feeding
of glucose at 22C.
55 g/L
(14)
0 Immobilization
carrageenan
with
beads
A.5 1 0
cells/mL
1 0
viable
(15)
0 Immobilization i n a
hollow-fiber bioreactor
10
12
cells/mL
(7)
0 Immobilization i n a dual
hollow-fiber bioreactor
550 g/L
This work
Figure 6. Expansion of s i l i c o n e tube and contraction of poly

propylene tubes by growing A. niger B-60. The length scale shown
i n the pictures i s 250 pm. ( a ) . Cross section of an empty
s i l i c o n e tube.
(b). Deformed bioreactor.



3.
CHANG ET AL.
This i s not small at a l l i f we consider that the residence time i n

the hollow-fiber reactor was 12 minutes based on the t o t a l f i b e r
lumen voume of 0.339 cm while that i n the flask culture was 8 days.
The volumetric productivity of the reactor was 125 pg/mL/h based on
the void voulme of the reactor where the c e l l s were a c t u a l l y immobil i z e d . This was about t h i r t y - f o l d as compared to that of the flask
culture. This number drops to 15 or 10 i f we include the reactor
volume or the volume of the glass tubing used. This high p r o d u c t i v i ty comes essentially from a highly dense c e l l mass i n the reactor
shown i n the electron micrograph ( F i g . 9). The measured dry c e l l
mass was 600 g/L.
The c e l l s neither penetrated into the propylene
f i b e r s nor expanded the tubes. This growth c h a r a c t e r i s t i c s i s i n
good contrast to that of _E. c o l i or A_. niger c e l l s . The c e l l s grew
uniformly along the f i b e r s , which made possible the successful longterm operation of the reactor.
The main advantages of a hollow-fiber reactor system are: very
l i t t l e energy w i l l be consumed i n the aeration; primary p u r i f i c a t i o n
i s accomplished concurrently with the production.
In conventional
a n t i b i o t i c s or c i t r i c acid fermentation with A_. niger much energy
i s consumed i n several days of continuous aeration and mixing of
viscous fermentation broths which adds up to a substantial portion
of f i n a l production costs. If t h i s membrane bioreactor i s ever successful i n a scale-up operation, there w i l l be a tremendous savings
in aeration and mixing costs. Also the benefit of primary separation can never be underestimated because simpler downstream processing i s a key to production cost reduction.
However, as yet, much
work needs to be done for t h i s reactor to become a t t r a c t i v e for
3

41
Figure 9. Electron micrograph of densely packed Nocardia mediterranei (ATCC 21789) c e l l s near the polypropylene hollow f i b e r
(magnification, 25,000X).

42
i n d u s t r i a l production of valuable materials. Achieving higher volu

metric productivity i s c e r t a i n l y an advantage, but the f i n a l product
concentration i s too low for any r e a l recovery process to be c o n s i
dered as compared to that i n batch system. Substrate d i f f u s i o n l i m i
t a t i o n through the membrane can be blamed f o r t h i s low product con
centration, but t h i s i s not the case considering that more than 80%
of glucose i s consumed during the 10 minutes residence time i n the
IS. c o l i reactor. Oxygen l i m i t a t i o n can be a cause f o r t h i s .
Perhaps
the most important reason i s that c e l l s i n a distressed state can not
function as well as the c e l l s i n suspension. The water content of
the c e l l s i n the hollow-fiber would be around 40% f o r the c e l l s to
a t t a i n such a high dry c e l l weight. Currently we are working on ways
of increasing the f i n a l product concentration of rifamycin comparable
to that i n the batch system and are trying to improve the s t a b i l i t y
i n the operation of reactor f o r A. niger c e l l s .

Literature Cited
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Rony, P. R. Biotechnol. Bioeng. 1971, 13, 431.

Waterland, L. R.; Michaels, A. S.; Robertson, C. R. AIChE J.
1974, 20, 50.
Knazek, R. .; Gullino, R. M.; Kohler, P. O.; Dedrick, R. L.
Science, 1972, 178, 65.
Kan, J. K.; Shuler, M. L. Biotechnol. Bioeng. 1978, 20, 217.
Vick Roy, T. B.; Blanch, H. W.; Wilke, C. R. Biotechnol. Lett.
1982, 4, 483.
Shuler, M. L. Ann. N.Y. Acad. Sci. 1981, 369, 65.
Inloes, D. S.; Smith, W. J.; Taylor, D. P.; Cohen, S. N.;
Michaels, A. S.; Robertson, C. R. Biotechnol. Bioeng. 1983,
25, 2653.
Inloes, D. S.; Taylor, D. P.; Cohen, S. N.; Michaels, A. S.;
Robertson, C. R. Appl. Environ. Microbiol. 1983, 46, 264.
Robertson, C. R.; Kim, I. H. Biotechnol. Bioeng. 1985, 27,
1012.
Habison, .; Kubicek, C. P.; Rhr, M. FEMS Microbial Lett.
1979, 5, 39.
Mischak, H.; Kubicek, C. P.; Rhr, M. Biotechnol. Lett. 1984,
6, 425.
Elsworth, R.; Miller, G. .; Whitaker, A. R.; Kitching, D.;
Sayer, P. D. J. Appl. Chem. 1968, 17, 157.
Phares, E. F. In "Methods in Enzymology"; Colowick, S. P.;
Kaplan, N. O., Ed; Academic Press: New York, 1971; vol. 22 p.
157.
Shiloach, J.; Bauer, S. Biotechnol. Bioeng. 1975, 17, 227.
Wada, M; Kato, J.; Chibata, I. Eur. J. Appl. Microbiol.
Biotech.
1979, 8, 241.
Park, J. K.; Chang, H. N. AIChE J. (accepted).
Chung, . H.; Chang, H. N.; Kim, I. H. Korean J. Appl.
Microbiol. Biotechnol. 1985, 13, 209.

4
A Membrane Reactor for Simultaneous Production
of Anaerobic Single-Cell Protein and Methane
R. K. Finn and E. Ercoli

School of Chemical Engineering, Cornell University, Ithaca, NY 14853
Single-cell protein can be produced from agricultural

residue anaerobically in yields of about 20% (wt. cells
per wt. substrate) by using a mixed culture of rumen
bacteria. Even higher cell yields should be possible.
To achieve high cell densities, it is proposed that
acidic end products be removed by cyclic microfiltration
into a methanogenic fermentor. Preliminary experiments
suggest that such a tandem fermentation should be feasible on a continuous basis. More data are needed for
an economic evaluation.
Very l i t t l e attention has been given to the p o s s i b i l i t i e s for anaerobic production of s i n g l e - c e l l protein (SCP) from cheap carbohydrate residues (1,2). The reason for dismissing any anaerobic process is that c e l l y i e l d s , according to c l a s s i c a l Embden-Meyerhof
catabolism, are only 10 to 15% of the substrate fermented.
In cont r a s t , aerobic c e l l yields of 50 to 60% are easily obtainable.
However, there are highly e f f i c i e n t anaerobic c e l l s .
These
include the acetogens, propionic bacteria, and above a l l the
various rumen bacteria. The l a t t e r can attain c e l l yields on carbohydrate of 30 to 35 dry weight (3^4).
Such y i e l d values are
already corrected for any polysaccharide formation, and in fact the
protein content of rumen bacteria is about 60% (2). We therefore
have been considering t h e i r use as a protein feecT supplement for
monogastric animals l i k e chickens or pigs.
The ruminant animal and rumen microorganisms exist in a r e c i procally beneficial relationship, in which cellulose and other
plant carbohydrates are fermented by the rumen microbes to form
c h i e f l y C0 and v o l a t i l e fatty acids (VFA).
The microorganisms are
adapted to l i v e between pH 5.5 and 7.0, in the absence of oxygen,
1
These high yields result from a combination of factors including

low maintenance energy, higher than normal c e l l yields per mol of
ATP, and f i n a l l y excess ATP production, which can involve "anaerobic respiration" with cytochrome b in a fumarate c y c l e .
x
0097-6156/ 86/ 0314-0043506.00/ 0


44

at temperatures of 39 to 40C, and in the presence of moderate

concentrations of fermentation products. These v o l a t i l e fatty
acids, c h i e f l y acetic and propionic, are absorbed through the rumen
wall to provide energy for the animal. Removal of the acids is
essential because at concentration above about 0.3% they do i n h i b i t
c e l l growth. Within the rumen though, microorganisms grow very
e f f i c i e n t l y , and thereby provide s i n g l e - c e l l protein for t h e i r host
animals. Our challenge as engineers is to duplicate in v i t r o such
performance.
Some of the basic ideas of the anaerobic SCP process we are
developing at Cornell are summarized below.
Anaerobic SCP Process
1)
2)
3)
4)
5)
Rapid growth of mixed rumen bacteria (e.g. y = 0.16 hr"" on

starch) to high c e l l densities at c e l l yields of 30-35%
Membrane removal of inhibitory acid products
The acids feed slower growing methanogenic bacteria in a separate fermentor
The CH^ generated thus can be used to dry the SCP product
Rapid interchange between the two fermentors is possible with
alternating pulsed m i c r o f i 1 t r a t i o n .
1
The key to economic c e l l production is rapid growth to cell d e n s i t i e s l i k e those in the rumen, namely 1 0 or 1 0 cells/ml. Acidic
end-products are used to feed a methane generator, so that most of
the carbon is recovered in a useful form. An unusual feature of
this process is that rapid u l t r a f i l t r a t i o n rather than slow d i a l y sis can be used to feed the methane fermentor.
Insoluble substrates such as starch, hemicel1ulose or cellulose are retained
within the rumen fermentor by appropriate membranes. The rapid i n terchange of soluble acids between the two fermentors allows only a
low steady-state concentration to develop in the rumen fermentor
because conversion to methane proceeds simultaneously in the second
fermentor.
Additional features of the process are l i s t e d below.
10
1)
2)
3)
4)
5)
11
Broad range of insoluble carbohydrates fermented...crude mixed

cultures of defined consortia of rumen bacteria.
S t e r i l e operation may be unnecessary because rumen conditions
select for a very specialized mixed population.
Safety and nutritional value of the SCP product for use as an
animal feed has already been proven by studies on ruminant
animals.
A lower productivity than in an aerobic process is to be expected because of a somewhat lower c e l l y i e l d .
Overall costs may
s t i l l be competitive because of simpler equipment and energy
savings.
Fed-batch or continuous operation seems f e a s i b l e .

4.
FINN AND ERCOLI
45
Simultaneous Production of SCP and Methane

We h a v e d o n e o n l y p r e l i m i n a r y e x p e r i m e n t a l w o r k u s i n g m a i n l y g l u
cose and sugar beet pulp as s u b s t r a t e s .
We h a v e n o t y e t c o m b i n e d
t h e m e t h a n o g e n i c s t e p ; i n s t e a d we h a v e u s e d a b u f f e r e d s a l t s m e d i u m
i n t h e s e c o n d chamber t o remove i n h i b i t o r y e n d p r o d u c t s .
Procedures.
T h e b a s a l medium c o n t a i n e d m i n e r a l s a l t s , y e a s t e x
t r a c t (0.1 g/Jl), t r y p t i c a s e ( 0 . 1 g/), c y s t e i n e h y d r o c h l o r i d e (0.4
g/) a s a r e d u c i n g a g e n t , a n d r e a z u r i n a s a r e d o x i n d i c a t o r . T h e
b a s e u s e d t o m a i n t a i n a c o n s t a n t pH w a s s o d i u m c a r b o n a t e .
Some
m e d i a i n c l u d e d h e m i n ( 2 t o 6 mg/V) b e c a u s e m o s t s t r a i n s o f B a c t e r o i d e s r u m i n i c o l a , a m a j o r t y p e o f rumen b a c t e r i a , a r e s t i m u l a t e d b y
t h e a d d i t i o n o f s m a l l amounts o f h e m i n t o t h e medium ( 5 ) . F o r
example, t h e molar growth y i e l d o f B a c t e r o i d e s f r a g i l e s subsp
f r a g i l i s i n c r e a s e d f r o m 17.9 t o 4 7 . 0 ( g d r y w e i g h t c e l l p e r m o l o f
g l u c o s e ) w h e n 2 mg/I o f h e m i n w e r e a d d e d ( 6 ) . M e d i a w e r e i n o c u
l a t e d w i t h f r e s h r u m e n f l u i d t a k e n f r o m a cow f e d w i t h g r a i n a n d
hay.
The samples were used w i t h i n two h o u r s a f t e r r e m o v a l .
P r o t e i n was d e t e r m i n e d b y t h e m e t h o d o f L o w r y ( 7 ) a f t e r h y d r o
l y s i s w i t h 0.2N NaOH (100C, 15 m i n ) .
T o t a l n i t r o g e n was m e a s u r e d
by t h e m i c r o - K j e l d a h l m e t h o d w i t h s u l f u r i c a c i d / h y d r o g e n p e r o x i d e
r e a g e n t j t h e a m m o n i a was d e t e c t e d w i t h N e s s l e r s r e a g e n t .
Glucose
was m e a s u r e d b y s t a n d a r d c o l o r i m e t r i c a s s a y u s i n g d i n i t r o s a l i c y l i c
acid.
S t a r c h was h y d r o l y z e d w i t h c o n c e n t r a t e d HC1 a n d t h e n d e t e r
mined as sugar.
f
Results.
T o e s t a b l i s h o p t i m u m g r o w t h c o n d i t i o n s , we u s e d i n t h e
e a r l y experiments a low c o n c e n t r a t i o n o f the carbon source. Re
moval o f t h e i n h i b i t o r y a c i d s i s then unnecessary.
T a b l e I shows r e s u l t s f o r a m i x e d p o p u l a t i o n o f rumen b a c t e r
ia.
T h e f e r m e n t a t i o n s w e r e c o m p l e t e ( e s s e n t i a l l y no r e s i d u a l g l u
cose) a f t e r 6 t o 7 hours.
Table I .
Substrate
Cone.
G r o w t h o f Rumen B a c t e r i a o n G l u c o s e
Hemin
(mg/*)
Growth r a t e
(h )
l
(g/
(g/)
5
5
15
Nitrogen*
Fixed
0.61
0.66
0.60
0.071
0.090
0.126
Est'd c e l l * *
yield
(g/g s u b s t r . )
0.15
0.19
0.08
(0.18)
*Net u t i l i z a t i o n o f s o l u b l e n i t r o g e n f r o m t h e medium, i . e . c o n v e r
ted i n t o biomass.
**The c e l l y i e l d w a s e s t i m a t e d f r o m t h e n i t r o g e n f i x e d , a s s u m i n g
50% p r o t e i n c o n t e n t f o r t h e c e l l s , 15% n u c l e i c a c i d s . The v a l u e
s h o w n i n p a r e n t h e s i s was m e a s u r e d d i r e c t l y b y d r y w e i g h t .
The r e s u l t s i n T a b l e I show t h a t c e l l y i e l d s a r e l o w e r t h a n e x p e c
t e d b u t t h a t added hemin s t i m u l a t e s g r o w t h .
The l o w y i e l d o f c e l l s
a t h i g h e r g l u c o s e c o n c e n t r a t i o n may b e c a u s e d b y a c c u m u l a t e d a c i d s
suppressing growth while a l l o w i n g fermentation t o proceed.

46
Using starch as substrate yields were also low.

An explanation may
be that Streptococcus bovis, a homofermentative l a c t i c acid
organism common in the rumen grows rapidly on starch with low y i e l d
of biomass (&). Table n shows some of the results for fermentation
of sugar beet pulp (SBP).
Table
II.

Substrate
Cone.
Growth of Rumen Bacteria on Sugar Beet Pulp, (5%

inoculum)
Hemi
(mg/)
(9/A)
5
5
5
5
10
20
2
4
6
6
Nitrogen
fixed
(g/A)
0.067
0.075
0.074
0.086
0.123
0.203
E s f d cell
yield
(g/g subst. supplied)
0.14
0.16
0.16
0.18
0.12
0.10
The y i e l d values in Table II are based on substrate supplied; the

quantity unfermented could not be readily measured. The incorpora
tion of nitrogen from the medium is similar to that observed for
glucose.
Growth rates on sugar beet pulp were slower than on glucose,
as indicated by comparison of the solid lines in Figure 1, where
nitrogen in the solids fraction provides a measure of biomass. The
rate of acid production (broken lines in Figure 1) was also slower
for the sugar beet pulp; from the slopes of such lines one can
estimate the rate at which i t will be necessary to remove acids
from a fermentation with higher substrate concentrations.
Early experiments were done using simple d i a l y s i s in a 2-chambered membrane apparatus constructed from 3-inch glass pipe (Figure
2).
Starch was added p e r i o d i c a l l y to the rumen bacteria so as to
simulate a fed-batch operation. A d i a l y s i s membrane was held
between flanges separating the two chambers. The rate of acid
removal was too slow, however, in this simple d i a l y s i s apparatus.
Consequently, a microfi1tration chamber was arranged as shown in
the diagram of Figure 3. Plugging or fouling of the m i c r o f i l t r a tion membrane can be avoided by changing the pressure periodically
on either side of the membrane. Flow then o s c i l l a t e s across the
membrane. Such cycling can be accomplished without changing the
pressure in either of the fermentors by using two small centrifugal
pumps and the valve arrangement shown at the top of the diagram.
Both pumps operated continuously.
The solenoid valves were a l t e r
nately opened and closed on 30-second repeat cycles. Back-pressure
valves were set to provide c i r c u l a t i o n through the membrane cham
bers while maintaining also a positive pressure for m i c r o f i l t r a tion.
Each of the fermentation chambers had a working volume of
1.5 l i t e r s and there was an additional holdup of 0.5 l i t e r s on each
side of the f i l t r a t i o n chamber. A single membrane of polytetraf1uoroethylene was used. It had a pore size of 0.22 micrometers
and an area of 155 cm . Preliminary experiments have indicated


FINN AND ERCOLI
Time, h
F i g u r e 1. T i m e c o u r s e o f t h e r u m e n f e r m e n t a t i o n .
Solid lines
show n i t r o g e n i n t h e s o l i d s f r a c t i o n f o r 5 g / l i t e r g l u c o s e
( s o l i d c i r c l e s ) and f o r 5 g / l i t e r sugar beet pulp (open
circles).
The h i g h i n i t i a l value f o r sugar beet pulp r e p r e sents i t s p r o t e i n content as r e c e i v e d .
B r o k e n l i n e s show t h e c o r r e s p o n d i n g a m o u n t s o f 2 5 % a q u e o u s
Na2C3 u s e d f o r m a i n t a i n i n g c o n s t a n t pH d u r i n g f e r m e n t a t i o n o f
the glucose o r sugar beet pulp.

Library
1155 16th St., N.W.
Washington,
D.C. 20036
48

STARCH
RUMEN
METHANOGENIC
BACTERIA
BACTERIA
F i g u r e 2. D i a g r a m o f a p p a r a t u s u s i n g t w o 3 - i n c h g l a s s e l b o w s
w i t h a d i a l y s i s membrane a t t h e f l a n g e s e p a r a t i n g t h e t w o
chambers.
No f o r c e d f l o w t h r o u g h t h e membrane.
BACK-PRESSURE VALVES
S GLENOID VALVES
METHANOGENIC
RUMEN
BACTERIA
BACTERIA
<y )
F i g u r e 3. P r o c e s s s c h e m e f o r m i c r o f i l t r a t i o n w i t h c y c l i n g
f l o w back and f o r t h between t h e twof e r m e n t a t i o n chambers.


4.
FINN AND ERCOLI
49
that i t is possible to get a net interchange of the total volume

ten times per hour, even with rumen bacteria and starch p a r t i c l e s
present.
Longer cycle times than 30 seconds are needed, however,
to allow for adequate mixing of the permeate into the bulk f l u i d .
From the slope of the curve for addition of sodium carbonate
to the rumen fermentation of starch, one can estimate tht the net
rate of acid formation is equivalent to 36 millimoles of acetic
acid per l i t e r per hour.
If a level of no more than 0.2% acid must
be maintained in the rumen fermentor, then the exchange rate across
the membrane must be at least one l i t e r per hour or half of the
total reactor volume each hour.
This is well below the observed
exchange rate of 20 l i t e r s per hour.
In summary, we have described an anaerobic process for s i n g l e c e l l protein from crude carbohydrates.
The inhibitory by-products
are simultaneously converted into methane. Mass transfer l i m i t a
tions can be avoided by using microfTitration rather than d i a l y s i s .
Further study of the kinetics and improvements in y i e l d w i l l be
necessary in order to make an economic comparison with other pro
cesses for s i n g l e - c e l l protein.
Acknowledgments
We thank Professor James B. R u s s e l l , Department of Animal Science
at C o r n e l l , for many helpful discussions and acknowledge support of
one of us (E.E.) from Consejo Nacional de Investigaciones C i e n t i f i cas y Tecnicas de la Republica Argentina.
Literature Cited
1. Rolz, C. and Humphrey, ., "Microbial Biomass from Renewables:
Review of Alternatives," Adv. Biochem. Eng. 21, 1-53 (1982).
2. Mehta, K.I. and Callihan, C.D., "Production of Protein and
Fatty Acids in the Anaerobic Fermentation of Molasses by E.
ruminantium," J. Am. Oil Chemists Soc. 61, 1728-1734 (1984).
3. Isaccson, H.R., Hinds, C., Bryant, M.P., and Owens, F.N.,
"Efficiency of Energy Utilization by Mixed Rumen Bacteria in
Continuous Culture," J. Dairy Sci. 58, 1645-1659 (1975).
4. Russell, J.B. and Baldwin, R.L., "Comparison of Maintenance
Energy Expenditures and Growth Yields Among Several Rumen Bac
teria Grown in Continuous Culture," Appl. Environ. Microbiol.
37, 537-543 (1979).
5. McCall, D. and Caldwell, D., "Tetrapyrrole Utilization by
Bacteroides ruminicola, J. Bacteriol. 131, 809-814 (1977).
6. Macy, J., Probst, I., and Gottschalk, G., "Evidence for Cyto
chrome Involvement in Fumarate Reduction and ATP Synthesis by
Bacteroides fragilis in the Presence of Hemin," J. Bacteriol.
123, 436-442 (1975).


Membrane Processes in the Separation, Purification, and

Concentration of Bioactive Compounds
from Fermentation Broths
Enrico Drioli
Instituto di Principi di Ingeneria Chimica, Universitdegli Studi di Napoli,
Piazzale Tecchio, 80125 Napoli, Italy
The potential of membrane separation techniques (such as cross-flow microfiltration(MF),

u l t r a f i l t r a t i o n (UF), Reverse Osmosis (RO)and
electrodialysis (ED) ) and membrane reactors
in the treatment of fermentation broths are
huge. The synergistic effects obtainable by
designing the overall b i t e c h n o l o g i c a l pro
cess combining various membrane technique are
particularly significant.
In this paper experimental results are descri
bed which refer to processes of industrial
interest studied assuming membrane technolo
gies as the best available.
The separation,purification and concentration
of a thermosensitive bioactive compound from
a lysate has been carried out combining UF,
ion exchange and RO with significant cost re
duction and productivity increase. Enzyme mem
brane reactors have been used for triglyceride
enzymatic hydrolysis and product separation.
Thermophilic,thermostable enzyme u l t r a f i l t r a
tion membrane have been prepared, and used in
high temperature lactose hydrolysis.
The
term
the
chain
Downstream
tem
f o r t h erecovery,
o fu n i t
Processing
operations
tration
o ft h ep r o d u c t s
possible
recovery
step
a r ecombined
purification,
highest
The
i nBiotechnology
that
separation
a tt h e l o w e s t
recovery
factor
generally
possible
refers t o
into
sys
and concen
cost and
and quality.
represents
a large
part
0097-6156/86/0314-0052$06.00/0

o f
the
5. DRIOLI
overall
its
capital
cost
investiment
efficiency
biotechnological
The
recovery
broth
complicated
considered

systems.
core
that
the
i nthese
b i o -
often un-
processing i s
o f biotechnology.
and particularly
as broad
the fermentation
The downstream
f o r f u r t h e r development
technologies
from
by t h ef a c t
lowconcentration
stable ,non-newtonian
brane
plant and
f o r t h ep r o d u c t i o n o f
compounds.
a r ei nvery
key area
i na fermentation
i sa key factor
o f bioactive materials
i sg e n e r a l l y
products
a
53
Membrane Processes for Bioactive Compounds
UF,MF
technologies
Mem-
a n d RO c a n b e
i nthis
industrial
segment ( 1 ) .
In
Table
I a r esummarized
ducts
o f interest
costs
o f production
t h emarket
on l a r g e
ficantly
and positively
In
Table
I I a r esummarized
of
interest
Those
in
cells;
tion;
development
Systems
i n methods
o f enzyme
fermentation
u t i l i z a t i o n
The
shown
c i a l l y
available
been
of
involves
problems
liquids
membranes
and ener-
m i c r o - f i l -
introduced,
significantly.
suspended
f i l t r a t i o n .
Commer-
and tubular
o f broths
f i l t r a t i o n
a n d amino
solved
process
i n terms o f
containing
o f membrane
vaccines
been
many
o f product,
correctly
and f o r s t e r i l e
have
immobiliza-
membrane
process
f o r theconcentration
p r o t e i n s , enzymes,
ing
membranes
o f bacteria,
o f solutions
a c i d s (3.).
by a p e r i o d i c
backflush-
o f t h e membrane.
Fluids
containing
volume
module
from
upwards
c a n be pumped
easily
whole
membranes
a
capillary
and moulds,
Fouling
by
used
t h escope
when
this
o f treating
broadens
improvements
o r whole
reactors.
cross-flow
t o improve
t o :
and uneconomical
and u l t r a f i l t r a t i o n ,
p o s s i b i l i t y
have
(_2.) .
processes
Processes
generally
Continuous
solids
yeasts
membrane
f o r biocatalyst
o f raw materials,recovery
consumption.
been
signi-
technology
o f enzymes
membrane
i nDownstream
areinefficient
which
tration
pro-
Their
be a f f e c t e d
i ngeneral
and reuse
steps
have
w i l l
membrane
t h ev a r i o u s
Traditional
gy
scale
by u s i n g
cancontribute
f o r recovery
development
Membrane
o f various
f o r biotechnology.
systems
methods
values
i nbiotechnological processes.
achieving
fermentation
a r eused
microfiltration
cromolecular
very
a well-designed
high
broths
solutes,
w i l l
ning
relatively
liquid-phase
(2).
on t h ebroths
stage),
material,
only
o f 50% t o 6 0 % suspended
through
When
solids
membrane
recoveries
u l t r a f i l t r a t i o n
( o r on t h epermeate
retention o f thes o l u b i l i z e d
as well
be accomplished,
as p a r t i c u l a t e
giving
lowmolecular
ma-
and c o l l o i d a l
a f i l t r a t e
weight
from
solutes.

contai-
54
Table
I.
Total
duct
market
values
Number
Product
f o r the various
of
Current

value
millions)
category
compounds
Amino
acids
($
Vitamins
Enzymes
1,703.0
667.7
11
217.7
Steroid
hormones
....
376.8
Peptide
hormones
....
263.7
Viral
antigens
Short
peptides
Nucleotides
proteins
Antibiotics
Gene
preparations
4.4
72.0
300.0
4,240.0
100.0
.. .
Pesticides
Aliphatics
Miscellaneous
:
1
Methane
12,572.0
Other
24
2,737.5
Aromatics
10
1,250.9
Inorganics
Mineral
leaching
Only
These
two
These
the
of a
numbers
actual
numbers
numbers
largest
2,681.0
107
Totals
2
....
Biodgradation
number
refer
of
compounds
to major
are considered
classes
Current
SOURCE
value
: Genex
27,186.7
here.
o f compounds;
not
compounds.
refer
market
of
IA
JA
only
to those
volume
compounds
i n classes
representing
specified
i n the
thext.
d
pro
categories.
excluding
Corp.
methane
$14,614,700.000
( J j.

5.
DRIOLI
55
Membrane P r o c e s s e s Used Today i n B i o t e c h n o l o g y

A r e a of A p p l i c a t i o n
D r i v i n g Force
Symmetric m i c r o porous polymer

membrane.
Pore
s i z e 0.05-10
Hydrostatic
p r e s s u r e 15 bar
S i e v i n g mechanism,
pore s i z e and p a r
t i c l e diameter de
termine s e p a r a t i o n
characteristics
Sterile filtration,
clarification, cell
harvesting bacteria,
viruses separation.
Ultrafil
tration
Asymmetric m i c r o porous polymer

membrane.
Pore
s i z e 1-50 nm
Hydrostatic
p r e s s u r e 210 b a r
S i e v i n g mechanism,
pore s i z e , and p a r
t i c l e diameter de
termine s e p a r a t i o n
characteristics
S e p a r a t i o n , concen
t r a t i o n and p u r i f i
c a t i o n o f macromolecular s o l u t i o n s
such a s p r o t e i n s ,
enzymes, p o l y p e p
tides, etc.
Reverse
Osmosis
Asymmetric mem
brane w i t h homo
geneous s k i n
and microporous
sub- o r support
structure
Hydrostatic
p r e s s u r e
00 b a r
Solution-diffusion
mechanism, s o l u
b i l i t y , and d i f f u s i v i t y of i n d i v
i d u a l components
i n t h e homogeneous
polymer m a t r i x
determine s e p a r a
tion character
istics.
Concentration of
m i c r o s o l u t e s , such as
s a l t s , s u g a r s , amino
acids, etc., recovery
of water from m i c r o
b i o l o g i c a l processes.
Membrane
Distilla
tion
Symmetric o r
asymmetric
m a i n l y hydro
phobic microporous membrane
P a r t i a l vapor
pressure gra
dient i n t r o
duced by a
temperature
difference
P a r t i a l vapor p r e s
sure, separation
mechanism i s t h e
same a s i n d i s
tillation.
Separation v o l a t i l e
o r g a n i c s o l v e n t s such
as acetone, e t h a n o l ,
e t c . from aqueous
fermentation s o l u t i o n .
Pervaporation
Asymmetric mem
brane w i t h homo
geneous s k i n
and microporous
substructure.
P a r t i a l vapor
pressure gra
d i e n t 0.001 t o
1 bar
Solution-diffusion
mechanism, s o l u
b i l i t y and d i f f u s i v i t y of i n d i v i d u
a l components i n
the polymer m a t r i x
determine s e p a r a
tion character
istics.
Separation of organic
s o l u t i o n s such a s
ethanol, butanol,
a c e t i c a c i d , e t c . from
aqueous s o l u t i o n s ,
especially separation
of a z e o t r o p i c m i x t u r e s .
Electrodialysis
C a t i o n - and
anion-exchange
membrane
Electrical
potential
difference
E l e c t r i c charges
of p a r t i c l e
Removing s a l t s , a c i d s ,
and bases from f e r
m e n t a t i o n b r o t h s , sep
a r a t i o n o f amino
acids, etc.
Chemical
potential
gradients
p.e. C a r r i e r
transport
Microfiltration

Mass S e p a r a t i o n
Mechanism
Membrane Type
Liquid
supported
membranes
Symmetric o r
asymmetric
microporous
membranes sup
porting l i q u i d
phase
S e l e c t i v e removing
of s a l t s , b i o a c t i v e
compounds, e t c .

56
This
f i l t r a t e
osmosis
pounds
Those
i f of
processes
can
osmosis
tion
antibiotics
of
removal
of
mixtures
v i a
An
interesting
al
scale
for
from
solid
The
study
also
(4).
of
The
in
h)
water;
g)
f i l t r a t i o n ;
m)
on
d)
i)
significantly
increasing
obtained,
using
ghof
with
atm
30
FDR)
applied
1/m
with
of
organic
97-98%
cut-off
of
and
were
normal
Pyrogens
were
completely
At
The
and
final
has
been
of
the
obtained
tion
raw
were
amount
step
of
higher
few
in
R0
than
a l l the
hundred
in
UF
of
the
orga1).
possibicosts,and
and
process
purification
methanol).
membranes
10.000
M.W.
of
order
the
with
was
(Berat
of
by
reverse
Figure
recovery
concentration
high.
columns
osmosis
2).
regeneration,
High
80
product.
particularly
ion-exchange
(see
column
permeate
was
eluate
and
factor
step,
waste
up
with
to
final
g/1.
carbon
The
an
activated
the
of
e)
into
state.
the
moreover
acid;
Figure
(e.g.
Fluxes
detail
for
the
on
quality
purity
observed.
treated.
required
of
of
in
activated
decreased
material
duction
in
order
absent
combining
costs
was
precipitation
production
capillary
C.
product
analyzed
concentrations
The
of
treatment
was
the
the
cen-
centri-
S-adenosy1-L-methionine
steady
concentration
also
Reduction
water
90%
HPLC
at
a)
c)
p i c r i c
showed
place
amino
process
of
lysis;
as
solvents
of
20
industri-
transformation
the
in
pressure
at
downstream
chemical
such
product
factor
example.
biologi-
unstable
consisted
process
decreasing
precipitation
developed
purification
operation,
recovery
an
in
l y o p h i 1 i z a t i o n (see
this
for
p u r i f i c a -
p r e c i p i t a t i o n s with
of
com-
immunocomplexation
and
of
using
RO,is
thermal
agents
l i t y
by
reverse
s u g g e s t e d ( 3_) .
fermentor
b)
and
present
particularly
purification
out
and
of
been
separation;
carried
by
by
weight
pretreatment
traditional
complexing
in
UF
use
separation;
f)
ideal
been
time
safety
treated
molecular
concentration
combined
has
part
soluble
1)
easily
low
contaminants
recovery
cells
solvent;
carbon;
The
a p p l i c a t i o n has
centrifugation;
nic
the
the
specific
compound
of
sequential
enrichment
for
for
using
more
considered
by
lysate
yeast
trifuge
fuge
be
antigenic
and
after
also
processes.
cal
acid
be
interest.
reverse
The

might
i f i t s concentration
used
in
the
Kg
to
that
no
organic
from
fact
process
dollars
gave
per
Kg
an
of
final
150
g.
overall
final
p u r i f i c a per
Kg
of
solvents
cost
re-
bioactive
product.


5. DRIOLI
Figure
1. T r a d i t i o n a l
2)
centrifugation;
5)
chemical
tion
10)
57
downstream
3) t h e r m a l
precipitation;
p r o c e s s :1 ) e n r i c h m e n t ;
lysis;
4)vacuum
6)centrifugation;
f i l t e r ;
7)purifica-
by s o l u b i l i z a t i o n ; 8) r e p r e c i p i t a t i o n ; 9 ) f i l t r a t i o n
active
Figure
carbonjll)
2. M o d i f i e d
exchange;
Process
9) Reverse
11)
lyophi1ization.
: 6 ) u l t r a f i 1 t r a t i o n;
8)
Osmosis.

i o n
58
Enzyme
Membrane
Reactors
In
previous
examples
the
red
of
g e n e r a l l y as
small
the
molecules
separation
solution
t i f i e d
a
or
as
Such
true
removal
of
loss
substrate
to
when
an
ous
fermentation
reactor. A
products
the
i n
separation
parallel
place
system
classical
connected
designed,
(or
of
membrane
or
from
the
to
in
the
may
be
bulk
iden-
example
by
the
unit.
continuous
bulk
or
i s
continuous
dialysis
permits
insoluble
separation
systems
i t i s p o s s i b l e to
increase
duct-inhibited
fermentation,
the
removal
of
used
higher
than
solution
macromolecular
component
i n
the
for
this
productivity
of
pro-
example,
weight
of
cellulose
to
inhibited
12%,
might
be
at
continuIn
molecular
are
of
interest.
low
degradation
croorganisms
as
i s growing
ous
tion
the
conside-
the
).
use
zymatic
When
takes
i t s e l f ,
been
for
well
enzyme
ones.
reactor
reaction
of
The
case
membrane
have
barriers
reaction
membrane
loop
the
membranes
bigger
enzymatic
system,
without
from
chemical
the
stirred-tank
recirculation

i n
the
semipermeable
by
the
alcohol,
an
continu-
products.
alcohol
improved
by
The
where
en-
the
mi-
concentra-
the
use
of
this
concept.
Recently,
similar
the
production
ids
are
of
obtained
produced
acetyl
by
previous
type
ted
acylase,
the
ble
form,
from
the
b i l i z i n g
Other
from
stage
pyrogens.
might
allow
in
This
that
the
the
apply
laboratory
(6J . T r a d i t i o n a l
operations
at
ried
out
room
possibility
to
in
step
the
same
i s
The
prepare
and
appears
study
natural olive
matic
membrane
of
o i l was
reactor
in
concepts
and
was
to
i s
at
the
can
be
conti-
obtained
mass
and
of
total
bacteria.
enzymatic
hydrolysis
make
i n
particular
used
as
raw
used
based
the
pro-
be
car-
pressure.
g l y c e r i n e and
interest.
material.
on
our
requiring
h y d r o l y s i s can
atmospheric
free
products
the
pressure
immo-
(JL) .
growth
to
solu-
enzyme
investigation
separate
Unlike
in
the
toxic
cell
for
under
and
enzyme
losses
of
ac-
carrier-loca-
content
removal
enzymatic
temperature
the
solution
chemical
temperature
non-competitive.
at
enzyme
those
enzymes.
consumption
increases
triglyceride
of
Degussa f o r
L-amino
synthetically-
separating
fermentation
of
of
with
avoids
product
continuous
cess
means
enzyme
hydrolysis
high
by
reactor
by
case,
division
for
batch
to
applied
this
employs
reduces
are
The
In
approach
solution.
and
been
membrane
significant
yield
acids
fixed-bed
new
and
adjusted
product
The
reagent
advantages
nually
of
uses
has
acids.
biocatalytic
DL-amino
the
and
process
L-amino
In
An
The
acids
our
enzy-

5.
DRIOLI
capillary
membranes.
dracea,2975
form
A continuous
the
capillary
applied
centration

membrane
and separation
and
In
The enzyme
Figure
membranes
pressure
typical
as f u n c t i o n o f time.
in
show
Microporous
be used
emulsion
rate
phase
Only
study
where
an high
interface
were o b flow
rate
enzyme
con-
(Figure3 ) .
results
are presen-
o f the t r i g l y c e r i -
g l y c e r i n e was p r e s e n t
capillary
t o distribute
t h e enzyme
c a n be c o n t r o l l e d
by changing
o f enzyme
outa t axial
o f conversion
hydrophobic
i nthis
t h ewater
Increase
t o mantein
experimental
t h edegree
cylin-
i na g e l
o f t h eo i l substrate i n
was c a r r i e d
des
also
surface.
a t t h emembrane-solution
4 some
Candida
o f t h er e a c t i o n products
useful
which
from
immobilized
recirculation
ted
permeate.
(lipase
U/mg) w a s d y n a m i c a l l y
on t h e i n t e r n a l
s t a b i l i t y
tained.
59
small
the
o i l droplets
i sdissolved.
i ndroplets
i n
membranes c a n
size
This
and formation
t h etransmembrane pressure
and axial
flow
c a n be a l s o
pro-
rate .
Enzyme
Membranes
Highly
duced
efficient
enzyme
by i m m o b i l i z i n g
fibers.
F o rexample,
support
matrix
enzymes
The dense
permeable
through
theinner
spongy
part,
The
development
design
could
to
denaturation.
In
most
ver,
o f improved
without
have
membranes
which
been
on a l a r g e
selective
branes
require
dures.
studied
very
mass
i scombined
l o w membrane
scale
loss
mobilization
where
exposed
procedures,howei n membrane
and standard
processes, i n
might
mem-
reactions,would
preparation
have
tech-
o f enzyme
thea r t i f i c i a l
chemical
proce-
been r e c e n t l y
accomplish
membranes,involving
a t t h emembrane-solution
per-
biocatalysis
and less
progress
across
specific
enzyme
a r e parameters
performance.
The p r e p a r a t i o n
i no u r laboratory, which
Gelled
into the
Applied
i nthe effluent
protected
Two i m m o b i l i z a t i o n p r o c e d u r e s
requirements.
rate
f o r industrial
transfer
cost
place.
immobilization
limited.
with
flow
reactors
enzyme
more
o f thetraditional
bei m -
diffuse
immobilization techniques
flow
a r ealso
should
t o t h e enzyme
takes
while
thefiber l u -
The l a t t e r
o f t h er e a c t o r
thecontributions o f recent
nology
through
o f thefiber
and axial
i n t h e porous
membrane,
a t t h elumen w a l l
o f continuous
Enzymes
flows
molecules.
t o control
be a c h i e v e d
stream.
capillary
theconversion
pressure
contribute
mits
layer
wall
where
transmembrane
that
skin
or i n hollow
c a n be c o n f i n e d
solution
t o t h e enzyme
reactors
i n membranes
o f an asymmetric
substrate-containing
men.
membrane
enzymes
interface,
those
labile imcan r e s u l t


60
FLOW
Figure
REM,
3. F l o w
capillary
servoir,
Figure
pH
OF
sheet
enzyme
LABORATORY
EXPERIMENTAL
o f laboratory
membrane
reservoir.
4. T r i g l y c e r i d e s
degree
o i l3%,lipase
PLANT
experimental
plant.
reactor ;SS,substrate re-
S P ,permeate
= 6, o l i v e
rate
SHEET
o fconversion
with
3 0 mg i n 5 0 0 m l ; a x i a l
960 ml/min.

time.
flow
5.
DRIOLI
from
concentration
unstirred
with
or p a r t i a l l y
surized
face
zyme
o f t h emembrane,
membrane
urease,
studied.
that
enzymes
has
From
been
a gel
cantly
forming
membrane
trary,
been
behaviour
specific
techniques
activity
a r ep a r t i c u l a r l y
induced
vironment;
this
The
represent
membrane
o f using
technology
f i l l e d
enzymes
vents,
h a s been
cent
limited
i nthecasting
nealing,
which
isolation
thermophile
zymes
a r eg e n e r a l l y
offers
an i n t e r e s t i n g
nes
technique
f i l l e d
with
S.Soifataricus
such
for
whole
prepared
membranes
by s e v e r a l
optimally
methods.
with
tech
sol
temperature an
properties.
(JL),
The r e
an
for
denaturating
using
ex
enzymes,
en
agents,
t h ephase
o f U F a n d RO
i n
membra
source.
o f industrial
and malic
f i l l e d
membranes
non-aqueous
a s t h e enzyme
enzymes
However, t h e
a t 8 0 C a n d w h o s e
t o protein
cells
a s -galactosidase
A r t i f i c i a l
for
t h ep r e p a r a t i o n
contains
favora
traditional
Solfatarieus"
opportunity
immobi
micro-en
b i o c a t a l y s t might
using
t h ec a t a l y t i c
stable
be
i nthegel.
o f U F a n d RO
by t h eneed
growing
transi
con
i nt h edevelopment o f
cells,
o f "Solfolobus
the
u l t r a f i l t r a t i o n and
with
engineering.
scale
o f
i sp a r t i c u l a r l y
solutions and high
destroys
treme
version
f i l l e d
o r whole
should
concentration
a n d enzyme
The more
enzymes,
i n t h e enzyme
improvement
a t industrial
technique
the kinetic
t o a decrease
traditional
preparation
niques,
on t h e con
The
(JL)
enzymes
t h e enzyme
enzyme
membranes
asignificant
with
not s i g n i f i
o r environmental
changes
mem
polymeric
t o conformational
thesituation
o f t h ehigh
osmosis
does
The
studying
lead
ligands
technique
moreover,
because
The
factors.
for
sensitive
significant
ble
also
retain
The method
and tubular
morphology,
allosteric
by s p e c i f i c
With
without
appears
o r t o a d e a c t i v a t i o n o f these
tions
straints.
etc., has
i t
system.
membrane
i nfact,
which
reverse
membranes
s t a b i l i t y .
t o be u s e f u l
o f immobilized
traditional
lized
flat
t o be t h e c o n t r o l l i n g
shown
gelled en
phosphatase,
i sincreased.
recirculating
t h e enzyme
formed
^ c i d
o n a UF m e m b r a n e
c u t - o f f a n d t h e membrane
appear
on t h e d e t a i l e d
results
s t a b i l i t y
c a nbe
on t h e pres
enzymes,1ipase,
t h esupporting
influence
for
layer
o u tusing
i na continuous
material
depending
a dynamically
malic
solution
o f enzyme
i n " g e l " form
technique
and t h e i r
carried
amount
theexperimental
forming
activity
branes
has
formation
-galactosidase,
been
their
Such
i n batch
processes
o f thesubstrate
immobilized
( JL ) .
Both
a n d i n UF
an appropriate
totally
dynamics
phenomena.
processes
recirculation
t h emembrane,
fluid

polarization
continuous
along
61
for
interest,
example.
S . S o i f a t a r i c u s have
Cellulose acetate
been
and poly-

62
sulfone
phase
were
were
used
used
for
crosslinking
A
obtain
cell
asymmetric
(10)
immobilization
hydrophilic polyisocyanate
liquid
two
structured
polyurethane
free
porous
suggested
films
containing
Physical
and
groups
as
an
tubes
and
by
the
glutaraldehyde
membranes
groups
and
used
in
per
thin
which
by
the
be
co-
protein
porous
The
use
at
with
has
material,
compounds
r e a c t i o n between
amino-groups
been
Hydrophilic
this
active
of
least
molecule,
agent.
prepared
specific
prepare
contains
polymer
biologically
and
to
films.
immobilyzing
can
immobilized
entrapment
cyanate
in
was
foams
prepolymer,
isocyanate
recently
membranes
Albumin
method.
polyurethane

to
inversion technique
(11).
free
contribute
iso
to
the
"immobilization".
The
physico-chemical
sidase
shown
activity
by
the
in
enzyme
the
-galactosidase
100
and
appeared
rature
and
lactosidase
nic
9
of
activity
cant
for
up
to
was
intact
free
by
ne
system
and
than
C.
p i l l a r y
res
of
Those
at
w/v,
was
the
Membranes
were
membrane
to
decrease
entrapment
may
be
were
a
The
the
dope
used
the
the
of
in
as
the
the
non-solvent
The
range
in
a
N-N
of
ca
mixtu
dimethyl-
bacteria,
5).
phase
the
in
conversion,
lyophilized
in
of
polyuretha
prepared
(Figure
to
dope
s i g n i f i
with
microorganism
c o n s i s t i n g of
mixture
according
preparations.
also
8-
enzymatic
increase
for
studied
dope
of
the
degree
been
orga
consequence
of
membrane
B-ga-
After
comparison
greater
have
tempe
with
imparted
in
procedures.
other
the
room
activity.
p o l y v i n y l p y r r o l i d o n and
of
on
wall
membranes
the
the
the
indications
The
capillaries
yer
typical
ger
structure
t i a l l y
at
pH
about
inversion
equipment
agent
to
in F i
promote
formation.
Microphotographs
of
no
activity
spinning
was
C,
those
trapped-cell
of
system
formed
: water
hr
at
loss
35-fold
t e c h n i q u e ^ (_12.) s p i n n i n g
gure
24
of
-galactosidase
the
added
to
to
optimal
any
effect
membranes
Before
were
up
the
activity
permeabi1ization
polysulphone,
acetamide.
At
temperature
c o n f i g u r a t i o n from
of
c e l l .
B-galacto-
similar
room
entrapment
in
were
at
Cell
This
rate,
s t a b i l i t y
70-85
3%
the
flow
hr
enzymatic
activity
permeate
for
cause
membrane
by
enzymatic
24
storage
cells.
cytoplasmic
caused
not
in
trapped-cell
Incubation
observed.
increase
free
stable
3-8.
wet
the
of
models
e x h i b i t e d maximal
of
did
above
in
pH
solvents
months
properties
the
of
membranes,
distribution
s t i l l
and
(1
10-20
Figure
the
where
the
dense
membranes,
c\m)
7,
give
b a c t e r i a both
underneath
exhibit
asymmetric
of
and
dense
internal
a
clear
in
skin l a
supporting
b a c t e r i a are
the
layer.
f i n
preferen
allocated.

DRIOLI
O*
w/foie ceils of

Solfaar/cus
qq fiiacrotnolecules

00
g-
Suhsirae
J Proc/ucis
)
Figure
red
Figure
ry
5. A s y m m e t r i c
by phase
6. F l o w
membrane
enzyme
invertion
sheet
capillary
membranes
prepa-
method.
o f thespinning
system
f o r capilla
formation.


F i g u r e 7 a . SEM p i c t u r e o f t h e d e n s e s k i n o f a n a s y m m e t r i c
c a p i l l a r y membrane w i t h e n t r a p p e d c e l l s .
Reproduced w i t h
p e r m i s s i o n f r o m R e f . 14.
F i g u r e 7 b . SEM p i c t u r e o f t h e p o r o u s s u b l a y e r o f a n
a s y m m e t r i c c a p i l l a r y membrane w i t h e n t r a p p e d c e l l s .
Reproduced w i t h p e r m i s s i o n f r o m R e f . 14.

5. DRIOLI
Mechanical
tested
fibres.
properties
strain
The
a
average
factor
o f t h emembranes
t o those
The Young
carried
2.5 r e l a t i v e
t o t h eaverage
fibres.
activity
evaluating
as permeate
rate
was found
entrapped
o f glucose
times
lactose
Tester.
lower
value
cells
by
o f
was
production,defi
glucose
permeate
concentration,
concen
and transmem
pressures.
t o product
cytoplasmatic
concentration
-galactosidase
Michaelis-Menten
parent
Michaelis
as
t h ep r e s s u r e
70
C r a n g e s
In
flow
a t different
Referring
atm,
o f membrane
t h er a t e
poly-
by a s t r e s s -
Universal
o f about
cell-free
ent
estimated
o u ton a Instrom
modulus
catalytic
brane
were
by c e l l - f r e e
wet, and t h e ultima
o f t h eYoung
the
tration
modulus,
were p r e l i m i n a r l y
value
The
ned
exhibited
o f t h emembranes
analysis
assessed

properties
and compared
sulphone
te
65
kinetic
i n t h epermeate
i ncells
behaviour,Figure
constant
increases
from
increases.
Maximum
glucose
from
2 0 . 4 t o 34 m o l e s / h r ,
stream,
e x h i b i t s an
appar
8. T h e
ap
3 . 7 t o 1 9 . 2 mM
production
a t
a t 0.04 a n d 0.055
respectively.
terms
ganisms
o f s t a b i l i t y
shows
preciable
months,
loss
t h e -galactosidase
t o be s t a b l e
o f activity,
a t least,
during
up t o o n e y e a r
when
stored,
continuous
= 78'C
o f the microor
without
any
ap
a n d up t o t h r e e
operation.
Qox = 3 . 8 U / h
50 mM acetate buffer pH 5
0.35 .
F i g u r e 8. G l u c o s e p r o d u c t i o n r a t e v s . l a c t o s e f e e d c o n c e n t r a t i o n .
= 70 *C. R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 1 3 .

66
From
the point
mances
most
comparable
resting
the
o f view
o f capillary
t o those
conversions
remarkable
tosidase
o f mechanical
membranes
i nl a c t o s e
o f immobilized
encourage
further
studies
membrane
reactor
oriented
an
enzyme
al
applications.
with
o f bacteria-free
observed
s t a b i l i t y
properties,
charged
perfor
cells
ones.
area l
The i n t e
hydrolysis and
bacterial
B-galac-
f o rt h e d e v e l o p m e n t o f
t o possible
industri

Literature Cited
1.
2.
3.
"Impact of Applied Genetics",OTA NR-132 April 1981.

Michaels,A.S. Desalination 1980,35,329.
Michaels,A.S.; Matson.S.L. Desalination 1985,53,231258.
4. Drioli,E.; Serafin,G.; Rigoli.A. presented at First
Engineering Foundation Conference " Advances in Fer
mentation Recovery Process Tech." Banff June 6-12
(1981) unpublished results.
5. Leuchtenberger,W.;Karrenbauer,M.;Picker,U. "Scale up
of an Enzyme Membrane Reactor Process for the Manu
facture of L-enantiomeric Compounds" Report from De
gussa AG, D-6450 Hanau I, FDR.
6. Molinari,R.; Drioli,. Proc.Nat.Congr.Ind.Chem.Div.
Sci., Siena 10-12 June 1985
7. Drioli,E.;Scardi,V. J.Mem.Sci. 1976,1,237-248.
8. Rossi,M.;Nucci,R.;Raia,C.A.;Molinari,R.;Drioli,.
J.of Mol.Cat. 1978,4,233.
9. De Rosa,M.;Gambacorta,A.;Esposito,.; Drioli,.;Gaeta,
S. Biochimie 1980,62, 517
10. Drioli,.;Iorio,G.; De Rosa,M.; Gambacorta,A.;Nicola
us,B. J.Mem.Sci. 1982,11,365-370
11. Drioli,.;Iorio,G.;Santoro,R.; De Rosa,M.;Gambacorta,
A.;Nicolaus,R. J . Mol.Cat.1982,14,247.
12. Catapano,G.;Iorio,G.;Drioli,.;Filosa,M. "Capillary
Membrane Bioreactors with Entrapped Whole Cells : a
Theoretical Model" submitted for publication.
13. Drioli,.;Iorio,G.; Catapano,G.; De Rosa,M.; Gamba
corta, . J.f Mem.Sci. in press.
14.
Drioli, E.; et al. La Chimica L'Industria 1985, (67)11,

617-622.

6
Liquid Emulsion Membranes and Their Applications
in Biochemical Separations

M . P. Thien, T. A. Hatton, and D. I. C. Wang

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge,
M A 02139
Applications of liquid emulsion membranes (LEMs) to

biomedical and biochemical systems are reviewed and
other potential applications identified. The LEM-mediated downstream processing of small, zwitterionic
biochemicals (e.g. amino acids) is examined using
chloride ion counter-transport to separate and
concentrate the amino acid phenylalanine from
stimulated fermentation broth. The effect of agitation rate and osmotic swelling of membranes on
separation is shown to be significant.
F e r m e n t a t i o n t e c h n o l o g y h a s p r o g r e s s e d much i n t h e p a s t d e c a d e .
Advances i ng e n e t i c e n g i n e e r i n g , coupled w i t h b e t t e r a n a l y t i c a l
t e c h n i q u e s a n d more e f f i c i e n t f e r m e n t a t i o n i n s t r u m e n t a t i o n , h a v e
made p o s s i b l e t h e l a r g e - s c a l e p r o d u c t i o n o f many c o m p l e x a n d e x o t i c
biochemicals.
U n f o r t u n a t e l y , advances i n s e p a r a t i o n and p u r i f i c a t i o n t e c h n o l o g y h a v e n o t k e p t a b r e a s t o f t h o s e made i n f e r m e n t a t i o n .
Most downstream p r o c e s s i n g r e q u i r e s a number o f t r a d i t i o n a l a n d i n herently batch unit operations, operations which are g e n e r a l l y
c o s t l y and i n e f f i c i e n t .
One n e w t e c h n i q u e , t h e u s e o f l i q u i d
e m u l s i o n m e m b r a n e s , p r e s e n t s g r e a t p o t e n t i a l a s a n e f f i c i e n t means
of s e p a r a t i n g and c o n c e n t r a t i n g fermentation products.
In this
p a p e r , a r e v i e w o f L E M t e c h n o l o g y i s p r e s e n t e d w i t h some e m p h a s i s
g i v e n t o t h e l i m i t e d work done i n t h e a r e a o f b i o t e c h n o l o g y .
As a
s p e c i f i c e x a m p l e o f t h e p o t e n t i a l f o r LEMs i n b i o t e c h n o l o g y , we
summarize o u r work on t h e r e c o v e r y o f t h e amino a c i d p h e n y l a l a n i n e
from simulated fermentation b r o t h .
Concept
S i n c e t h e y w e r e f i r s t d e v e l o p e d i n 1 9 6 7 (jL) , l i q u i d e m u l s i o n
membranes (LEMs) h a v e b e e n u s e d i n a v a r i e t y o f s e p a r a t i o n s ( s e e
r e v i e w s b y F r a n k e n f e l d a n d L i (2) , M a r r a n d Kopp (3), a n d Way et_ a l .
(4)).
C o n c e p t u a l l y , LEMs a r e q u i t e s i m p l e .
They c o n s i s t o f an
0097-6156/ 86/0314-0067$06.00/ 0

68

e m u l s i o n o f two i m m i s c i b l e p h a s e s w h i c h , once f o r m e d , i s subsequently d i s p e r s e d i n a t h i r d (continuous o r " e x t e r i o r " ) phase ( s e e

F i g u r e 1 ) . A s i s s e e n i n F i g u r e 1, o n e o f t h e t w o p h a s e s w h i c h
comprise t h e e m u l s i o n i s c o m p l e t e l y e n c a p s u l a t e d by t h e o t h e r phase.
T h i s e n c a p s u l a t e d " i n t e r i o r " o r " d i s p e r s e d " phase thus n e v e r
d i r e c t l y c o n t a c t s t h e continuous o r e x t e r n a l phase.
I n most a p p l i c a t i o n s t h e i n t e r i o r and e x t e r i o r phases a r e aqueous s o l u t i o n s . The
n o n - d i s p e r s e d e m u l s i o n phase ( u s u a l l y an o i l , making t h e r e s u l t i n g
e m u l s i o n a " w a t e r - i n - o i l , " W/0, e m u l s i o n ) a c t s a s a membrane b e t w e e n
t h e i n t e r i o r a n d e x t e r i o r p h a s e s a n d i s t h u s c a l l e d t h e membrane
phase.
S e p a r a t i o n s u s i n g LEMs c a n b e e f f e c t e d i n t w o d i f f e r e n t w a y s
(.5).
The f i r s t o f t h e s e ( c a l l e d TYPE I t r a n s p o r t ) i s a s i m p l e d i f f u s i o n p r o c e s s i n w h i c h t h e s o l u t e p a r t i t i o n s i n t o t h e membrane
p h a s e f r o m t h e e x t e r i o r p h a s e , d i f f u s e s a c r o s s t h e membrane t o t h e
d i s p e r s e d i n t e r i o r phase d r o p l e t s , and p a r t i t i o n s i n t o t h e i n t e r i o r
phase.
A r e a c t i o n takes p l a c e i n t h e i n t e r n a l phase which converts
the s o l u t e i n t o a species which i s incapable of p a r t i t i o n i n g back
i n t o t h e membrane p h a s e .
TYPE I t r a n s p o r t i s l i m i t e d t o u n c h a r g e d
s o l u t e s o n l y , s i n c e o n l y uncharged s o l u t e s w i l l be a b l e t o f a v o r a b l y
p a r t i t i o n i n t o t h e membrane p h a s e .
The s e c o n d t y p e o f t r a n s p o r t p r o c e s s , T Y P E I I o r f a c i l i t a t e d
t r a n s p o r t , i s shown i n F i g u r e 2 u s i n g L - p h e n y l a l a n i n e , an amino a c i d ,
as t h e s o l u t e t o b e s e p a r a t e d a n d c o n c e n t r a t e d .
The s o l u t e , due t o
i t s charge (the phenylalanine i s separated w h i l e i n i t s a n i o n i c
form), cannot p a r t i t i o n i n t o t h e o i l phase by i t s e l f .
Consequently,
a n o n - w a t e r s o l u b l e " c a r r i e r " m o l e c u l e , u s u a l l y an i o n i c s u r f a c t a n t
c o n s i s t i n g o f a h y d r o p h o b i c s e c t i o n and an u n i v a l e n t l y c h a r g e d
f u n c t i o n a l g r o u p , i s a d d e d t o t h e membrane p h a s e .
The example i n
F i g u r e 2 u s e s a q u a t e r n a r y ammonium s a l t a s t h e c a r r i e r .
In order
to remain i n t h e oil/membrane phase, t h e c a r r i e r coordinates w i t h a
counter-ion (chloride, i n this case).
I f t h e c a r r i e r i s a l r e a d y complexe! w i t h t h e i n t e r i o r p h a s e c o u n t e r - i o n b e f o r e s e p a r a t i o n b e g i n s ,
t h e c a r r i e r - i o n c o m p l e x d i f f u s e s a c r o s s t h e membrane t o t h e
exterior/membrane phase i n t e r f a c e .
A t t h i s i n t e r f a c e an i o n exchange r e a c t i o n takes p l a c e i n which t h e s o l u t e i s exchanged f o r
the c o u n t e r - i o n .
T h i s r e a c t i o n i s d r i v e n e i t h e r b y "mass a c t i o n "
(i.e.
t h e r e i s v e r y l i t t l e c o u n t e r - i o n i n t h e e x t e r i o r p h a s e compared t o the s o l u t e ) , o r by t h e c a r r i e r s h i g h a f f i n i t y f o r t h e
solute.
The c a r r i e r - s o l u t e c o m p l e x t h e n d i f f u s e s b a c k a c r o s s t h e
membrane t o t h e i n t e r i o r p h a s e w h e r e t h e s o l u t e i s e x c h a n g e d f o r t h e
counter-ion ( u s u a l l y present i n great excess).
I f the counter-ion
i n t h e membrane i s c o n c e n t r a t e d e n o u g h , t h e t r a n s p o r t o f s o l u t e w i l l
be d r i v e n b y t h e c o u n t e r - i o n g r a d i e n t ( i n a p r o c e s s a k i n t o a c t i v e
t r a n s p o r t a c r o s s c e i l membranes).
S i n c e t h e volume o f t h e i n t e r i o r
p h a s e c a n b e made much s m a l l e r t h a n t h a t o f t h e e x t e r i o r p h a s e ,
s e p a r a t i o n and c o n c e n t r a t i o n o c c u r s i m u l t a n e o u s l y as t h e s o l u t e i s
t r a n s p o r t e d from e x t e r i o r t o i n t e r i o r phase.
1
W h i l e p r o v i d i n g a means o f s e p a r a t i n g a n d c o n c e n t r a t i n g a g i v e n
s o l u t e , LEMs a r e n o t w i t h o u t p o t e n t i a l p r o c e s s d i f f i c u l t i e s .
One
such d i f f i c u l t y i s that o f mechanical s t a b i l i t y .
While considered
as a s i g n i f i c a n t c o n c e r n b y some ( 2 , 7 , 8 ) , p r o p e r f o r m u l a t i o n o f t h e
membrane p h a s e c a n a l m o s t e r a d i c a t e a n y p r o c e s s d i f f i c u l t i e s
a s s o c i a t e d w i t h membrane b r e a k a g e .
A n o t h e r , a n d p e r h a p s much m o r e


THIEN E T A L .
Liquid Emulsion Membranes for Biochemical Separations
Droplets of Internal
Reagent Phase
External,
F i g u r e 1.
system.
Exterior
C
Continuous
Schematic diagram o f a l i q u i d
Phase
= (C - C ) 8
10 3
Membrane P h a s e
Phase
e m u l s i o n membrane ( L E M )
Interior
Phase
- CH
3
F i g u r e 2. M e c h a n i s m f o r t y p e I I ( f a c i l i t a t e d ) t r a n s p o r t
p h e n y l a l a n i n e / c h l o r i d e system.

i n a
70

o f a s i g n i f i c a n t c o n c e r n , i s t h a t o f membrane s w e l l . O s m o t i c s w e l l
i s a p r o c e s s by w h i c h w a t e r i s t r a n s f e r r e d i n t o t h e i n t e r i o r a q u e o u s
phase v i a the d i f f u s i o n of hydrated s u r f a c t a n t molecules.
Much l i k e
a r e g u l a r TYPE I I t r a n s p o r t p r o c e s s , i t i s b e l i e v e d (6) t h a t w a t e r
i s t r a n s p o r t e d a c r o s s t h e membrane due t o t h e o s m o t i c g r a d i e n t
e s t a b l i s h e d by t h e c o n c e n t r a t i o n o f i n t e r n a l phase r e a g e n t .
Although s e l d o m m e n t i o n e d i n the l i t e r a t u r e ( 7 - 9 ) , s w e l l i n g has b e e n
r e p o r t e d b y some t o b e as h i g h as 1 5 0 % o f t h e i n t e r i o r p h a s e v o l u m e
(10).
C o l i n a r t e t a l . (6) u n d e r t o o k a m e c h a n i s t i c s t u d y s h o w i n g
t h a t , s i n c e p r a c t i c a l l y a l l s u r f a c t a n t s h y d r a t e t o some e x t e n t ,
s w e l l i n g c a n be a n t i c i p a t e d f o r a l l LEM f o r m u l a t i o n s a n d c o u l d be a
s i g n i f i c a n t p r o b l e m i n LEM
separations.
Applications
I n s p i t e o f t h e p o t e n t i a l p r o b l e m s o f s w e l l i n g a n d b r e a k a g e , LEMs
h a v e b e e n t e s t e d on t h e p i l o t p l a n t s c a l e w i t h good r e s u l t s ( 2 , 9 , 1 1 ,
1 2 ) , and w i l l s o o n be u s e d f o r t h e c o m m e r c i a l s e p a r a t i o n o f z i n c
f r o m V i s c o s e w a s t e w a t e r f r o m r a y o n and c e l l o p h a n e p r o c e s s i n g ( 1 3 ) .
I t s h o u l d be n o t e d t h a t t h e s e p i l o t p l a n t s t u d i e s i n d i c a t e d t h a t
LEM
p r o c e s s e s w e r e as e c o n o m i c a l l y a d v a n t a g e o u s , i f n o t m o r e s o , t h a n
c u r r e n t l y e m p l o y e d s o l v e n t e x t r a c t i o n and c o n v e n t i o n a l i o n e x c h a n g e
techniques.
U n f o r t u n a t e l y , LEM-mediated s e p a r a t i o n s of
biochemicals
h a v e n o t b e e n c a r r i e d o u t on a p i l o t p l a n t s c a l e .
LEMs h a v e b e e n a p p l i e d f o r t h e s e p a r a t i o n a n d r e c o v e r y o f a
h o s t o f d i f f e r e n t compounds.
P r e v i o u s e f f o r t s have been p r i m a r i l y
f o c u s e d on t h e r e c o v e r y o f m e t a l i o n s f r o m a q u e o u s s o l u t i o n s ( i n c l u d i n g c o p p e r , z i n c , chromium, m e r c u r y , u r a n i u m , n i c k e l , and i r o n ; ( 3 ) )
and t h e r e m o v a l o f o r g a n i c compounds f r o m w a s t e w a t e r
(14-17).
I n t e r m s o f t h e amount o f l i t e r a t u r e d e v e l o p e d , b i o c h e m i c a l
s e p a r a t i o n s h a v e b e e n l a r g e l y i g n o r e d by t h o s e i n t h e f i e l d o f
LEMmediated separations.
One a p p l i c a t i o n t h a t h a s e n j o y e d some e x p e r i m e n t a l s c r u t i n y i s t h a t o f t h e u s e o f LEMs i n d r u g d e l i v e r y a n d
overdose p r e v e n t i o n systems.
They have been used t o s e p a r a t e o r r e l e a s e
s e v e r a l d i f f e r e n t types of drugs i n c l u d i n g a c e t y l s a l i c y c l i c a c i d
( 1 8 ) , phnobarbital ( 1 9 ) , a n d s e v e r a l b a r b i t u r a t e s
(20,21).
LEM s y s t e m s h a v e a l s o b e e n s h o w n t o be s u c c e s s f u l i n s e p a r a t i n g
c o m m o d i t y - t y p e b i o c h e m i c a l s s u c h as p r o p i o n i c a c i d ( 1 0 ) a n d a c e t i c
a c i d ( 1 0 , 2 2 ) and h a v e b e e n u s e d f o r t h e p r e p a r a t i o n o f L - a m i n o a c i d s
f r o m r a c e m i c D,L m i x t u r e s b y means o f e n z y m a t i c h y d r o l y s i s o f a m i n o
acid esters (23).
In a d d i t i o n to b i o c h e m i c a l s e p a r a t i o n s , the work
o f M o h a n a n d L i s h o w e d t h a t e n z y m e s c o u l d be e n c a p s u l a t e d i n l i q u i d
e m u l s i o n m e m b r a n e s w i t h no d e l e t e r i o u s e f f e c t on enzyme a c t i o n ( 2 4 ) .
L a t e r w o r k by t h e s e a u t h o r s i n d i c a t e d t h a t e n c a p s u l a t e d
live cells
c o u l d r e m a i n v i a b l e a n d f u n c t i o n i n t h e LEM i n t e r i o r p h a s e f o r p e r i o d
as l o n g as f i v e d a y s ( 2 5 ) .
T h e s e v a r i o u s u s e s o f l i q u i d e m u l s i o n membranes show t h e
v e r s a t i l i t y o f L E M - m e d i a t e d s e p a r a t i o n s and p o i n t t o p o s s i b l e a p p l i c a t i o n s o f l i q u i d e m u l s i o n membranes i n t h e b i o c h e m i c a l s
field.
These a p p l i c a t i o n s i n c l u d e the i n - s i t u r e c o v e r y of f e r m e n t a t i o n
prod u c t s . T h i s w o u l d be p a r t i c u l a r l y u s e f u l i n f e r m e n t a t i o n s w h e r e t h e
major product i n h i b i t s growth or p r o d u c t i o n r a t e s .
Examples i n c l u d e
t h o s e s y s t e m s w h i c h p r o d u c e o r g a n i c a c i d s s u c h as a c e t a t e
and
lactate.
P e r h a p s t h e m o s t u s e f u l a p p l i c a t i o n w o u l d be t h e down-

6.
I EN ET AL.
71
stream p r o c e s s i n g o f biochemicals that canonly be economically

separated by c o s t l y i o n exchange t e c h n i q u e s .
One e x a m p l e o f t h i s
l a s t category o f a p p l i c a t i o n s i s t h a t o f the downstream p r o c e s s i n g o f
amino a c i d s .
T h i s a p p l i c a t i o n has b e e n t h o r o u g h l y i n v e s t i g a t e d b y
the authors andour s t u d i e s are summarized below.
A more e x t e n s i v e
a n a l y s i s o f our r e s u l t s w i l l be given i n a l a t e r p u b l i c a t i o n (26).

Amino A c i d R e c o v e r y U s i n g L i q u i d
Emulsion
Membranes
Amino a c i d s f i n d a v a r i e t y o f c o m m e r c i a l a p p l i c a t i o n s , i n c l u d i n g u s e
as l i v e s t o c k f e e d s u p p l e m e n t s , n u t r i t i o n a l s u p p l e m e n t s , a n d r a w
m a t e r i a l s f o r s p e c i a l i t y c h e m i c a l s ( e . g . g l u t a m i c a c i d f o r monosodium
g l u t a m a t e , MSG).
M o s t o f t h e s e a m i n o a c i d s a r e p r o d u c e d i n commer
c i a l q u a n t i t i e s by m i c r o b i a l fermentation.
U n f o r t u n a t e l y , some a m i n o
a c i d s must b e s e p a r a t e d b y c o m p l i c a t e d a n d c o s t l y i o n - e x c h a n g e
schemes due t o l o w f e r m e n t a t i o n t i t e r s and v e r y l o w s o l u b i l i t y i n
o r g a n i c media ( s o l u b i l i t i e s l o w enough t o p r e c l u d e the u s e o f s o l v e n t
e x t r a c t i o n a s a means o f s e p a r a t i o n ) . S u b s e q u e n t s e p a r a t i o n a n d
p u r i f i c a t i o n c o s t s may c o m p r i s e a s much a s 8 0 % o f t o t a l p r o d u c t i o n
c o s t s f o r t h e s e a m i n o a c i d s . One s u c h p r o d u c t , p h e n y l a l a n i n e , h a s
r e c e n t l y e n j o y e d g r e a t c o m m e r c i a l s u c c e s s as one o f the raw m a t e r i a l s
of the n o n - n u t r i t i v e sweetner aspartame (Nutrasweet).
Phenylalanine
h a v i n g somewhat l o w e r f e r m e n t a t i o n t i t e r s t h a n t y p i c a l b i o c h e m i c a l
commodities, coupled w i t h the c u r r e n t commercial i n t e r e s t s i n p h e n y l
alanine
a n d i t s a b i l i t y t o b e e a s i l y m e a s u r e d b y UV s p e c t r o p h o t o
m e t r y , make p h e n y l a l a n i n e a g o o d m o d e l c o m p o u n d f o r t e s t i n g n e w b i o
chemical separation techniques.
T h i s b e i n g t h e c a s e , we h a v e c h o s e n
to t e s t the a p p l i c a b i l i t y o f LEM-mediated s e p a r a t i o n s t o the problem
of the downstream p r o c e s s i n g o f p h e n y l a l a n i n e from f e r m e n t a t i o n
broth.
Experimental
System
The s y s t e m e x a m i n e d i n t h i s s t u d y w a s t h a t o f a s i m p l e p h e n y l
a l a n i n e / c h l o r i d e c o u n t e r - t r a n s p o r t system i n which the i n t e r i o r
p h a s e was c o n c e n t r a t e d i n c h l o r i d e a n i o n a n d t h e e x t e r i o r p h a s e c o n
t a i n e d c o n c e n t r a t i o n s o f p h e n y l a l a n i n e i n the range o f commercial
fermentation t i t e r s .
S i n c e p h e n y l a l a n i n e ( l i k e a l l -amino a c i d s ) i s
p r e d o m i n a n t l y a z w i t t e r i o n a t p H ' s b e t w e e n 3.5 a n d 9.0 ( a n d i s t h u s
u n a b l e t o b e t r a n s p o r t e d t h r o u g h t h e membrane), t h e pH o f t h e e x t e r i o r
p h a s e w a s k e p t a b o v e p H 10.0 t o i n s u r e t h a t t h e a m i n o a c i d w a s
p r e s e n t i n i t s t r a n s p o r t a b l e a n i o n i c form.
As mentioned above, t h e
m e c h a n i s m f o r t h i s s y s t e m i s shown i n F i g u r e 2.
E x p e r i m e n t s t y p i c a l l y c o n s i s t e d o f d i s p e r s i n g 70 m i l l i l i t e r s o f
a 2 m o l a r s o l u t i o n o f s o d i u m c h l o r i d e (NaCl) i n 100 m i l l i l i t e r s o f an
o i l phase.
This o i l phase c o n s i s t e d o f a p a r a f f i n i e s o l v e n t (Solvent
100 N e u t r a l , E x x o n C h e m i c a l C o m p a n y ) , a n o n i o n i c e m u l s i o n - s t a b i l i z i n g
s u r f a c t a n t (Paranox 100, Exxon C h e m i c a l Company), a c a t i o n i c
" c a r r i e r " - a t r i - c a p r y l q u a t e r n a r y ammonium s a l t ( A l i q u a t 3 3 6 ,
Henkel C o r p o r a t i o n ) , anda c o - s u r f a c t a n t f o r the c a r r i e r molecule
( d e c y l a l c o h o l , Sigma C h e m i c a l ) .
The aqueous phase was s l o w l y added
t o t h e o i l p h a s e u n d e r i n t e n s e s h e a r p r o v i d e d b y a s t a t o r - t y p e homogenizer.
A l l emulsions had s i m i l a r i n t e r n a l droplet s i z e and s i z e
d i s t r i b u t i o n s a s a n a l y z e d b y a H o r i b a CAPA 5 0 0 p a r t i c l e s i z e


72
d i s t r i b u t i o n analyzer.
The e x t e r i o r p h a s e c o n s i s t e d o f 700
millil i t e r s o f 11 g/1 L - p h e n y l a l a n i n e
( S i g m a C h e m i c a l ) b r o u g h t t o pH
11.0
b y p o t a s s i u m h y d r o x i d e a d d i t i o n . The e m u l s i o n f o r m u l a t i o n a n d p h a s e
volumes were not o p t i m i z e d f o r s e p a r a t i o n performance.
Batch s e p a r a t i o n e x p e r i m e n t s were c a r r i e d out i n a b a f f l e d
g l a s s 2 l i t e r v e s s e l a t 25C.
A m e a s u r e d a m o u n t o f e m u l s i o n was
poured i n t o the r e a c t i o n v e s s e l , a measured q u a n t i t y of e x t e r i o r
p h a s e was a d d e d t o t h e v e s s e l a n d t h e a g i t a t i o n a n d t i m e r w e r e t u r n e d
on.
V e s s e l contents were a g i t a t e d u s i n g s i x m a r i n e - t y p e i m p e l l e r s .
I m p e l l e r RPM was m o n i t o r e d b y s t r o b e l i g h t .
Samples were t a k e n
t h r o u g h o u t t h e e x p e r i m e n t v i a a s a m p l i n g p o r t a t t h e b o t t o m o f the.
vessel.
E m u l s i o n was q u i c k l y s e p a r a t e d f r o m t h e e x t e r i o r p h a s e o f
s a m p l e s and d i s c a r d e d .
The e x t e r i o r p h a s e was a n a l y z e d f o r p h e n y l a l a n i n e
concentration
a n d pH.
A l l s a m p l e v o l u m e s w e r e r e c o r d e d and u s e d f o r mass b a l a n c e
determination.
P h e n y l a l a n i n e was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y a t
^max
2 5 7 . 5 nm.
Changes i n i n t e r i o r phase volume were c a l c u l a t e d
using material balances.
A l l m a t e r i a l b a l a n c e s c l o s e d t o w i t h i n 2%.
I n t e r i o r phase c o n c e n t r a t i o n s were e s t i m a t e d by the use o f m a t e r i a l
b a l a n c e s and e x t e r i o r p h a s e c o n c e n t r a t i o n s .
The i n t e r i o r p h a s e comp o n e n t s o f s e v e r a l r e p r e s e n t a t i v e e m u l s i o n s were measured by a n a l y z ing the i n t e r i o r phase components a f t e r t h e r m a l l y d e m u l s i f y i n g the
emulsion samples.
These measurements agreed w i t h e s t i m a t e s t o
w i t h i n 10%.
=
Results
S e p a r a t i o n r e s u l t s f o r a t y p i c a l membrane f o r m u l a t i o n u s e d i n o u r
s t u d i e s a r e s h o w n i n F i g u r e 3.
The r e s u l t s , p r e s e n t e d i n a c o n c e n t r a t i o n p r o f i l e format, i n d i c a t e that i n i t i a l separation i s fast
(when t h e d r i v i n g f o r c e , t h e c h l o r i d e g r a d i e n t , i s g r e a t e s t ) b u t
s l o w s down a s t r a n s p o r t c o n t i n u e s a n d c h l o r i d e i s t r a n s p o r t e d t o t h e
e x t e r i o r phase.
I t s h o u l d be n o t e d t h a t , a l t h o u g h t h e p r o f i l e g i v e s
a rough i d e a of s e p a r a t i o n performance, i t does not i n d i c a t e the
r e s p e c t i v e v o l u m e s o f t h e v a r i o u s p h a s e s and t h u s does n o t a c c o u n t
f o r t h e e f f e c t s o f membrane s w e l l o r b r e a k a g e .
The i n c i d e n c e a n d
r a m i f i c a t i o n s o f t h e s e e f f e c t s w i l l be d i s c u s s e d l a t e r .
The a g i t a t i o n d e l i v e r e d t o t h e r e a c t i o n v e s s e l i s an
important
p a r a m e t e r i n LEM s e p a r a t i o n s .
The a g i t a t i o n r a t e n o t o n l y i s t h e
d o m i n a n t f a c t o r i n d e t e r m i n i n g t h e s i g n i f i c a n c e o f mass t r a n s f e r
r e s i s t a n c e e x t e r n a l to the emulsion g l o b u l e s , but i t a l s o determines
t h e e m u l s i o n g l o b u l e s i z e , and t h u s t h e a r e a a v a i l a b l e f o r t r a n s p o r t .
The e f f e c t s o f a g i t a t i o n o n LEM a m i n o a c i d s e p a r a t i o n s a r e s h o w n i n
F i g u r e s 4 a n d 5.
The t r a d i t i o n a l p r o f i l e s i n F i g u r e 4 i n d i c a t e t h a t
t h e mass t r a n s f e r r a t e i s i n c r e a s e d w i t h i n c r e a s e s i n a g i t a t i o n .
F i g u r e 5 s h o w s c h a n g e s o f i n t e r i o r p h a s e v o l u m e ("%
swell")
b a s e d on i n i t i a l i n t e r i o r p h a s e v o l u m e ) a n d t h e e s t i m a t e d
internal
p h a s e p h e n y l a l a n i n e c o n c e n t r a t i o n as a f u n c t i o n o f a g i t a t i o n s p e e d .
T h i s f i g u r e i n d i c a t e s t h a t , c o n t r a r y t o e x p e c t a t i o n s b a s e d on t h e
r e s u l t s g i v e n i n F i g u r e 4, t h e b e s t s e p a r a t i o n ( i n t e r m s o f c o n c e n t r a t i n g p h e n y l a l a n i n e ) o c c u r s a t 400 RPM w h e r e c h a n g e s i n membrane
v o l u m e a r e a t a minimum.

THIEN E T A L .

12.0
10.0

8.0
c 6.0
4.0
0.0
20
Time (min)
F i g u r e 3. C h l o r i d e
separation.
countertransport
system:
t y p i c a l LEM
12.0
10.0
9.0
7.0
20
Time ( m i n )
Figure
4.
Effect
ofagitation
speed
on LEM s e p a r a t i o n .



6.
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75

Discussion
The a b o v e w o r k s h o w s t h a t a m i n o a c i d s c a n i n d e e d b e s e p a r a t e d a n d
c o n c e n t r a t e d u s i n g l i q u i d e m u l s i o n membranes.
The r e s u l t s a l s o
i n d i c a t e some v e r y i m p o r t a n t c o n c e r n s w h e n c o n s i d e r i n g t h e u s e o f
LEM-based s e p a r a t i o n p r o c e s s e s .
C o n t r a r y t o t r a d i t i o n a l methods o f
e v a l u a t i n g LEM p e r f o r m a n c e , the p a r a m e t e r o f d i r e c t i n t e r e s t , t h a t i s
t h e i n t e r i o r p h a s e c o n c e n t r a t i o n , must b e e x a m i n e d .
The e f f e c t s o f
s w e l l i n g a n d b r e a k a g e , due t o t h e i r p o t e n t i a l s i g n i f i c a n c e , must
always be considered s e r i o u s l y .
Several different strategies could
be f o r m u l a t e d t o m i n i m i z e t h e d e l e t e r i o u s e f f e c t s o f s w e l l i n g i n t h e
above c o u n t e r - i o n p r o c e s s .
F u r t h e r i m p r o v e m e n t s , such a s an amino
a c i d - s p e c i f i c r e a c t i o n i n t h e i n t e r i o r p h a s e ( t o m a i n t a i n a maximum
d r i v i n g f o r c e ) c o u l d be used t o s i g n i f i c a n t l y improve the performance
o f LEM systems f o r amino a c i d s e p a r a t i o n s .
A s c h e m a t i c o f one p o s s i b l e c o m m e r c i a l LEM r e c o v e r y scheme f o r
amino a c i d s w h i c h t a k e s advantage o f t h e i r u n i q u e s o l u b i l i t y p r o p e r
t i e s i s shown i n F i g u r e 6. Once t h e a m i n o a c i d h a s b e e n c o n c e n
t r a t e d i n a b a s i c i n t e r i o r phase, the emulsion i s broken and t h e
i n t e r i o r phase n e u t r a l i z e d w i t h the a c i d o f the c o u n t e r - i o n t o
a p p r o x i m a t e l y p H 7.0.
I t h a s been found t h a t amino a c i d s have a
much h i g h e r s o l u b i l i t y i n a q u e o u s s o l u t i o n s o f e x t r e m e p H t h a n i n
t h e b r o a d i s o e l e c t r i c r a n g e c e n t e r e d a r o u n d p H 7.0 ( 2 7 ) .
Neutral
i z i n g t h e pH o f t h e i n t e r i o r p h a s e s h o u l d c a u s e t h e s p o n t a n e o u s
c r y s t a l l i z a t i o n o f t h e amino a c i d , l e a v i n g o n l y t h a t amount o f amino
a c i d t h a t i s s o l u b l e i n the i s o e l e c t r i c range i n s o l u t i o n . The
r e s u l t i n g n e u t r a l i z e d s o l u t i o n may t h e n b e u s e d a g a i n a s t h e
i n t e r i o r phase a f t e r s a l t and base i s added.
Conclusions
The v e r s a t i l i t y o f LEMs i s c l e a r .
From the e n c a p s u l a t i o n o f l i v i n g
c e l l s t o the removal o f t o x i c o r i n h i b i t i n g s u b s t a n c e s , and i n t h e i r
use a s a d o w n s t r e a m p r o c e s s , l i q u i d e m u l s i o n membranes r e m a i n a
p o w e r f u l and, a s o f y e t , v i r t u a l l y u n t a p p e d r e s o u r c e f o r b i o c h e m i c a l
engineers.
T h e a b i l i t y o f LEMs t o s e p a r a t e a n d c o n c e n t r a t e a m i n o
acids demonstrated here gives strength t o t h i s observation, and i t
i s a n t i c i p a t e d t h a t these systems w i l l enjoy i n c r e a s i n g a t t e n t i o n i n
t h e y e a r s t o come.
Acknowledgments
T h i s work was f u n d e d i n p a r t b y a Monsanto J u n i o r F a c u l t y Award t o
T.A. H a t t o n .
We e x p r e s s o u r g r a t i t u d e t o K a r e n L e e , L i n d a M a r i n i l l i ,
and Todd Renshaw f o r t h e i r v a l u a b l e t e c h n i c a l a s s i s t a n c e .

76

Replenished Interior
Phase
Membrane Make-up
Membrane Phase
Phenylalanine
Crystals
"Mother Liquor"
F i g u r e 6.
Process
d i a g r a m f o r LEM-based r e c o v e r y
o f amino

acids.
6. THIEN ET AL.
11
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L i , . N.; Shrier, A. L. Rec. Dev. in Sep. Tech. 1972, 1, 163.
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Chilamarki, R. N.; Rhodes, C. T. J. Appl Biochem. 1976, 2, 405.
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1974, 60, 2, 258.

7
Use of Aqueous Two-Phase Systems for Recovery and
Bo Mattiasson and Rajni Kaul

Department of Biotechnology, Chemical Center, University of Lund, S-221 00 Lund,
Sweden
Aqueous two-phase systems are generated by mixing

aqueous solutions of two water-soluble polymers, or
a polymer and a salt. These systems offer extremely
mild conditions for separation of cells, organelles,
proteins and other biomolecules, in biochemical processes. Considerable attention has been directed
towards the use of the two-phase systems in several
areas of biotechnology. The present paper summarizes
the state of the art concerning extractive bioconversions for production of small as well as macromolecules, and protein purification using aqueous
two-phase system.
The w e l l d o c u m e n t e d p h e n o m e n o n o f s e p a r a t i o n o f a n a q u e o u s s o l u t i o n
o f two d i f f e r e n t w a t e r - s o l u b l e p o l y m e r s i n t o i n d i v i d u a l p h a s e s ,
d u r i n g r e c e n t y e a r s , h a s shown w i d e s p r e a d p o t e n t i a l i n b i o t e c h n o l o g y
(1) A n u m b e r o f p o l y m e r s h a v e b e e n e m p l o y e d f o r t h e p r e p a r a t i o n o f
t h e s e b i - p h a s i c s y s t e m s . ( 2 ) . The m o s t c o m m o n l y u s e d s y s t e m s h a v e
b e e n t h o s e o f p o l y ( e t h y l e n e g l y c o l ) ( P E G ) a n d d e x t r a n . The m o l e c u l a r
weight o f t h e polymers used p l a y s an i m p o r t a n t r o l e i n d e t e r m i n i n g
the c h a r a c t e r i s t i c s o f t h e phase system. Phase systems formed b y
m i x i n g one polymer and a h i g h c o n c e n t r a t i o n o f c e r t a i n s a l t s i n
aqueous s o l u t i o n s , have a l s o been r e p o r t e d . ( 2 ) .
Aqueous two-phase systems a r e c h a r a c t e r i z e d b y a h i g h water cont e n t , w h i c h makes t h e m c o m p a t i b l e w i t h t h e b i o l o g i c a l m a t e r i a l . T h e
d i s t r i b u t i o n o f b i o m o l e c u l e s between the phases i s determined
mainly
by t h e i r s u r f a c e p r o p e r t i e s a n d t h e c o m p o s i t i o n o f t h e phase s y s t e m ,
and i s d e n o t e d b y p a r t i t i o n c o e f f i c i e n t , K
t , d e f i n e d as t h e
r a t i o o f i t s c o n c e n t r a t i o n i n the top and bottom phase, r e s p e c t i v e l y .
The p a r t i t i o n i n g i s i n d e p e n d e n t o f t h e a b s o l u t e c o n c e n t r a t i o n o f t h e
s u b s t a n c e o v e r a f a i r l y wide range. Any s u b s t a n c e p r e f e r s a phase
w h e r e a maximum number o f i n t e r a c t i o n s a r e p o s s i b l e ; t h e s e c o u l d b e
r e l a t e d to e l e c t r i c a l , h y d r o p h i l i c , hydrophobic and c o n f o r m a t i o n a l
f o r c e s . R e p o r t s i n t h e l i t e r a t u r e h a v e shown t h a t d i f f e r e n t c h a r a c t e r i s t i c s o f t h e system c a n be m a n i p u l a t e d i n o r d e r t o a c h i e v e t h e
p
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7.
MATTIASSON A N D K A U L
d e s i r e d p a r t i t i o n i n g e f f e c t (2).Though, as a general r u l e , small

uncharged molecules are d i s t r i b u t e d q u i t e e v e n l y between the phases
(i.e. K p
^ 1.0). P a r t i c l e s , such as c e l l o r g a n e l l e s o r
c e l l s , n o r m a l l y p a r t i t i o n between the i n t e r f a c e and one o f the b u l k
p h a s e s . The d i s t r i b u t i o n o f m a c r o m o l e c u l e s l i k e p r o t e i n s , i s q u i t e
v a r i a b l e , a n d a l s o shows g r e a t s e n s i t i v i t y t o c h a n g e s i n t h e c o m p o s i t i o n o f the phase system.
The u s e o f t h e a q u e o u s t w o - p h a s e s y s t e m s i n b i o t e c h n o l o g y
basically exploits this varying distribution of biomaterials
between the p h a s e s . These systems can be b u f f e r e d and are s u i t a b l e
f o r c a r r y i n g o u t b i o c o n v e r s i o n s . The p h a s e p o l y m e r s h a v e a l s o b e e n
s h o w n t o h a v e a s t a b i l i z i n g i n f l u e n c e o n b i o c a t a l y s t s (_3,4_); t h e
l a t t e r a r e , i n a way, t e m p o r a r i l y i m m o b i l i z e d w i t h i n l i q u i d
d r o p l e t s . The d i f f e r e n t a r e a s i n w h i c h t h e t w o - p h a s e s y s t e m s h a v e
shown p o t e n t i a l i n c l u d e e x t r a c t i v e f e r m e n t a t i o n s , p u r i f i c a t i o n o f
b i o m o l e c u l e s , c e l l s , membranes and o r g a n e l l e s , a n d b i o l o g i c a l
b i n d i n g a s s a y s (I)
However, most o f the s y s t e m s r e p o r t e d so f a r
a r e b a s e d o n h i g h l y p u r i f i e d p h a s e c o m p o n e n t s , t h e c o s t s o f some o f
w h i c h p o s e s a s e v e r e l i m i t a t i o n o n t h e s c a l e up o f t h e p r o c e s s e s .
N e v e r t h e l e s s , t h e r e has been a s e r i o u s attempt i n employing cheap
phase components, f o r example, i n p u r i f i c a t i o n o f p r o t e i n s on l a r g e
s c a l e (_5,6) .
T h i s p a p e r i s meant t o b e a n o v e r v i e w o f t h e d e v e l o p m e n t s i n
the u s e o f aqueous two-phase systems i n the r e c o v e r y o f p r o d u c t s o f
f e r m e n t a t i o n , and a l s o p u r i f i c a t i o n o f p r o t e i n s and other
biomolec u l e s . I t a l s o t o u c h e s u p o n t h e means t o i m p r o v e t h e e c o n o m i c s o f
t h e p r o c e s s t o make i t i n d u s t r i a l l y f e a s i b l e . I t h a s b e e n r e a l i z e d
t h a t the c l o s e r i n t e g r a t i o n o f b i o c o n v e r s i o n p r o c e s s e s w i t h downs t r e a m t e c h n o l o g y i s e s s e n t i a l f o r q u i c k e r a n d more e c o n o m i c a l e n r i c h m e n t o f the p r o d u c t . I n i t i a l e f f o r t s have a l r e a d y been t a k e n i n
t h i s d i r e c t i o n , some o f w h i c h a r e d i s c u s s e d h e r e .
a

79
Aqueous Two-Phase Systems
Extractive bioconversions
i n aqueous two-phase
systems
P r o d u c t i n h i b i t i o n seems t o b e a g e n e r a l p h e n o m e n o n i n t r a d i t i o n a l
fermentation processes, which are, t h e r e f o r e , c h a r a c t e r i z e d by d i l u t e p r o d u c t s t r e a m s . The n e e d t o o b t a i n h i g h p r o d u c t
concentrat i o n s i n the f e r m e n t a t i o n b r o t h i s e s s e n t i a l both f o r i t s y i e l d a n d
r e c o v e r y . The m a j o r i m p r o v e m e n t t o w a r d s a c h i e v i n g t h i s g o a l h a s
been f o c u s e d , i n r e c e n t y e a r s , on g e n e t i c m a n i p u l a t i o n o f m i c r o o r g a n i s m s , a s a r e s u l t o f w h i c h s t r a i n s have been o b t a i n e d t h a t a r e
t o l e r a n t t o h i g h c o n c e n t r a t i o n s o f t h e p r o d u c t . A t t h e same t i m e ,
however, the i n t e n s i f i c a t i o n o f the p r o d u c t r e c o v e r y p r o c e s s e s i s
a l s o r e q u i r e d , w h i c h h a s u n t i l now b e e n c a r r i e d o u t s e p a r a t e l y i n
s e v e r a l s t a g e s , o f t e n g i v i n g l o w y i e l d s . The c o n t i n u o u s r e m o v a l o f
the p r o d u c t b y c o u p l i n g the e x t r a c t i o n s t e p w i t h t h a t o f
fermentation ( e x t r a c t i v e bioconversion) would help i n
m i n i m i z i n g t h e i n h i b i t o r y e f f e c t s a n d / o r d e g r a d a t i o n phenomena. T h i s
may a l s o l e a d t o t h e p a r t i a l e n r i c h m e n t o f t h e p r o d u c t , w h i c h w i l l
f a c i l i t a t e the e v e n t u a l p u r i f i c a t i o n s t e p s as w e l l .
In an aqueous two-phase s y s t e m , i f the b i o c a t a l y t i c r e a c t i o n
t a k e s p l a c e i n one o f the p h a s e s , w h i l e the p r o d u c t s are e i t h e r
e v e n l y d i s t r i b u t e d , o r p r e f e r e n t i a l l y p a r t i t i o n e d t o the o t h e r

80

phase, an i d e a l s i t u a t i o n f o r e x t r a c t i v e c o n v e r s i o n c o u l d be v i s u a l i z e d ( F i g u r e 1 ) . Aqueous two-phase systems a r e c h a r a c t e r i z e d by the

requirement o f minimal energy to c r e a t e a f i n e l y d i s p e r s e d e m u l s i o n
r e s u l t i n g i n a l a r g e i n t e r f a c e and h i g h mass t r a n s f e r , which i s
e s p e c i a l l y advantageous i n r e d u c i n g m i c r o e n v i r o n m e n t a l product i n h i b i t i o n (_1) R e c e n t l y , s t u d i e s have been performed on the e x t r a c t i v e
f e r m e n t a t i o n o f c h e m i c a l s , o f i n t e r e s t to the b i o l o g i c a l and c h e m i c a l i n d u s t r y , i n two-phase systems.
P r o d u c t i o n o f b u l k c h e m i c a l s . The p r o d u c t i o n of s o l v e n t s i s n o r m a l l y
c h a r a c t e r i z e d by a g e n e r a l i n h i b i t i o n phenomenon which has been
m a i n l y a t t r i b u t e d to the changes i n membrane p e r m e a b i l i t y , o r to the
t o x i c e f f e c t s on the m e t a b o l i c pathway. Aqueous two-phase systems
have been shown to be e f f e c t i v e as media f o r the e x t r a c t i v e ferment a t i o n of a number of s o l v e n t s which i n c l u d e e t h a n o l , a c e t o n e - b u t a n o l and a c e t i c a c i d ( 3 ) . Improved p r o d u c t i v i t y has been a c h i e v e d i n
most of the c a s e s as compared to the c o n v e n t i o n a l f e r m e n t a t i o n s ,
which i s s i g n i f i c a n t l y due to the e l i m i n a t i o n o f product i n h i b i t i o n .
However, t h e r e i s an i n d i c a t i o n t h a t changes i n the microenvironment
of the m i c r o b i a l c e l l s due to the presence o f n o n - m e t a b o l i z a b l e
polymers c o u l d a l s o c o n t r i b u t e , i n the i n i t i a l phases, to the
i n c r e a s e d p r o d u c t i o n . The a d d i t i o n o f PEG and d e x t r a n to a growth
medium, f o r i n s t a n c e , was shown to g i v e i n c r e a s e d i n i t i a l e t h a n o l
y i e l d s , as a r e s u l t o f d e c r e a s e i n the c h e m i c a l p o t e n t i a l o f water
(8).
Much e f f o r t has gone, i n r e c e n t y e a r s , i n s e t t i n g up a l c o h o l i c
f e r m e n t a t i o n s based on i m m o b i l i z e d c e l l t e c h n o l o g y (9) Some o f the
systems have proved to be h i g h l y p r o d u c t i v e , b u t a r e f a c e d w i t h
drawbacks o f leakage of c e l l s , and s t e r i c a l h i n d r a n c e s . F e r m e n t a t i o n
i n two-phase system, on the o t h e r hand, has been s u c c e s s f u l l y c a r r i e d out w i t h macromolecular s u b s t r a t e s such as s t a r c h and c e l l u l o s e
(_7,TO ) . I t i s a l s o e a s i e r to c o n t r o l a r e a c t i o n system i n v o l v i n g a
number o f enzymes, i n a two-phase system as compared to the immobil i z e d systems; f o r example, t h e r e i s a p o s s i b i l i t y to add more of
the l a b i l e c a t a l y s t d u r i n g the c o n t i n u o u s o p e r a t i o n s .
A l l the e x t r a c t i v e f e r m e n t a t i o n p r o c e s s e s s t u d i e d i n the
two-phase system have employed PEG and d e x t r a n as the phase polymers
(Table I ) .
The m i c r o b i a l c e l l s employed f o r the c o n v e r s i o n have been seen
to be e n r i c h e d i n the d e x t r a n r i c h bottom phase and a l s o , the i n t e r f a c e . The macromolecular s u b s t r a t e s a r e a l s o found l o c a t e d i n the
bottom phase. In f a c t , i n one o f the e a r l i e r s t u d i e s on e t h a n o l f e r m e n t a t i o n , s t a r c h a l o n e c o n s t i t u t e d the lower phase of a two-phase
system ( 3 ) . The s o l v e n t m o l e c u l e s a r e r a t h e r e v e n l y d i s t r i b u t e d b e tween the two phases. However, the p a r t i t i o n i n g b e h a v i o u r of the
s o l v e n t m o l e c u l e s can somewhat be changed by v a r i a t i o n s i n the phase
c o m p o s i t i o n . Furthermore, a s i g n i f i c a n t e x t r a c t i o n of the s o l v e n t
i n t o the upper phase c o u l d be a c h i e v e d by i n c r e a s i n g the top to
bottom phase volume r a t i o s ( 1 , 1 1 ) . Semicontinuous
batch
f e r m e n t a t i o n s have been performed w i t h the c e l l s b e i n g r e c i r c u l a t e d
i n the bottom phase. A f t e r the c o n v e r s i o n , the top phase w i t h the
p r o d u c t i s removed and r e p l a c e d by a f r e s h one a l o n g w i t h more
s u b s t r a t e . A l t e r n a t i v e l y , the phase i s r e t u r n e d to the system a f t e r
r e m o v a l o f the s o l v e n t e.g. b y d i s t i l l a t i o n (12,13). The

7.

Top
phase

1 |

Agitation
Bottom
phase
c = catalyst
s = substrate
= product
F i g u r e 1. P r i n c i p l e f o r e x t r a c t i v e b i o c o n v e r s i o n i n aqueous twophase systems, where the b i o c a t a l y s t i s t e m p o r a r i l y i m m o b i l i z e d

i n the d r o p l e t s o f one of the phases. Reproduced w i t h p e r m i s s i o n
from R e f . 7. C o p y r i g h t 1984. S o c i e t y o f C h e m i c a l I n d u s t r y .

82
i n t e g r a t i o n o f membrane t e c h n o l o g y w i t h t h e p h a s e s y s t e m h a s b e e n
s h o w n t o be a d v a n t a g e o u s f o r t h e e f f i c i e n t r e c o v e r y o f t h e p r o d u c t
d u r i n g s t a r c h b i o c o n v e r s i o n (_7). I n t h i s m a n n e r , a n y l o s s e s o f t h e
u p p e r phase p o l y m e r and t h e b i o c a t a l y s t a r e a l s o r e d u c e d t o a
m i n i m u m . The r e p e a t e d u s e a n d t h e i n c r e a s e d o p e r a t i o n a l s t a b i l i t y o f
the b i o c a t a l y s t helps i n improving the economics of the process.
Table
I . Bulk
chemical

Bioconversion
production
i n aqueous two-phase
Biocatalyst
Phase System
systems
Reference
Glucose
> ethanol
Saccharomyces
cerevisiae
6% PEG 8 0 0 0 2% D e x t r a n T 5 0 0
(12)
Starch
* ethanol
P< - a m y l a s e ;
glucoamylase
S.cerevisiae
5% PEG 20 M3% c r u d e d e x t r a n
d>
Cellulose
êthanol
PEG 8 0 0 0 cellulase,
Dextran T40
/3-glucosidase
+ S.cerevisiae
acetonebutanol
Clostridium
acetobutylicum
Glucose
Glucose
>acetic
acid
2 5 % PEG 8 0 0 0 6% D e x t r a n T 4 0
Escherichia
coli
Reproduced w i t h p e r m i s s i o n
Chemie.
(10)
from Ref. 11.
6% PEG 8 0 0 0 7.5%
Dextran
Copyright
(13)
(3)
1985, V e r l a g
One o f t h e common s i d e e f f e c t s o b s e r v e d d u r i n g e x t r a c t i v e
b i o c o n v e r s i o n i s t h e a c c u m u l a t i o n o f unwanted b y - p r o d u c t s i n t h e
s y s t e m w h i c h may a f f e c t t h e p r o d u c t i v i t y d u r i n g
continuous
o p e r a t i o n ( 1 4 ) . The b u i l d up o f g l y c e r o l a n d o t h e r n o n - v o l a t i l e
p r o d u c t s was shown t o d e c r e a s e t h e e t h a n o l y i e l d s d u r i n g
r e p e a t e d f e r m e n t a t i o n s i n a t w o - p h a s e s y s t e m ( 1 2 ) . The p r o b l e m
was, h o w e v e r , s o l v e d b y d i a l y s i n g t h e f e r m e n t a t i o n b r o t h and
a l s o a d d i n g more y e a s t c e l l s . I t a p p e a r s t h a t t h e c o m b i n a t i o n o f
u l t r a f i l t r a t i o n w i t h t h e p h a s e s y s t e m may c i r c u m v e n t t h e p r o b l e m
o f b y - p r o d u c t i n h i b i t i o n i n most o f t h e c a s e s .
Even though aqueous two-phase sytems h o l d promise f o r b u l k
c h e m i c a l p r o d u c t i o n , t h e i r a p p l i c a b i l i t y on a l a r g e s c a l e i s n o t
a s s u r e d u n l e s s t h e phase components, a t l e a s t , t h e f r a c t i o n a t e d
d e x t r a n , a r e r e p l a c e d by cheaper polymers, o r technology i s
developed p e r m i t t i n g f u l l r e c y c l i n g o f t h e polymers. These
aspects are discussed l a t e r i n the paper.
P r o d u c t i o n o f f i n e c h e m i c a l s . I n s p i t e o f t h e i n t e r e s t shown i n t h e
p r o d u c t i o n o f b u l k c h e m i c a l s i n aqueous two-phase systems, t h e p o t e n t i a l of these systems f o r f i n e c h e m i c a l p r o d u c t i o n has not y e t
b e e n e x p l o i t e d . The o n l y b i o c o n v e r s i o n r e p o r t e d h a s b e e n t h e d e a c y l a t i o n o f b e n z y l p e n i c i l l i n t o 6-amino p e n i c i l l a n i c a c i d ( 1 5 ) .
Today, i n d u s t r i a l d e a c y l a t i o n i s performed by p e n i c i l l i n a c y l a s e i n
a n i m m o b i l i z e d f o r m . The p r o d u c t i v i t y o f t h e r e a c t i o n i n a


7. MATTIASSON A N D K A U L
83
t w o - p h a s e s y s t e m w a s f o u n d t o b e i n t h e same r a n g e a s t h a t o f
i m m o b i l i z e d s y s t e m . B u t , t h e u s e o f p h a s e s y s t e m may h a v e a n
a d v a n t a g e o v e r i m m o b i l i z e d r e a c t o r s , i n t h a t t h e i n a c t i v a t e d enzyme
may b e r e p l a c e d a t a n y t i m e d u r i n g o p e r a t i o n .
The s l o w d e v e l o p m e n t i n t h e a r e a o f f i n e c h e m i c a l
production
by e n z y m a t i c / m i c r o b i a l methods c o u l d be due t o t h e f a c t t h a t most
o f t h e compounds o f c o m m e r c i a l i n t e r e s t have poor s o l u b i l i t i e s i n
a q u e o u s s o l u t i o n s . On t h i s s c o r e , e n z y m a t i c s y n t h e s i s h a s n o t b e e n
a b l e t o compete w i t h t r a d i t i o n a l t e c h n o l o g y . However, r e c e n t s t u d i e s o n a f e w s y s t e m s h a v e shown t h a t t h e p r e s e n c e o f a n o r g a n i c
s o l v e n t i n a b i o c a t a l y t i c r e a c t i o n medium o f f e r e d some a d v a n t a g e s
w i t h r e s p e c t t o s o l u b i l i t y and downstream p r o c e s s i n g ( 1 6 ) . The
a q u e o u s - o r g a n i c s o l v e n t two-phase systems have p r o v i d e d e f f e c t i v e
r e a c t i o n media f o r t h e e x t r a c t i v e t r a n s f o r m a t i o n o f s t e r o i d s ( 1 7 ) .
B u t t h i s a p p r o a c h may n o t b e a p p l i c a b l e f o r p r o c e s s e s i n v o l v i n g
l i v i n g c e l l s b e c a u s e o f t h e d e n a t u r i n g e f f e c t o f most o f t h e s o l v e n t s . T h i s i s i n d i r e c t c o n t r a s t t o t h e two-phase systems, which
are b i o c o m p a t i b l e and a r e c h a r a c t e r i z e d by a low s u r f a c e t e n s i o n
between the phases.
The p o l y m e r s g e n e r a l l y e m p l o y e d i n f o r m i n g a q u e o u s t w o - p h a s e
systems a r e q u i t e h y d r o p h i l i c as compared w i t h o r g a n i c s o l v e n t s ;
t h e r e i s , however, a marked d i f f e r e n c e i n h y d r o p h o b i c i t y o f t h e
p h a s e p o l y m e r s . T h u s , b y e x t r a c t i o n i n t o t h e more h y d r o p h o b i c p h a s e
i t may b e p o s s i b l e t o d e s i g n b i o c o n v e r s i o n i n a q u e o u s t w o - p h a s e
systems o f substances o f low water s o l u b i l i t y .
To t h i s a i m , we
examined the t r a n s f o r m a t i o n o f h y d r o c o r t i s o n e t o p r e d n i s o l o n e b y
A r t h r o b a c t e r s i m p l e x c e l l s i n 2 5 % PEG 8000 - 6% D e x t r a n T 4 0 s y s t e m
( 1 8 ) . Our s t u d i e s showed t h a t w h i l e t h e c e l l s p r e f e r r e d t h e b o t t o m
phase, the K
o f t h e s t e r o i d was i n f a v o u r o f t h e P E G r i c h
upper phase ( K
~ 3.0 a n d W . O f o r h y d r o c o r t i s o n e a n d p r e d n i s o l o n e r e s p e c t i v e l y ) . T h e r e a c t i o n r a t e was f o u n d t o b e c o m p a r a b l e w i t h those o f systems i n which t h e o r g a n i c s o l v e n t had been
i n c l u d e d , w h i c h c o u l d b e d u e t o t h e e f f i c i e n t mass t r a n s f e r b e t w e e n
t h e t w o p h a s e s . Due t o t h e h i g h t o p t o b o t t o m p h a s e v o l u m e r a t i o
( 8 . 5 : 1 ) a r e c o v e r y o f 98-99% c o u l d be o b t a i n e d i n t h e t o p p h a s e .
p
A f t e r t h e c o m p l e t e t r a n s f o r m a t i o n , t h e PEG r i c h phase c o u l d
be p a s s e d o v e r a c o l u m n o f h y d r o p h o b i c s o r b e n t f o r t h e e x t r a c t i o n
of p r e d n i s o l o n e , and then r e c i r c u l a t e d t o the r e a c t o r w i t h a d d i t i o n a l s u b s t r a t e . The b a c t e r i a l c e l l s c o u l d b e u s e d r e p e a t e d l y f o r t h e
b a t c h c o n v e r s i o n s o f h y d r o c o r t i s o n e , and a l s o had t h e p o s s i b i l i t y
of being r e a c t i v a t e d by supply o f n u t r i e n t s .
These s t u d i e s , t h e r e f o r e , s e t t h e b a s i s f o r a c o n t i n u o u s
system
f o r e x t r a c t i v e b i o c o n v e r s i o n o f s t e r o i d s . The i n t e r e s t i n g p o i n t t h a t
comes t o n o t i c e i s t h a t t h e c o m b i n a t i o n o f two t e c h n i q u e s s u c h a s
aqueous two-phase s e p a r a t i o n , and a d s o r p t i o n , which a r e g e n e r a l l y
used i n d i v i d u a l l y f o r p e r f o r m i n g e x t r a c t i v e b i o c o n v e r s i o n s , c o u l d be
advantageous f o r e f f i c i e n t product recovery i n case o f f i n e
chemicals as w e l l .
S i m i l a r i l y , a q u e o u s t w o - p h a s e s y s t e m s may f i n d u s e i n o t h e r
areas l i k e the e x t r a c t i v e conversion o f b i o s u r f a c t a n t s , which a r e
a s s u m i n g i n c r e a s e d i m p o r t a n c e i n b i o t e c h n o l o g y . These compounds
g e n e r a l l y a f f e c t t h e c e l l s and have t o be removed c o n t i n u o u s l y
during c u l t i v a t i o n .


84
P r o d u c t i o n o f m a c r o m o l e c u l e s . At t i m e s , the macromolecules produced

d u r i n g f e r m e n t a t i o n s may be t o x i c to the c e l l s s y n t h e s i z i n g them,
thus p r e v e n t i n g f u r t h e r s y n t h e s i s . In o t h e r s i t u a t i o n s , they are so
f r a g i l e , t h a t they may be degraded i n the u n f a v o u r a b l e c o n d i t i o n s
p r e v a i l i n g i n the f e r m e n t o r . The macromolecules b e l o n g i n g to both
the c l a s s e s are g e n e r a l l y p r o t e i n i c i n n a t u r e . In e i t h e r c a s e , i t i s
d e s i r a b l e to remove the product as i t i s formed. Aqueous two-phase
systems o f f e r a p r o m i s i n g t e c h n o l o g y to do the same. Advantage i s
a g a i n taken of v a r y i n g volume r a t i o s to o b t a i n an e f f i c i e n t
e x t r a c t i o n of the macromolecular product i n t o the top phase, i f the
p a r t i t i o n c o e f f i c i e n t s are not f a v o u r a b l e . Recovery i n t o the top
phase c o u l d a l s o be enhanced by the m o d i f i c a t i o n of the phase
polymers depending on the n a t u r e of the p r o d u c t .
The t o x i c e f f e c t of macromolecules on the b a c t e r i a l c e l l s
p r o d u c i n g them i s r e p r e s e n t e d v e r y w e l l i n case of C l o s t r i d i u m
s p e c i e s . The p r o d u c t i o n o f c e r t a i n s u i c i d a l e x t r a c e l l u l a r p r o t e i n s
( a u t o l y s i n s ) have been r e p o r t e d d u r i n g c o n t i n u o u s c u l t u r e of
a c e t o b u t y l i c u m ( 1 9 ) , which a f f e c t s the biomass c o n c e n t r a t i o n and
a l s o the s o l v e n t p r o d u c t i o n . The t o x i n produced by C_* t e t a n i i s a l s o
a p r o t e o l y t i c enzyme c a p a b l e of d e g r a d i n g the c e l l w a l l of the
b a c t e r i u m ; the p r o t o p l a s t s formed as a r e s u l t , a r e e x t r e m e l y l a b i l e .
In a p i o n e e r i n g study on the p r o d u c t i o n of t h i s t o x i n , P u z i s s and
Hedn (20) showed t h a t e x t r a c t i v e b i o c o n v e r s i o n c o u l d be used when
growing . t e t a n i . In an aqueous two-phase system c o n s i s t i n g o f 12%
PEG 4000 and 2% D e x t r a n (mol.wt.480 000), the t o x i n was e q u a l l y
d i s t r i b u t e d i n the two phases, w h i l e the m a j o r i t y of the c e l l s were
i n the lower phase. Because of the h i g h top to bottom phase volume
r a t i o (15:1) the c o n c e n t r a t i o n of the p r o t e i n was s u b s t a n t i a l l y
reduced i n the immediate v i c i n i t y of the c e l l s . As a r e s u l t , more
than 1000 f o l d i n c r e a s e i n the t o t a l y i e l d of t o x i n was o b t a i n e d , as
compared to p r o d u c t i o n i n c o n v e n t i o n a l medium. A c c o r d i n g to the
a u t h o r s , the phase polymers had a s t a b i l i z i n g e f f e c t on the
p r o t o p l a s t s i n the o l d e r c u l t u r e s , which are known to c o n t i n u e some
m e t a b o l i c a c t i v i t i e s of the c e l l s . T h i s c o u l d , i n p a r t , c o n t r i b u t e
to the t o x i n output i n the phase system.
The two-phase systems have a l s o shown p o t e n t i a l i n the
p r o d u c t i o n of p r o t e c t i v e a n t i g e n s ( 2 0 ) . The e x t r a c t i v e phenomenon,
t h u s , may be used to advantage i n the p r o d u c t i o n of o t h e r i m p o r t a n t
c e l l u l a r p r o d u c t s such as v a c c i n e s .
The s e m i c o n t i n u o u s p r o d u c t i o n o f <-amylase and c e l l u l a s e
has been s t u d i e d i n PEG-Dextran systems u s i n g B a c i l l u s s u b t i l i s and
T r i c o d e r m a r e e s e i , r e s p e c t i v e l y (21, 22). Some improvements i n the
y i e l d s have been observed i n both the c a s e s . In case of c e l l u l a s e
p r o d u c t i o n , the economics of the p r o c e s s c o u l d be improved by u s i n g
a cheap, l i g n o c e l l u l o s i c waste as the s u b s t r a t e , and r e p l a c i n g the
f r a c t i o n a t e d d e x t r a n w i t h a crude one.
O p t i m i z i n g the phase system i s an e s s e n t i a l p r e r e q u i s i t e f o r
the enzyme p r o d u c t i o n , and s t i l l much work i s to be done r e g a r d i n g
t h i s a s p e c t . Some methods f o r the removal of phase polymers from the
end product have been r e p o r t e d as d e s c r i b e d l a t e r , which have to be
a p p l i e d f o r the e c o n o m i c a l r e c o v e r y of the pure enzyme.

7.
MATTIASSON AND K.AUL

Purification
of
p r o t e i n s by p a r t i t i o n
85
i n aqueous two-phase systems
Removal of c e l l d e b r i s , DNA and p r o t e a s e s , c o n s t i t u t e the major

problems f a c e d d u r i n g the l a r g e - s c a l e i s o l a t i o n o f i n t r a c e l l u l a r
p r o t e i n s . T h i s has emphasized the need f o r r a p i d prodedures f o r p r o
t e i n p u r i f i c a t i o n . The fundamental s t u d i e s on s e p a r a t i o n s i n aqueous
two-phase systems suggest t h a t i t i s p o s s i b l e to s p o n t a n e o u s l y p a r
t i t i o n the p r o t e i n o f i n t e r e s t i n t o one o f the phases by s e l e c t i n g
the s u i t a b l e
c o n d i t i o n s . The method i s v e r y e f f i c i e n t and conve
n i e n t when s e t up. However, i t may be v e r y l a b o r i o u s to f i n d t h e
p r o p e r c o n d i t i o n s f o r a f a v o u r a b l e p a r t i t i o n i n g . Furthermore, such a
system may be i n f l u e n c e d by v a r i a t i o n s i n parameters such as compo
s i t i o n of the c e l l homogenate. S e v e r a l examples on l a r g e s c a l e
i s o l a t i o n o f p r o t e i n s have been r e p o r t e d . Some o f these a r e l i s t e d
i n Table I I .
T a b l e I I . I s o l a t i o n of enzymes i n aqueous two-phase
systems.
Reference
(23)
Enzyme
Formaldehyde
dehydrogenase
Organism
Candida
boidinii
Phase system
PEG/crude
dextran
-Galactosidase
E.coli
PEG/salt
(24)
Fumarase
Brevibacterium
ammoniagenes
PEG/salt
(25)
Aspartase
E.coli
PEG/salt
(25)
Leucine
dehydrogenase
Bacillus
sphaericus
PEG/crude
dextran
(25)
A f f i n i t y p a r t i t i o n i n g . In some c a s e s , i t may not be p o s s i b l e to

s e p a r a t e one p r o t e i n out o f a complex m i x t u r e by means of s p o n t a
neous p a r t i t i o n i n g i n an aqueous two-phase system. Then a f f i n i t y i n
t e r a c t i o n s may be u t i l i z e d . When f i r s t d e s c r i b e d , a f f i n i t y p a r t i
t i o n i n g was used f o r p u r i f i c a t i o n of membrane v e s i c l e s ( 2 6 ) , and has
s i n c e been e x p l o i t e d i n a broad spectrum o f a p p l i c a t i o n s .
The g e n e r a l s t r a t e g y i s to d e s i g n a system where a l l , o r a t
l e a s t , most c e l l components p a r t i t i o n to the bottom phase. By s p e c i
f i c i n t e r a c t i o n w i t h an a f f i n i t y l i g a n d , h a v i n g a s t r o n g p r e f e r e n c e
f o r the top phase, the p r o t e i n forms an a f f i n i t y complex t h a t p a r t i
t i o n s a c r o s s the phase boundary. The a f f i n i t y bound m a t e r i a l i s then
r e c o v e r e d from the top phase. I n T a b l e I I I a r e l i s t e d examples o f
some groups o f l i g a n d s used i n t h i s c o n t e x t .
The l i g a n d s a r e o f t e n m o d i f i e d by c o u p l i n g PEG t o them. The
c o u p l i n g c h e m i s t r y when d e a l i n g w i t h P E G - m o d i f i c a t i o n i s the same a s
i n c o n v e n t i o n a l a f f i n i t y chromatography. U s u a l l y , a c o n j u g a t e
between PEG and one l i g a n d m o l e c u l e i s formed. The l i f t i n g power o f
such a l i g a n d may v e r y w e l l be enough when p u r i f y i n g m u l t i v a l e n t
s t r u c t u r e s , e . g . membrane v e s i c l e s o r o l i g o m e r i c p r o t e i n s . In o t h e r

86
c a s e s , e . g . when t h e l i g a n d i s a m a c r o m o l e c u l e t h a t p e r s e p r e f e r s
the bottom phase, then a h i g h e r degree of m o d i f i c a t i o n i s needed.
L e c t i n s and a n t i b o d i e s u s e d f o r s e l e c t i v e e x t r a c t i o n o f g l y c o s y l a t e d
s t r u c t u r e s and a n t i g e n s r e s p e c t i v e l y , a r e a m o n g s t t h e l i g a n d s
b e l o n g i n g to t h i s c a t e g o r y .
Table I I I . Examples of
systems.

Ligand
Cibacron
blue
affinity
partitioning
i n aqueous
two-phase
Compound p u r i f i e d
Source
Reference
Formate dehydrogenase Candida b o i d i n i i
(27)
Concanavalin
A
Peroxidase
Antibodies
^2~
gl b l
Erythrocytes
Coenzyme
(NADH)
Dehydrogenases
Horseradish
(28)
Human p l a s m a
Human b l o o d
(29)
(30)
Candida b o i d i n i i
(27)
H o w e v e r , a p r o b l e m e n c o u n t e r e d when u s i n g h e a v y m o d i f i c a t i o n i s
t h a t t h e b i n d i n g c a p a c i t y may go down. T h i s c a n be a t t r i b u t e d t o a
c h e m i c a l m o d i f i c a t i o n of groups e s s e n t i a l f o r the a f f i n i t y
i n t e r a c t i o n , o r may be due t o s t e r i c a l h i n d r a n c e b y t h e p o l y m e r
c h a i n s bound to the l i g a n d . I f t h i s d e c r e a s e i s s e v e r e , the w h o l e
p r o c e d u r e may be u s e l e s s u n l e s s a s e c o n d s e p a r a t o r i s u s e d a s a
m o d i f i e d group t h a t b i o s p e c i f i c a l l y b i n d s the l i g a n d which, i n t u r n ,
i n t e r a c t s w i t h t h e s t r u c t u r e t o be i s o l a t e d . E x a m p l e s o f s u c h
secondary separators are c e l l s of Staphylococcus aureus c a r r y i n g
p r o t e i n A on t h e i r s u r f a c e s t h a t b i n d s to the F
p a r t of the
immunoglobulin
G, l e a v i n g t h e F , p a r t f r e e f o r t h e b i n d i n g o f
antigen.
a u r e u s c e l l s c a n be h e a v i l y m o d i f i e d b y m e t h o x y
p o l y e t h y l e n e g l y c o l (MPEG) t o make t h e m p a r t i t i o n t o t h e t o p p h a s e
and s t i l l m a i n t a i n t h e i r b i n d i n g c a p a c i t y f o r I g G - m o l e c u l e s .
Another
such s e p a r a t o r i s a v i d i n that i s MPEG-modified, r e t a i n i n g i t s
b i n d i n g c a p a c i t y f o r b i o t i n . H e n c e , a g e n e r a l scheme f o r
p a r t i t i o n i n g of m o l e c u l e s of i n t e r e s t to the top p h a s e , i s o b t a i n e d
i f t h e y a r e l a b e l l e d w i t h b i o t i n . I n T a b l e I V a r e l i s t e d some d a t a
on p a r t i t i o n b e h a v i o u r o f t h e a b o v e d i s c u s s e d s e c o n d a r y s e p a r a t o r s .
c
A q u e o u s t w o - p h a s e s y s t e m s a r e r a t h e r s u b t l e . The s e n s i t i v i t y o f
such s y s t e m s i s v e r y w e l l d e m o n s t r a t e d b y V e i d e et^ a_l. (31) i n t h a t
a m i x t u r e of
PEG a n d p o t a s s i u m p h o s p h a t e g i v i n g a h o m o g e n e o u s s o l u t i o n , s u d d e n l y s t a r t e d t o f o r m a t w o - p h a s e s y s t e m when c e l l h o m o g e n a t e was l o a d e d i n t h e s y s t e m . M i n o r v a r i a t i o n i n i o n i c c o m p o s i t i o n
may a l s o g i v e d r a m a t i c c h a n g e s i n p a r t i t i o n b e h a v i o u r . T h i s may
be
r e g a r d e d as a n a d v a n t a g e when d e a l i n g w i t h w e l l k n o w n , s t a b l e
s y s t e m s , w h e r e a s i n o t h e r c a s e s , i t may l e a d t o u n p r e d i c t a b l e b e h a v i o u r . Hence,
i t may be d e s i r a b l e t o d e s i g n t h e a f f i n i t y p a r t i t i o n i n g i n s u c h a way t h a t t h e p a r t i t i o n b e h a v i o u r o f t h e a f f i n i t y
c o m p l e x c a n be p r e d i c t e d , and i s s t a b l e w i t h i n a w i d e r a n g e o f e x t e r n a l c o n d i t i o n s . The a p p r o a c h d e s c r i b e d f o r S t a p h y l o c o c c u s a u r e u s

7.
87
above r e p r e s e n t s such a r o b u s t p r e d i c t a b l e p a r t i t i o n b e h a v i o u r . A
s t a b l e system i s e a s i e r to o p e r a t e , b u t may prove
quite expensive,
thus l i m i t i n g i t s a p p l i c a t i o n to a n a l y t i c a l p u r p o s e s .
T a b l e I V . D i s t r i b u t i o n i n two-phase systems of s e p a r a t o r s ,
S t a p h y l o c o c c u s aureus and a v i d i n .
S t a p h y l o c o c c u s aureus
Native
MPEG-modified

Top
Avidin
MPEG-modified
90
phase
0 %
80 %
Interface
10 %
20 %
0 %
Bottom phase 90 %
0 %
10 %
Note: A phase system c o n s i s t i n g o f 0.15 g/g PEG-4000 and

0.15 g/g MgS0^-7H 0 was used.
2
Source:
Reproduced w i t h p e r m i s s i o n from Ref. 29.
1983, E l s e v i e r B i o m e d i c a l P r e s s .
Copyright
R e c e n t l y , another v a r i a t i o n on t h i s theme was p r e s e n t e d . By

i n t r o d u c i n g PEG-modified a f f i n i t y s o r b e n t s (Sepharose beads c a r r y i n g
the i m m o b i l i z e d l i g a n d ) a new p r o c e s s c o n f i g u r a t i o n f o r p r o t e i n
p u r i f i c a t i o n was a c h i e v e d as shown i n F i g u r e 2 ( 3 2 ) . The beads were
exposed to the c e l l homogenate b e f o r e the phase components were
added. A f t e r phase s e p a r a t i o n the p a r t i c l e s were r e c o v e r e d from t h e
top phase, as a l a y e r on t o p o f the i n t e r f a c e . The beads were
c o l l e c t e d and t r a n s f e r r e d to a column, where they were washed f r e e
o f the phase polymers, and then the e l u t i o n was c a r r i e d out
a c c o r d i n g to c o n v e n t i o n a l p r o c e d u r e s .
By t h i s t e c h n i q u e of combining a f f i n i t y p a r t i t i o n i n g w i t h a f f i n i t y c h r o m a t o g r a p h i c e l u t i o n , the advantages o f the two p r o c e d u r e s
were combined. The r a p i d and e f f e c t i v e removal of c e l l d e b r i s i n the
e x t r a c t i o n p r o c e d u r e , and f i n a l l y , the e f f i c i e n t e l u t i o n procedure
o f the a f f i n i t y chromatographic s t e p was a c h i e v e d .
E x t r a c t i o n i n aqueous two-phase systems i s s a i d to be an o p e r a t i o n t h a t i s easy to s c a l e up ( 3 3 ) . T h i s i s a l s o t r u e f o r a f f i n i t y
p a r t i t i o n i n g and i s c l e a r l y i l l u s t r a t e d by the p r o c e s s f o r p u r i f i c a t i o n of formate dehydrogenase (34, 35). In t h i s s t u d y , s m a l l s c a l e
experiments gave an o v e r a l l enzyme y i e l d o f 74%. When s c a l e d up by a
f a c t o r of 40 000, the y i e l d was 70%, thus d e m o n s t r a t i n g the
f e a s i b i l i t y o f e v a l u a t i n g the performance o f e x t r a c t i o n p r o c e s s on a
small scale.
Removal o f polymers
A g e n e r a l problem t h a t i s s p e c i f i c f o r the two-phase e x t r a c t i o n
t e c h n i q u e i s the removal o f polymers from the p u r i f i e d p r o t e i n . I n
the case o f P E G - s a l t systems, a g e n e r a l s t r a t e g y has been to e x t r a c t
one p r o t e i n to t h e P E G - r i c h phase and then r e p l a c e the s a l t phase by
a f r e s h s a l t phase under c o n d i t i o n s i n which the p r o t e i n p r e f e r s the
s a l t phase. T h i s phase i s then s e p a r a t e d and the s a l t removed by


88
Cells
Add
Add
phase
heads
system
Homogenate
Figure
2. P a r t i t i o n i n g
affinity
Copyright
sorbent.
i n two-phase
Reproduced
system w i t h
with permission
1986, W i l e y - I n t e r s c i e n c e
t h e use o f an
f r o m R e f . 2.

7. MATTIASSON A N D K A U L
89
u l t r a f i l t r a t i o n o r d i a l y s i s ( 3 5 ) . However, one h a s t o t a k e i n t o
c o n s i d e r a t i o n t h e f a c t t h a t t h e r e w i l l a l s o b e some P E G i n t h e s a l t
p h a s e a n d t h a t t h i s amount i n r e l a t i o n t o t h e a m o u n t o f p r o t e i n i s
q u i t e l a r g e . Low m o l e c u l a r w e i g h t PEG c a n be removed i n t h e d i a l y s i s
s t e p . A l t e r n a t i v e methods t o remove t h e phase components have b e e n
p r e s e n t e d . Ion exchange (2) o r hydrophobic chromatography (37) g i v e s
o p p o r t u n i t i e s t o adsorb the charged p r o t e i n s and l e t the uncharged
p o l y m e r s p a s s . The u s e o f p a r t i c l e b o u n d l i g a n d s m e n t i o n e d a b o v e
e n a b l e s one t o wash o f f t h e p o l y m e r s p r i o r t o e l u t i o n o f t h e
affinity-bound material.

Economy
A hampering f a c t o r i n the e x p l o i t a t i o n o f aqueous two-phase systems
i n b i o t e c h n o l o g y has been t h e c o s t o f t h e phase c o m p o n e n t s . The c o s t
o f P E G / s a l t systems have been r e g a r d e d a s a c c e p t a b l e , whereas
polymer/polymer systems, u s u a l l y c o n s i s t i n g of f r a c t i o n a t e d d e x t r a n
as b o t t o m phase p o l y m e r , have been too e x p e n s i v e t o u s e i n l a r g e
s c a l e a p p l i c a t i o n s . E f f o r t s t o use c r u d e d e x t r a n l e d t o a r e d u c t i o n
i n c o s t , b u t n o t t o a n e x t e n t t h a t made i t w o r t h w h i l e t o p u r s u e . T h e
crude d e x t r a n produced b y L e u c o n o s t o c m e s e n t e r o i d e s i s too v i s c o u s
t o be u s e d d i r e c t l y . P a r t i a l h y d r o l y s i s h a d t o be a p p l i e d and t h a t
i n c r e a s e d the cost s u b s t a n t i a l l y .
A r e a s o n why t h e P E G / s a l t s y s t e m s h a v e n o t b e e n u s e d more
e x t e n s i v e l y i s that the h i g h i o n i c s t r e n g t h s e v e r e l y i n f l u e n c e s t h e
a f f i n i t y i n t e r a c t i o n s when a f f i n i t y p a r t i t i o n i n g i s t o b e e x p l o i t e d .
However, i n a p p l i c a t i o n s where spontaneous p a r t i t i o n i n g i s s u f f i c i e n t , these systems are o f t e n used. R e c e n t l y , the useo f s t a r c h
d e r i v a t i v e s named a s R e p p a l PES h a s b e e n r e p o r t e d ( 3 2 ) t h a t a r e u s e f u l i n f o r m i n g aqueous two-phase s y s t e m s . The p r o p e r t i e s o f t h e s e
new p o l y m e r s r e s e m b l e t h o s e o f d e x t r a n .
B a s e d o n t h e c o s t s f o r t h e p o l y m e r s o f 2.5 $/kg a n d 7 $ / k g
f o r PEG and R e p p a l , r e s p e c t i v e l y , one c a n c a l c u l a t e t h e c o s t o f t h e
phase s y s t e m . F o r two e q u i v a l e n t phase s y s t e m s h a v i n g d e x t r a n a n d
R e p p a l as b o t t o m phase p o l y m e r s r e s p e c t i v e l y , the c o s t i s r e d u c e d
from
5.5 $/L f o r P E G / d e x t r a n t o 0 . 5 0 $/L f o r P E G / R e p p a l .
A c r u c i a l p o i n t when d i s c u s s i n g e c o n o m i c s o f p h a s e s y s t e m s i s
t h e l o a d i n g o f t h e s y s t e m . The more b i o l o g i c a l m a t e r i a l t h a t c a n b e
l o a d e d , l e s s e r i s the c o s t o f phase s y s t e m per u n i t p r o c e s s e d . F u r t h e r m o r e , e x t e n s i v e s t u d i e s have been c a r r i e d out c o n c e r n i n g r e c i r c u l a t i o n o f t h e p h a s e c o m p o n e n t s . The l a t t e r e f f o r t s h a v e m a i n l y
been focused on P E G / s a l t systems, and a l s o on the polymer/polymer
phase systems e m p l o y i n g a f f i n i t y l i g a n d s . K u l a a n d c o w o r k e r s
r e p o r t e d t h e r e u s e o f t h e s a l t p h a s e when c a r r y i n g o u t e x t r a c t i v e
p u r i f i c a t i o n ( 3 7 ) . They showed t h a t t h e p h a s e s c o u l d be r e c y c l e d 4
t i m e s w i t h o u t any marked l o s s i n p e r f o r m a n c e o f t h e phase s e p a r a t i o n
and i n s p e c i f i c a c t i v i t y o f the p u r i f i e d p r o t e i n .
An a d d i t i o n a l a s p e c t o f t h e p h a s e c o m p o n e n t s t o be c o n s i d e r e d
i s t h e i r p o l l u t i n g e f f e c t s . I f t h e u s e d p h a s e s a r e pumped down t h e
d r a i n , then the c o s t o f the waste water treatment p l a n t has t o be
i n c l u d e d i n the p r o c e s s economics. Phosphate i s r e g a r d e d as one o f
t h e more d i f f i c u l t n u t r i e n t s t o b e r e m o v e d e f f i c i e n t l y i n w a s t e
w a t e r t r e a t m e n t p r o c e s s . B i o d e g r a d a b l e p o l y m e r s seem much more

90
a c c e p t a b l e from the p o l l u t i o n p o i n t of view. PEG i s b i o d e g r a d a b l e ,

though by a slow p r o c e s s . R e c y c l i n g of phase components w i l l thus
reduce the d i r e c t c o s t s and a l s o the l o a d i n g on the r e c i p i e n t
systems.
The systems are e c o n o m i c a l l y a c c e p t a b l e when s e p a r a t i n g p r o t e i n s out of a c e l l homogenate. On the o t h e r hand, when c a r r y i n g out
b i o c o n v e r s i o n p r o c e s s e s , the phase system has to be c o n t i n u o u s l y
r e u s e d , hence, t h e r e must be a method to remove the p r o d u c t s from
the phase system. T h i s can be performed i n a number of ways l i k e
a d s o r p t i o n , membrane f i l t r a t i o n , e t c .

Equipment f o r c o n t i n u o u s
extraction
I t has been demonstrated t h a t i f an e f f i c i e n t s e p a r a t i o n of d i f f e r ent s u b s t a n c e s i n an aqueous two-phase system, cannot be a c h i e v e d i n

a s i n g l e s t e p , a m u l t i s t a g e procedure c o u l d be r e s o r t e d to (2).
This
method, well-known as c o u n t e r c u r r e n t d i s t r i b u t i o n (CCD), i n v o l v e s
l a r g e number o f e x t r a c t i o n s t e p s i n o r d e r to s e p a r a t e s u b s t a n c e s
d i f f e r i n g o n l y s l i g h t l y i n p a r t i t i o n c o e f f i c i e n t s . Thus, i t has been
p o s s i b l e to f r a c t i o n a t e r e d b l o o d c e l l s based on t h e i r age (38,39);
i t has a l s o been demonstrated t h a t the wing buds i n c h i c k embryos,
at a v e r y e a r l y stage of development c o n t a i n c e l l p o p u l a t i o n s w i t h
d i f f e r e n t s u r f a c e p r o p e r t i e s , and a l s o , l a t e r d u r i n g the d e v e l o p ment, show s t r u c t u r a l d i f f e r e n c e s (39, AO). Another i l l u s t r a t i v e
example of the extreme r e s o l v i n g power i n such m u l t i s t a g e e x t r a c t i v e
p r o c e s s e s i s the d e m o n s t r a t i o n t h a t enzymes t h a t o p e r a t e i n sequence
i n the c e l l but so f a r , have never been shown to express any a f f i n i t y f o r each o t h e r , p a r t i t i o n t o g e t h e r i n the phases ( A l ) . I f t h i s
h i g h r e s o l v i n g power were e x p l o i t e d , a v e r y e f f i c i e n t p u r i f i c a t i o n
p r o c e s s would be s e t up.
The CCD machines are c h a r a c t e r i z e d by the presence of a s e r i e s
o f compartments c o n t a i n i n g d e f i n e d volumes of top and bottom phases,
i n which a substance p a r t i t i o n s i t s e l f depending on i t s p a r t i t i o n
c o e f f i c i e n t . The phase m i x i n g i s f o l l o w e d by the s e t t l e m e n t of the
phases, and moving the top phases to the a d j a c e n t bottom phases.
T h i s p r o c e s s i s r e p e a t e d depending on the number of compartments i n
the machine. Normally, a CCD machine has a low c a p a c i t y , d e s p i t e a
h i g h r e s o l v i n g power ( 2 ) .
A c e n t r i f u g a l s t e p can be i n t r o d u c e d i n the p r o c e s s to reduce
the phase s e t t l i n g time. T h i s has been made p o s s i b l e by the use of
c o u n t e r - c u r r e n t c o n t i n u o u s f l o w c e n t r i f u g a l s e p a r a t o r s developed by
S a n k i E n g i n e e r i n g L t d . ( J a p a n ) , which a l s o enable the CCD to be
d r i v e n c o n t i n u o u s l y , w i t h h i g h t h r o u g h p u t s . In these c o n t i n u o u s
p r o c e s s e s i t i s , o f c o u r s e , s u i t a b l e a l s o to i n t r o d u c e a f f i n i t y
interactions.
Conclusion
The v e r s a t i l i t y and the p o t e n t i a l of aqueous two-phase systems i n
f u t u r e b i o t e c h n o l o g y has been amply demonstrated. The a p p l i c a t i o n s
d e s c r i b e d here d e a l w i t h e x t r a c t i v e b i o c o n v e r s i o n s , i s o l a t i o n and
p u r i f i c a t i o n of p r o t e i n s . B i o c h e m i c a l a n a l y s e s i n terms of b i n d i n g
a s s a y s have a l s o been s u c c e s s f u l l y a p p l i e d i n the two-phase systems
(1).

7.
91
The b i o c o m p a t i b i l i t y , f a s t s e p a r a t i o n p r o c e s s and the ease to

s c a l e up a r e some o f the
c h a r a c t e r i s t i c s t h a t make aqueous
two-phase systems an a t t r a c t i v e a l t e r n a t i v e to o t h e r known
t e c h n i q u e s when b i o c h e m i c a l s e p a r a t i o n i s to be c a r r i e d o u t .
Acknowledgments

The a u t h o r s a r e g r a t e f u l to the N a t i o n a l Swedish Board f o r

T e c h n i c a l Development f o r f i n a n c i a l s u p p o r t .
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3.
4.
5.
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11.
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Mattiasson, B. Trends Biotechnol. 1983, 1, 16-20.

Albertsson, P.-. "Partition of Cell Particles and
Macromolecules"; 3rd ed., Wiley-Interscience: New York,
1986.
Mattiasson, B.; Hahn-Hgerdal, . In "Immobilized Cells and
Organelles"; Mattiasson, B., Ed.; CRC: Florida, 1983; Vol. I,
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Monsan, P.; Combes, D. Ann. NY Acad. Sci. 1984, 434, 48-60.
Tjerneld, F.; Johansson, G.; Berner, S.; Persson, L. 4th Int.
Conf. Partition in Aqueous Two-Phase Systems, Lund, Aug 1985,
Abstract p 25.
Ling, T.G.I.; Mattiasson, B. 4th Int. Conf. Partition in
Aqueous Two-Phase Systems, Lund, Aug 1985, Abstract p. 45
Larsson, M.; Mattiasson, B. Chemist.Indust. 1984, 12,
428-431.
Hahn-Hgerdal, B.; Larsson, M.; Mattiasson, B. Biotechnol.
Bioeng. Symp. No. 12, 1982, 199-202.
Mattiasson, B. In "Immobilized Cells and Organelles";
Mattiasson, B., Ed.; CRC: Florida, 1983; Vol. II, p. 23.
Hahn-Hgerdal, B.; Mattiasson, B.; Albertsson, P.-.
Biotechnol. Lett. 1981, 3, 53-58.
Mattiasson, B.; Larsson, M. Proc. 3rd Eur. Cong. Biotechnol.
Verlag Chemie: Weinheim, 1985, Vol. IV, p. 375.
Kuhn, I. Biotechnol. Bioeng. 1980, 22, 2393-2398.
Mattiasson, B.; Suominen, M.; Andersson, E.; Hggstrm, L;
Albertsson, P.-.; Hahn-Hgerdal, B. In "Enzyme Engineering";
Chibata, I.; Fukui, S.; Wingard, L.B., Jr., Eds.; Plenum: New
York, 1982, Vol. 6, p. 153.
Mattiasson, B.; Larsson, M. In "Biotechnology and Genetic
Engineering Reviews"; Russel, G.E., Ed.; Intercept:England,
1985, Vol. 3, p. 137.
Andersson, E.; Mattiasson, B.; Hahn-Hgerdal, B. Enzyme
Microb. Technol. 1984, 6, 301-306.
Proc. Symp. on Biocatalysts in Organic Syntheses; Tramper,J.;
Van Der Plas, H.C.; Linko, P., Eds.; Elsevier: Amsterdam,
1985.
Antonini, E.; Carrea, G.; Cremonesi, P. Enzyme Microb.
Technol. 1981, 3, 291-296.
Kaul, R.; Mattiasson, B., unpublished data.
Soucaille, P.; Minier, M.; Ferras, E.; Goma, G. Preprint 3rd
Eur. Cong. Biotechnol., Munich, Sept 1984, Vol. II, p. 85.


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20. Puziss, M.; Hedn, C.-G. Biotechnol. Bioeng. 1965, 7,

355-366.
21. Andersson, E.; Johansson, A.-C.; Hahn-Hgerdal, . Ann. NY
Acad. Sci. 1984, 434, 115-118.
22. Persson, I.; Tjerneld, F.; Hahn-Hgerdal, B. 4th Int. Conf.
on Partition in Aqueous Two-Phase Systems, Lund, Aug 1985,
Abstract p. 20.
23. Kroner, K.M.; Hustedt, H.; Kula, M.-R. Biotechnol. Bioeng.
1982, 24, 1015-1045.
24. Veide, .; Smeds, A.-L.; Enfors, S.-O. Biotechnol. Bioeng.
1983, 25, 1789-1800.
25. Hustedt, H.; Kroner, K.H.; Menge, U.; Kula, M.-R. Trends
Biotechnol. 1985, 3, 139-144.
26. Flanagan, S.D.; Barondes, S.H. J. Biol. Chem. 1975, 250,
1484-1489.
27. Kula, M.-R.; Johansson, G.; Buckmann, A.F. Biochem. Soc.
Transac. 1979, 7, 1-5.
28. Mattiasson, B.; Ling, T.G.I. J. Immunol. Methods 1980, 38,
217-223.
29. Ling, T.G.I.; Mattiasson, B. J. Immunol. Methods 1983, 59,
327-337.
30. Sharp, K.A.; Yalpani, M.; Howard, S.J.; Brooks, D.E. 4th Int.
Conf. Partition in Aqueous Two-Phase Systems, Lund, Aug 1985,
Abstract p. 38.
31. Veide, .; Lindback, T.; Enfors, S.-O. Enzyme Microb.Technol.
1984, 6, 325-330.
32. Mattiasson, B.; Ling, T.G.I. J. Chromat. (in press).
33. Kula, M.-R.; Buckmann, .; Hustedt, H.; Kroner, K.H.; Morr,
M. In "Enzyme Engineering"; Broun, E.B.; Manecke, G.;
Wingard, L.B.,Jr., Eds; Plenum: New York, 1978, Vol. 4, p.
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34. Kroner, K.H.; Schtte, H.; Starch, W.; Kula, M.-R. J. Chem.
Tech. Biotechnol. 1982, 32, 130-137.
35. Cordes, .; Flossdorf, J.; Kula, M.-R. Preprint 3rd Eur.
Cong. Biotechnol. Munich, Sept 1984, Vol. III, p.557.
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1st Eur. Cong. Biotechnol., Interlaken, Sept 1978, Part 1, p.
48.
37. Kula, M.-R. Lecture presented at 4th Int. Conf. Partition in
Aqueous Two-Phase Systems, Lund, Aug 1985.
38. Martin, M.; Tejero, C.; Galvez, M.; Pinilla, M.; Luque, J.
Acta Biol. Med. Ger. 1981, 40, 979-984.
39. Walter, H.; Pangburn, M.K. 4th Int. Conf. Partition in
Aqueous Two-Phase Systems, Lund, Aug 1985, Abstract p.4.
40. Sharpe, P.T.; Cottrill, C.P.; Wolpert, L. 4th Int.
Conf.Partition in Aqueous Two-Phase Systems, Lund, Aug 1985,
Abstract p. 7.
41. Bachman, L.; Johansson, G. FEBS Lett. 1976, 65, 34-43.

8
Recovery of Proteins from Polyethylene Glycol-Water
Solution by Salt Partition

G. B. Dove and G. Mitra

Cutter Laboratories, Berkeley, CA 94710
Addition of salts (e.g. potassium phosphate

dibasic) partitions an aqueous system containing 20%
w/v polyethylene glycol (PEG) into two liquid
phases: a PEG enriched phase and a salt enriched
phase. Proteins and polymers (e.g. DNA, albumin,
immunoglobulins, alpha-1 antitrypsin, and PEG)
distribute unevenly between the two phases.
Partition coefficients (concentration in PEG phase /
concentration in salt phase = K) are influenced by
physical parameters, such as salt composition and
concentration, pH (ion ratios) and temperature.
Specific proteins (e.g. alpha-1 antitrypsin) exhibit
low values in a wide range of conditions.
Higher salt concentrations and pH yield higher
partition coefficients.
In a plasma source, the
of alpha-1 antitrypsin is 0.0006 at 0.5 M salt and
increases to 0.0062 at 1.6 M salt. The of PEG
increases to 200+ in 1.0 M salt. Proteins in
general exhibit values of 0.01-100. Altering pH
to make proteins or other partitioned materials
more/less
hydrophilic
induces
greater/lower
solubility. A pH change from 5 to 9 increases in
general by 100-fold+.
Further, the trends
demonstrated by increasing salt concentration are
amplified. Lower temperatures (5 to -5 C) increase
the PEG by two to ten-fold with little change in
protein distribution. Conditions may be tailored to
optimize isolation of specific proteins to permit
recoveries of 90% from mixed systems, such as plasma
or fermentation broths.
T h i s paper i s o r g a n i z e d i n t o t h r e e p a r t s .
P u r i f i c a t i o n techniques
a r e o u t l i n e d b r i e f l y i n c o m p a r i s o n t o aqueous e x t r a c t i o n ,
followed
by
a review
o f p r o p e r t i e s and work i n m u l t i p h a s e
systems
with
emphasis
on t h e p u r i f i c a t i o n o f p r o t e i n s .
Finally,
recent
work
0097-6156/ 86/ 0314-0093S06.00/ 0


94
undertaken
by
the
authors i s presented,
involving
proteins of
pharmaceutical importance.
Various
methods
are
available
f o r the
separation
of
biochemicals.
These i n c l u d e p h y s i c a l methods o f c e n t r i f u g a t i o n and
filtration,
c h e m i c a l methods o f p r e c i p i t a t i o n and e x t r a c t i o n ,
and
i n t e r a c t i v e t e c h n i q u e s , s u c h a s e l e c t r o p h o r e s i s and chromatography.
T h e s e methods a r e employed t o p e r f o r m t h e s t e p s n e c e s s a r y t o p u r i f y
biological
m a t e r i a l s from complex s o l u t i o n s .
The i s o l a t i o n o f
a
specific
component
( e . g . a p r o t e i n ) from a plasma s o u r c e
or
a
fermentation broth r e q u i r e s s e v e r a l stepss
a)
removal
of c e l l
particles
(disruption
i f
intracellular
p r o d u c t ) , e.g. c e n t r i f u g a t i o n .
b)
p r e l i m i n a r y p u r i f i c a t i o n , e.g c o n c e n t r a t i o n , p r e c i p i t a t i o n .
c)
s e c o n d a r y p u r i f i c a t i o n , e.g. high-rsolut!on chromatography.
d)
finishing.
Through
these
steps, the
necessary p u r i t y
and
yield
are
achieved.
Requirements f o r p u r i t y and y i e l d a r e d i c t a t e d by f i n a l
use.
P u r i t y may range as low a s 10% f o r b u l k enzymes t o v i r t u a l l y
100%
f o r t h e r a p e u t i c use.
The f i n a l y i e l d a f f e c t s
directly
the
cost
of
the
finished
product
and
i s of c r i t i c a l
economic
importance,
as
feed stocks
f o r the
processes
are
typically
expensive.
Final
y i e l d s a r e c o n s i d e r e d i n terms o f
biologically
active material,
as many o f t h e components a r e l a b i l e and u s e l e s s
i n a denatured s t a t e .
S p e c i f i c a l l y , the c o n s t r a i n t s of high p u r i t y
and
biologically-active
y i e l d i n the
production of therapeutic
products
limit
the
alternatives
available
for
purification
processes.
Extractions
and
precipitations
i n chemistry
are
welle s t a b l i s h e d f o r o r g a n i c systems.
F o r example, n u c l e i c a c i d s may be
extracted
in a
phenol/water m i x t u r e (J.).
The
use
of
aqueous
e x t r a c t i o n s has s e v e r a l advantages o v e r t h e s e and o t h e r w i d e l y used
methods o f s e p a r a t i o n . 1) C h e m i c a l components,
s u c h as polymers
and/or
salts,
may
be
chosen t o m i n i m i z e
dnaturt!on due
to
solvency or i n t e r f a c i a l tension ( 2 ) .
Solvent/water m i x t u r e s , such
as
phenol/water,
produce i n t e r f a c i a l t e n s i o n s i n t h e range o f
50
dyne/cm, compared
to
0.1
dyne/cm
i n aqueous s y s t e m s ^ ) .
2)
Physical
sources
o f dnaturt!on a r e m i n i m a l ,
with v i r t u a l l y
no
shear.
S i m p l e m i x i n g o n l y i s r e q u i r e d ; c e n t r i f u g a t i o n may be used
to
hasten
separation (4).
3)
C o n d i t i o n s may
be
tailored
to
satisfy
s p e c i f i c i s o l a t i o n r e q u i r e m e n t s by b a s i n g
s e p a r a t i o n s on
dissimilar
s o l u b i l i t i e s and a f f i n i t i e s ,
which a r e dependent on pH
and s a l t s .
These e f f e c t s a r e not a p p l i c a b l e t o o t h e r methods.
4)
The
process
i s e a s i l y s c a l e d t o any
volume o f m a t e r i a l ,
with
minimal c a p i t a l i n v e s t m e n t 4 ) .
Beyond c o n v e n t i o n a l p r o d u c t s ,
the
technique
i s applicable to biotechnical separations with
unique
p o s s i b i l i t i e s , which w i l l be a d d r e s s e d below.
P r o p e r t i e s and A p p l i c a t i o n s o f Aqueous Systems
The
methodology
of
aqueous e x t r a c t i o n s i s a d a p t a b l e
to
the
requirements
o f i s o l a t i n g b i o l o g i e s due t o t h e h i g h water
content
of
the
system.
The a d d i t i o n o f w a t e r - s o l u b l e polymers
and/or
salts
t o water produces s p o n t a n e o u s l y two o r more l i q u i d
phases.
The
d e n s e r s o l u t i o n ( u s u a l l y t h e s a l t - r i c h one) forms t h e
bottom
phase.
Each
phase i s c o m p r i s e d p r i m a r i l y o f w a t e r
(80-95%X.2).


8.
DOVE A N D M I T R A
95
Recovery of Proteins by Salt Partition
The
t o p and bottom l i q u i d r e g i o n s a r e s e p a r a t e d by an i n t e r f a c e
(P1).
P r e c i p i t a t e s may form a t t h i s l i q u i d - l i q u i d i n t e r f a c e o r may
settle
t o t h e bottom o f t h e v e s s e l <P2), f o r m i n g a s o l i d - l i q u i d
interface.
Any m a t e r i a l
i n t h e m i x t u r e may d i s t r i b u t e
t o any
r e g i o n , dependent on t h e combined p r o p e r t i e s o f a l l components.
Multiphase
l i q u i d systems a r e a n a l o g o u s t o p r e c i p i t a t i o n s i n
that
precipitations
consist
o f one l i q u i d phase
and one s o l i d
region,
whereas m u l t i p h a s e l i q u i d systems p o s s e s s two o r more
l i q u i d r e g i o n s and a range o f s o l i d r e g i o n s .
The i n c r e a s e d number
of
environments
available
t o a material
make i s o l a t i o n
more
amenable t o o p t i m i z a t i o n .
In a g i v e n system o f a polymer,
a s a l t and water,
two l i q u i d
phases
e x i s t with the corresponding equilibrium concentrations of
each component.
T y p i c a l l y , t h e t o p phase i s e n r i c h e d w i t h polymer
and
t h e bottom phase i s e n r i c h e d w i t h s a l t .
The phases p a r t i t i o n
because o f mutual " i n c o m p a t i b i l i t y " (j>).
A p r o t e i n i n t h e system
will
d i s t r i b u t e between t h e two phases a c c o r d i n g t o t h e p r o p e r t i e s
of
the partitioning
a g e n t s and o t h e r m a t e r i a l s
present.
A
p a r t i t i o n c o e f f i c i e n t ( K ) may be d e f i n e d a s ( 2 ) s
Ct/Cb
<1 )
where C t and Cb a r e t h e c o n c e n t r a t i o n s o f p r o t e i n i n t h e t o p and

bottom phases,
respectively.
I f a component has an e q u a l a f f i n i t y
for
b o t h phases,
t h e c o n c e n t r a t i o n s i n each phase a r e e q u a l and
i s equal t o unity.
The
partition
coefficient
o f any component
i n t h e system
(polymer,
salt,
p r o t e i n ) may be m a n i p u l a t e d by s e v e r a l p a r a m e t e r s
(4):
a)
C h e m i c a l b a s i s and m o l e c u l a r w e i g h t o f p r i m a r y polymer ( e . g .
p o l y e t h y l e n e g l y c o l s PEG).
b)
C h e m i c a l b a s i s o f second agent ( e . g . polymer, s a l t ) .
c) Concentrations of additives (e.g. proteins).
d)
pH ( o r i o n i c r a t i o s ) ,
e ) Temperature.
The
number o f c o n t r o l l a b l e p h y s i c a l
parameters
i s large.
Parameters may be v a r i e d i n d i v i d u a l l y i n m e t h o d i c a l f a s h i o n , a s was
done i n t h i s work.
E x p e r i m e n t s n e c e s s a r y t o o p t i m i z e a system
for
a s p e c i f i c component may be r e d u c e d by t h e s i m p l e x method ( 5 )
or f r a c t i o n a l f a c t o r i a l design ( 6 ) .
In
the last
thirty
years,
substantial
data has
been
accumulated
f o r aqueous m u l t i p h a s e systems.
The d a t a may be
classified
by t h e components n e c e s s a r y t o form m u l t i p l e
phases.
N o n - i o n i c polymers, p o l y e l e c t r o l y t e s , low m o l e c u l a r w e i g h t o r g a n i c s
and
s a l t s have been s t u d i e d t o e s t a b l i s h phase diagrams f o r two o r
more components i n d e f i n e d systems
(Albertsson,
2 ) . The most
common systems i n v e s t i g a t e d have been m i x t u r e s o f PEG and d e x t r a n
o r a phosphate s a l t .
S e p a r a t i o n s by t h e s i m p l e p a r t i t i o n i n g a g e n t s
can
be enhanced
by changes
i n t h e chemical
and
physical
environment.
T h e a d d i t i o n o f minor c o n t a m i n a n t s ( e . g . d e t e r g e n t s
or other s u r f a c t a n t s ) a l t e r s the surface tension ( 7 ) .
Modification
of
polymers,
such
a s a t t a c h i n g a c h a r g e d group t o PEG ( 8 ) o r
dextran
( 9 ) , and a p p l i c a t i o n s t o a f f i n i t y l i g a n d systems (9j. 10)
and
a s s a y s (JJ.) a r e under i n v e s t i g a t i o n .
Mass t r a n s p o r t
across
t h e i n t e r f a c e has been s t u d i e d ( 1 2 ) .


96
Partitioning
o f plasma p r o t e i n s ( 2 ^ 6 1 3 ) ,
enzymes
(see
below), c e l l s and c e l l p a r t i c l e s (2j_ 14, 15) and n u c l e i c a c i d s ( 16,
1 7 ) i n a v a r i e t y o f systems have been r e p o r t e d .
Large
scale
purification
of
enzymes i n PEG/dextran
and
P E G / s a l t have been r e p o r t e d and r e v i e w e d by K u l a
et. al.(18,
19,
20).
Examples i n c l u d e p u l l u l a n a s e and 1,4-a-glucan
phosphorylase
a-amylase ( 2 2 ) ,
b - g a l a c t o s i d a s e ( 2 3 ) , and
the
general
economics o f e x t r a c t i v e enzyme r e c o v e r y ( 2 4 ) . P r o c e s s s t u d i e s have
covered
the
use
of
continuous
centrifuges
(18,
19)
and
c o u n t e r c u r r e n t d i s t r i b u t i o n t r a i n s (2 2 5 ) .
P u r i f i c a t i o n o f b - g a l a c t o s i d a s e from E.
c o l i may be compared:
Higgins
(6) o u t l i n e s a process of succeeding
centrifugations
to
remove c e l l
debris,
nucleic
acids precipitate,
and
protein
precipitate
(product).
Veide
(23)
outlines
a single
aqueous
e x t r a c t i o n w i t h PEG and s a l t i n which b - g a l a c t o s i d a s e p a r t i t i o n s t o
t h e P E G - r i c h phase.
C e l l s , n u c l e i c a c i d s , and a m a j o r p a r t o f t h e
c o n t a m i n a t i n g p r o t e i n s p a r t i t i o n t o t h e s a l t - r i c h phase.
Several
applications
i n process
purification
serve
to
illustrate
the
d i v e r s i t y of uses.
The
partitioning
of
phases
allows
components
t o be s e p a r a t e d w i t h i n t h e c o n f i n e s of
another
operation.
Production of
a material (e.g.
fermentation
and
subsequent
cell
s e p a r a t i o n ) may be
simplified.
Tissue
culture
b r o t h w i t h c e l l s may be s o n i c a t e d . The c e l l w a l l s a r e d i s r u p t e d t o
r e l e a s e i n t r a c e l l u l a r p r o d u c t s and d e g r a d i n g enzymes. The b r o t h i s
p a r t i t i o n e d q u i c k l y with PEG/salt i n bulk.
In
an
unmodified
PEG/dextran
system,
cells
and
cellular
components p a r t i t i o n t o t h e d e x t r a n - r i c h bottom phase ( 2 ) ,
leaving
the
top
phase a v a i l a b l e
f o r product.
a-amylase has
been
partitioned
t o t h e t o p phase and B a c i l l u s s u b t i l i s c e l l s
to
the
bottom phase ( 2 2 ) . P r o d u c t i o n o f b i o l o g i c a l l y a c t i v e m a t e r i a l s may
be enhanced by removal o f p r o d u c t from c e l l s o r c e l l u l a r components
for
two r e a s o n s :
1 ) D e g r a d a t i o n o f p r o d u c t by
extra- or
intra
cellular
enzymes s t i l l
present i n the broth
i s prevented.
2)
Removal
o f p r o d u c t r e d u c e s n e g a t i v e feedback i n h i b i t i o n o f
growth
or production of c e l l s .
The method i s u n i q u e i n speed and e a s e f o r
handling
b u l k q u a n t i t i e s which i s c r i t i c a l f o r s e n s i t i v e
systems.
A
d i f f e r e n t approach i n v o l v e s t h e use o f
ligands covalently
attached
t o PEG,
a s n o t e d above ( 8 ) .
The l i g a n d / P E G complex
is
introduced
t o the
b r o t h , where t h e
ligand
binds
i t s target
complement,
a product.
PEG and s a l t a r e added;
t h e PEG/1igand/complement complex p a r t i t i o n s p r e f e r e n t i a l l y t o t h e PEG phase.
The
complement
has
been
isolated
i n the
PEG
phase,
whereas i t s
i n t r i n s i c d i s t r i b u t i o n would f a v o r t h e s a l t phase. The s e p a r a t i o n /
purification
i s h i g h where c o n d i t i o n s a r e such
that
a l l other
m a t e r i a l s p a r t i t i o n t o t h e s a l t phase.
This
study
was
initiated
to explore the
applications
of
partitioned
phases t o s e p a r a t i o n s o f
therapeutically
active
materials.
P o s s s i b i l i t i e s include:
1 ) i s o l a t i o n and p u r i f i c a t i o n
o f one component from a complex m i x t u r e ( e . g . alpha-1 a n t i t r y p s i n ,
immunoglobulins
from plasma s o u r c e s o r t i s s u e c u l t u r e
broth),
2)
removal o f a s e c o n d a r y p r o d u c t o r contaminant ( e . g .
DNA,
residual
PEG
from
a p r o c e s s s t r e a m ) and 3) s i m u l t a n e o u s
fermentation
and
i s o l a t i o n o f p r o d u c t from c e l l u l a r components.
A
system o f P E G / s a l t was chosen because t h e s e t t l i n g
time
is
s h o r t (10-60 min.) compared t o polymer/polymer systems ( P E G / d e x t r a n

8.
DOVE A N D
MITRA
is
30-180 min.)Typically,
PEG l e v e l s
are
lower
i n s a l t systems.
Further, i
levels
could
be
substantially
reduced
optimization.
The d a t a p r e s e n t e d i n t h i s
t h e work i n p r o g r e s s .
97
under s i m i l a r c o n d i t i o n s
t was o b s e r v e d
that
PEG
i n the s a l t
phase
by
paper r e p r e s e n t p a r t
of

M a t e r i als/Methods
Polyethylene
glycol
(PEG
3350),
H0(CH2-CH20)x-CH2-CH20H,
was
obtained
from Union C a r b i d e .
PEG 3350 has a m o l e c u l a r weight
of
3300-3400. Reagents ( p o t a s s i u m phosphate d i b a s i c , p h o s p h o r i c a c i d )
were o b t a i n e d from J.T. Baker, "Baker a n a l y z e d " r e a g e n t grade.
Simple
systems ( w i t h a s i n g l e d e f i n e d a d d i t i v e ) were produced
w i t h each o f t h e f o l l o w i n g m a t e r i a l s . C a l f thymus DNA, p o l y m e r i z e d ,
was
o b t a i n e d from Sigma.
P r o t e i n s o u r c e s were p r e p a r e d
in-house
and
s u b s e q u e n t l y d i a l y z e d i n t o low s a l t
solutions.
Human serum
albumin
and
immunoglobulin G ( l g G ) were p l a s m a - d e r i v e d .
Human
immunoglobulin
M (IgM) was produced by t i s s u e c u l t u r e f e r m e n t a t i o n
and p u r i f i e d .
A d e f i n e d complex system c o n s i s t e d o f b o t h
albumin
and
IgM t o g e t h e r .
An u n d e f i n e d complex system was s e t up w i t h an
intermediate material
o f Cohn plasma
fractionation
containing
alpha-1 a n t i t r y p s i n ( a l p h a - 1 ) , albumin, and o t h e r c o n t a m i n a n t s .
A s t o c k s o l u t i o n o f 40% PEG was s t o r e d a t 5 C.
S i m p l e systems
were
formed by t h e a d d i t i o n o f PEG s o l u t i o n ,
salt,
and water
to
give
20% w/v PEG and a p p r o p r i a t e s a l t .
S o l u t i o n s were mixed
and
adjusted
t o pH
with
phosphoric a c i d .
DNA
was
added
to
an
approximate
c o n c e n t r a t i o n o f 1 mg/ml.
Protein
concentrates i n
unbuffered
s o l u t i o n s were added t o an a p p r o x i m a t e c o n c e n t r a t i o n o f
1-10
mg/ml.
To t h e plasma f r a c t i o n ,
PEG and s a l t c r y s t a l s
were
added.
Systems were g e n t l y mixed by
rocking
i n polypropylene
c e n t r i f u g e t u b e s . The m i x t u r e s were a l l o w e d t o s e t t l e o v e r n i g h t a t
-4, 5 o r 20 C.
Tubes were c e n t r i f u g e d a t 2000 RCF f o r 30 min.
Samples were a s s a y e d by absorbance a t 280 nm, B r a d f o r d p r o t e i n
a s s a y ( 2 7 ) , and r a d i a l i m m u n o d i f f u s i o n p l a t e s ( H e l e n a L a b o r a t o r i e s ) .
Alpha-1
antitrypsin
was
assayed
for biological
activity
by
competitive
assay with e l a s t a s e (28).
DNA was
assayed
by
the
diphenylamine
methods o f B u r t o n ( 2 9 ) and G i l e s and M y e r s ( 3 0 ) ,
with
m o d i f i c a t i o n s due t o t h e p r e s e n c e o f PEG and s a l t s ( 3 1 ) . M a t e r i a l s
were a l s o a s s a y e d by s i z e e x c l u s i o n chromatography on
Superose
6
( P h a r m a c i a FPLC), w i t h peak i n t e g r a t i o n a t 280 nm.
PEG was a s s a y e d
by HPLC.
Results
Data
a r e p r e s e n t e d i n s e v e r a l forms f o r many o f
the
partitioned
materials.
The c o n c e n t r a t i o n o f m a t e r i a l i n t h e s a l t phase and t h e
partition
coefficient
(concentration
i n the
PEG
phase
/
concentration
i n t h e s a l t phase = K) a r e p l o t t e d as
functions
of
s a l t c o n c e n t r a t i o n and pH on s e m i - l o g s c a l e .
On t h e l o g a x i s , 1E0
r e p r e s e n t s 1 10(0) o r 1;
2E3 r e p r e s e n t s 2 10(3) o r 2,000. A
value
of
1 indicates
no
preference for either
phase;
the
concentration
i s t h e same i n b o t h .
I t i s t h i s value of unity that
is critical.
Values g r e a t e r than 1 i n d i c a t e a p r e f e r e n c e f o r the
top
(PEG) phase and v a l u e s l e s s than 1 i n d i c a t e t h e bottom
(salt)
phase.
In s e v e r a l c a s e s ,
p r e c i p i t a t i o n of p r o t e i n occurs a t the

98
S E P A R A T I O N , RECOVERY, A N D P U R I F I C A T I O N I N B I O T E C H N O L O G Y

i n t e r f a c e o r a t t h e bottom o f t h e t u b e .
As p r e c i p i t a t i o n
occurs,
t h e c o n c e n t r a t i o n o f p r o t e i n i n t h e s a l t phase may d e c r e a s e w i t h o u t
a concurrent increase i n the p a r t i t i o n c o e f f i c i e n t .
Centrifugation
after
s e t t l i n g f o r 24 h o u r s had no e f f e c t
on
t h e volume r a t i o s (PEG phase / s a l t p h a s e ) . G e l a t i n o u s p r e c i p i t a t e s
were o b s e r v e d
i n systems a t h i g h e r s a l t and l o w e r
pH due t o
saturation
o f t h e system w i t h r e s p e c t t o t h e p r o t e i n a t t h e g i v e n
salt/pH
environment.
C e n t r i f u g a t i o n compressed t h i s p r e c i p i t a t e
l a y e r t o l e s s t h a n 10% o f t h e phase volume.
In
general,
t h e volume r a t i o
(PEG phase / s a l t
phase)
d e c r e a s e s a s t h e s a l t c o n c e n t r a t i o n o r pH i n c r e a s e s .
Table
I.
R e l a t i v e volumes o f phases a s
c o n c e n t r a t i o n i n a t y p i c a l system a t 5 C.
S a l t Concentration
0.5
0.6
0.8
0.8 (-5 C)
1.0
1.2
1.6
(M)
PEG
78
61
48
47
44
44
44
( % vol.)
function
S a l t (%
of
salt
vol.)
22
39
52
53
56
56
56
The g r o s s p h y s i c a l c h a r a c t e r i s t i c s o f t h e system c a n be i n f l u e n c e d
by
relatively
m i n o r changes i n c o m p o s i t i o n .
For
example,
adjustment
o f pH w i t h h y d r o c h l o r i c a c i d i n s t e a d
of
phosphoric
p r e v e n t s an i n t e r f a c e from f o r m i n g below pH 5.5.
DNA, IgM, IgG, a l b u m i n , and alpha-1 a n t i t r y p s i n f o l l o w s i m i l a r
trends
(Table
II,
Figures
1-4).
As t h e s a l t
concentration
increases,
the concentration
of material
i n the s a l t
phase
decreases
and t h e p a r t i t i o n c o e f f i c i e n t i n c r e a s e s .
IgG e x h i b i t s
the
same p a t t e r n w i t h i n c r e a s i n g pH ( F i g u r e 5 ) .
Other
materials
are not as c o n s i s t e n t .
The t r e n d s appear t o be somewhat a d d i t i v e s
increasing
the s a l t
c o n c e n t r a t i o n and pH l e a d
t o the greatest
partition
coefficients.
Temperature
changes g i v e mixed
results
with
little
change ( F i g u r e s 2, 4, 5 ) .
In s e v e r a l
instances,
systems a t t h e extreme v a l u e s o f t h e parameter r a n g e s i n d i c a t e an
a m p l i f i c a t i o n o f trends observed i n the middle ranges.
Various
forms o f DNA
behave d i f f e r e n t l y .
In PEG/dextran
systems, n a t i v e DNA e x h i b i t s h i g h e r v a l u e s o f t h a n d e n a t u r e d DNA.
Both m a t e r i a l s e x h i b i t h i g h e r v a l u e s a s t h e pH i s i n c r e a s e d ( i . e .
(H2P04)- i s s h i f t e d t o ( P 0 4 ) 3 - ) ( J 6 ) .
A
summation
o f p a r t i t i o n c o e f f i c i e n t s a t pH 9 i s g i v e n i n
F i g u r e 6. A l l m a t e r i a l s e x h i b i t a tendency toward t h e s a l t phase a t
low s a l t c o n c e n t r a t i o n s .
As t h e s a l t c o n c e n t r a t i o n i n c r e a s e s , DNA,
albumin,
IgM, and IgG a r e r e p e l l e d from t h e s a l t phase t o t h e PEG
phase. Alpha-1 remains i n t h e s a l t phase e x c l u s i v e l y . The p a t t e r n
at
pH 9 s t a n d s i n c o n t r a s t t o t h e r e s u l t s a t pH 5, i n F i g u r e
7.
M a t e r i a l s do n o t m i g r a t e t o t h e PEG phase. v a l u e s remain below 1
even i n h i g h s a l t c o n c e n t r a t i o n s .


99
Salt ConcQntration (M)

Figure
1.
Albumin
concentration
function of s a l t concentration.
i nthe s a l t
phase
as a
4->

Figure
2.
Albumin p a r t i t i o n c o e f f i c i e n t
(concentration i n
PEG phase / c o n c e n t r a t i o n i n s a l t phase) a s a f u n c t i o n o f s a l t
concentration.


100
Figure
4.
Immunoglobulin
G
function of salt concentration.
partition
coefficient

as

8.
101
Recovery of Proteins by Sait Partition
DNA
Figure
6.
Summary o f p a r t i t i o n c o e f f i c i e n t s
a t pH 8 - 9 a s

102

AlbiMin
Salt Concentration (M)

Figure
7.
Summary o f p a r t i t i o n c o e f f i c i e n t s
a t pH 5-6 as

8.
103
Table
I I . Concentration
of materials
i n the s a l t
phase and
p a r t i t i o n c e f f i c i e n t a s f u n c t i o n s o f s a l t c o n c e n t r a t i o n and pH.
Material
t= 5 C
Salt
Cone*
(M)
PH
0.5
0.8
0.8
0.8
1.6
9
6
7
9
9
0.5
0.8
0.8
0.8
1.6
9
6
7
9
9
0.5
0.6
0.8
1.0
1.2
1.6
8
8
8
8
8
8

DNA
IgM
Alpha-1
P a r t i t i o n Coeff.
Cone *n
( S a l t phase) ( P E G / S a l t )
(ug/ml )
23.5
337
5.6
5.4
1
( mg/ml )
1.0
1.4
0.080
0.035
0.0019
( mg/ml )
17.8
10.1
6.3
5.5
4.7
3.3
0.043
0.004
0.179
0.185
538
0.0019
0.0013
0.024
0.054
n.a.
0.00056
0.0025
0.0033
0.0038
0.0044
0.0062
The
d i f f e r e n c e between alpha-1 and o t h e r
p r o t e i n s may be
attributed
t o e i t h e r t h e i n t r i n s i c n a t u r e o f alpha-1
(e.g.
low
hydrophobicity)
o r t h e p r e s e n c e o f m i s c e l l a n e o u s plasma
fraction
contaminants
i n t h e s a l t phase a t t r a c t i n g alpha-1 o r i n t h e PEG
phase r e p e l l i n g
alpha-1.
A mixture of albumin
and IgM show
q u a l i t a t i v e l y t h e same v a l u e s a s t h e r e s p e c t i v e s i m p l e systems. As
the
s a l t concentration increases,
the concentration of protein i n
the
s a l t phase d e c r e a s e s and t h e p a r t i t i o n c o e f f i c i e n t
increases.
Again,
a t l o w e r pH, m a t e r i a l s do n o t m i g r a t e t o t h e PEG phase and
partition
coefficients
do n o t exceed
1.
Further
work i s i n
p r o g r e s s t o d e f i n e t h e s e p a r a t i o n between m u l t i p l e components based
on e x p e r i m e n t s w i t h d e f i n e d systems.
The
effects
o f s a l t c o n c e n t r a t i o n and pH have been
studied
p r e v i o u s l y (2j. 16, 3 2 ) . I t h a s been found t h a t t h e c o n c e n t r a t i o n
and
pH a r e n o t a s c r i t i c a l a s t h e r a t i o o f i o n s .
The pH i s a
measure o f t h e i o n i c environment;
t h a t i s , t h e r a t i o o f charged
i o n s d e r i v e d from t h e s a l t , K2HP04 and a c i d ,
H3P04.
Compared t o
effects
o f small ions,
t h e c o n c e n t r a t i o n o f p r o t e i n s has l i t t l e
effect
on p a r t i t i o n c o e f f i c i e n t s ( 3 2 ) . I n
general,
higher
valent
anions
yield
higher
partition
coefficients:
o f (P04)3- >
(HP04)2- > (H2P04)-.
The e f f e c t s
of cations
and a n i o n s a r e
cumulative.
Ionic
effects
c a n i n c l u d e t h e a d d i t i o n o f NaCl t o
PEG/dextran systems ( 2 ) .
The
d i s t r i b u t i o n may be d e f i n e d i n terms o f a model
From t h e B r o n s t e d f o r m u l a <2 33, 3 4 ) :
equation.
(M x ) / ( R )
= e

(2)
104
where M i s t h e m o l e c u l a r w e i g h t o f t h e p a r t i t i o n e d component,
is
a f a c t o r dependent on t h e component and system o t h e r t h a n
size,
R
is
t h e gas c o n s t a n t
and i s t h e t e m p e r a t u r e .
To p r e d i c t
qualitatively
t h e b e h a v i o r o f t h e system,
e x i s t i n g data
may
be
extrapolated.
C a l c u l a t i n g w i t h IgG d a t a and e x t r a p o l a t i n g i t t o
IgM, t h e v a l u e s o f T a b l e I I I a r e g e n e r a t e d .

Table
I I I . C a l c u l a t i o n o f Bronsted
d a t a and e x t r a p o l a t i o n t o IgM a t 5 C.
S a l t Conc'n
PH
IgG
IgG
IgM
IgM
<M)
(K, d a t a )
<x)
(K, c a l c . )
(K, d a t a )
formula
parameters with
0.5 M
9
0.8 M
6
0.8 M
7
0.8 M
9
5E-2
-4.3E-4
3E-7
2E-3
1-2
-6.6-4
1-10
1-3
2E-1
-2.3E-4
3E-4
2E-2
6E-1
-7.3E-5
8E-2
7E-2
IgG
values
For
IgM, ( c a l e . ) / ( d a t a ) i s l e s s t h a n
The
1.
converge only
a t h i g h e r s a l t and pH.
Data
indicate a
higher
affinity
f o r t h e PEG phase t h a n p r e d i c t e d by e x t r a p o l a t i o n o f IgG
v a l u e s i n t h e Bronsted formula.
I f t h e PEG phase t e n d s t o a t t r a c t ,
o r t h e s a l t phase r e p e l s ,
hydrophobic s p e c i e s ,
t h e n IgM e x h i b i t s
g r e a t e r h y d r o p h o b i c i t y than e x p e c t e d .
Conversely, c a l c u l a t i o n s t o
predict
t h e b e h a v i o r o f IgG based on IgM would
indicate
greater
hydrophilicity.
I t may be h y p o t h e s i z e d t h a t t h e g l o b u l a r n a t u r e o f
IgM
s h i e l d s t h e Fc t e r m i n a l c a r b o x y l group from b u l k i n t e r a c t i o n s ,
reducing the net charge d e n s i t y o f the molecule i n s o l u t i o n .
A change i n t e m p e r a t u r e o f 15 C y i e l d s a change i n c a l c u l a t e d
values
o f o f l e s s t h a n 5%.
Data i n d i c a t e no s i g n i f i c a n t change
i n p r o t e i n v a l u e s over t h e observed temperature range.
Applications
The p r a c t i c a l consequences o f a p r o c e s s s e p a r a t i o n i n v o l v i n g a l p h a 1 a n t i t r y p s i n a r e i n d i c a t e d i n F i g u r e s 8 and 9.
The y i e l d ( F i g u r e
8) i s t h e p r o d u c t o f t h e c o n c e n t r a t i o n o f alpha-1 i n t h e s a l t phase
and t h e volume o f t h e phase.
Y i e l d s a r e above 9 0 % i n t h e r a n g e o f
0.5-0.8 M s a l t .
The s p e c i f i c a c t i v i t y
( a l p h a - 1 a c t i v i t y / A 2 8 0 ) , an
i n d i c a t i o n o f p u r i t y , i n c r e a s e s by 20-40% o v e r t h e i n i t i a l a c t i v i t y
at
pH 8 w i t h no dependence on s a l t
concentration
(Figure 9).
A d j u s t m e n t o f pH i n f u t u r e e x p e r i m e n t s may g i v e i n c r e a s e d p u r i t y .
The
concentration
o f PEG i n t h e s a l t phase i s p l o t t e d i n
F i g u r e 10. The l o w e s t v a l u e s a r e o b t a i n e d a t 1.2 M s a l t .
Reducing
the
t e m p e r a t u r e t o -5 C f u r t h e r r e d u c e s t h e PEG l e v e l s by a f a c t o r
o f 2-10, g i v i n g a minimum o f 0.06 mg/ml.
A t 0.8 M
salt,
higher
c o n c e n t r a t i o n s o f PEG a r e measured i n 25 m l . v e s s e l s a s compared t o
100
m l . I t was o b s e r v e d t h a t s m a l l beads o f PEG adhered
to the
w a l l s o f t h e s m a l l e r v e s s e l s . Thus, t h e i n c r e a s e d r a t i o o f s u r f a c e
area
/ volume i n c r e a s e d t h e a p p a r e n t PEG c o n c e n t r a t i o n .
It i s
e x p e c t e d t h a t a l l PEG v a l u e s would d e c r e a s e as t h e v e s s e l volume i s
increased.
Other
materials
( p r o t e i n s ) were n o t s u b j e c t t o
carryover.


8.
105
.4
.6
.8
1.2
1.4

Figure
8.
Recovery y i e l d o f Alpha-1
function o f salt concentration.
.S
i.9-|
i.H
.e
i n the s a l t
phase a s
vol. =100 i l .
vol. =25 ml.
t=5 C, pH 8
1.7i
1.6H
1.5H
5 i.2H
;
ti
en
1 , 1
1-
?8
1
7^2
L4
L6
^"
Figure
9.
Change
i n specific activity
o f Alpha-1
phase/initial) as a function o f salt concentration.

(salt

106
F i g u r e 10.
PEG c o n c e n t r a t i o n i n t h e s a l t phase as a f u n c t i o n
of s a l t concentration.


8.
107
The
a b i l i t y t o distribute proteins
and polymers,
including
nucleic
acids,
between two i m m i s i b l e phases h a s been shown.
The
liquid
system
c o n s i s t s o f a PEG r i c h t o p phase and a s a l t
rich
bottom
phase.
Proteins o f i n t e r e s t i n i n d u s t r i a l pharmaceuticals
i n c l u d e n u c l e i c a c i d s (DNA),
a l b u m i n , immunoglobulins, and alpha-1
antitrypsin.
The i n i t i a l
purification
of a single
component
( a l p h a - 1 ) from a complex m i x t u r e ( p l a s m a ) has been
demonstrated.
Further,
r e s i d u a l PEG i n t h i s s t e p h a s been s e v e r e l y r e d u c e d .
An
i n c r e a s i n g number o f new p r o c e s s e s a r e u t i l i z i n g PEG a s an a g e n t i n
precipitation
or other operation.
Removal o f PEG from
aqueous
solutions
by column
chromatography,
diafiltration,
and o t h e r
c o n v e n t i o n a l methods i s d i f f i c u l t .
I t has been shown t h a t t h e
concentration
o f PEG may
be r e d u c e d
by c o n t r o l l i n g
physical
p a r a m e t e r s , even i n complex m i x t u r e s .
The
t e c h n i q u e i s a p p l i c a b l e t o an i n f i n i t e v a r i e t y o f complex
s e p a r a t i o n s w i t h unique a t t r i b u t e s .
The p r o p e r t i e s
forming t h e
basis
of separation
( s u r f a c e charge, r e l a t i v e
a f f i n i t i e s ) are
unlike
t h o s e o f o t h e r commonly
used
p r o c e s s e s and
thereby
complement o t h e r t e c h n i q u e s t o p r o v i d e i n c r e a s e d s e l e c t i v i t y .
High
yield,
low c a p i t a l
r e q u i r e m e n t s and s i m p l e p r o c e s s i n g
steps
f a c i l i t a t e i n c o r p o r a t i o n i n t o a d e t a i l e d p u r i f i c a t i o n scheme.
Legend
o f Symbols
s p a r t i t i o n c o e f f i c i e n t = C(t) / C(b).
C(t)s
C o n c e n t r a t i o n i n t o p phase, t y p i c a l l y o f a p r o t e i n .
C(b)s
C o n c e n t r a t i o n i n bottom phase.
PEG:
polyethlylene glycol.
as
alpha
jfcs b e t a
IgGs
immunoglobulin G.
IgMs
immunoglobulin M.
A-1, alpha-1s
alpha-1 a n t i t r y p s i n .
Ms m o l e c u l a r w e i g h t ( g / gmole).
xs
f a c t o r i n Bronsted formula (atmospheres l i t e r s / g ) .
Rs
gas c o n s t a n t s (0.08205) (atmosphers l i t e r s / gmoles o K ) .
s
t e m p e r a t u r e ( K ) .
Literature Cited
1. Maniatis, T., Fritsch, E. F., Sambrook, J. "Molecular Cloning:
A Laboratory Manual"; Cold Spring Harbor Laboratory; 1982; p. 458.
2.
Albertsson,
P.A. "Partition of
Cell
Particles and
Macromolecules"; Wiley Intersciences New York; 1960.
3.
Ryden, J., Albertsson, P. A. J. Coll. Interface Sci. 1971, 37,
219.
4. Hustedt, H., Kroner, K.H., Menge, U., and Kula, M-R., Trends in
Biotechnology 1985, 3, no. 6, 139-141.
5. Backman, L., Shanbhag, V. P., Anal. Biochem. 1984, 138, 372.
6. Menge, U., Morr, M., Mayr, U., Kula, M. R., J. Appl. Biochem.
1983, 5, 75.
7. Albertsson, P. ., Biochemistry 1973, 12, 2525.
8. Johansson, G., Biochim. Biophys. Acta 1970, 222, 381.
9. Chaabouni, ., Dellacherie, E., J. Chrom. 1979, 171, 135.
10.
Flanagan, S. D., Barondes, S. H., J. Biol. Chem. 1975, 250,
1484.


108
11. Mattiasson, B., Ling, T. G. I., J. Immunol. Methods 1980, 38,

217.
12. Shanbhag, V. P., Biochim. Biophys. Acta 1973, 320, 517.
13. Busby, T. F., Ingham, K. C., Vox Sang. 1980, 39, 93.
14.
Bungay, H. R., "Enzyme Engineering"; . K. Pye and H. H.
Weethall, ed.; Plenum: New York; 1978, vol. 3, p. 225.
15. Hofsten, B. v., Baird, G. D., Biotech. Bioeng. 1962, 4, 403.
16. Albertsson, P. ., Biochim. Biophys. Acta 1965, 103, 1.
17.
Alberts, . ., "Methods in Enzymology, Nucleic Acids"; Wiley
Interscience: New York, 1967, vol. 12, 566.
18.
Kroner, . H., Hustedt, H., Granda, S., Kula, M. R., Biotech.
Bioeng. 1978, 20, 1967.
19. Kula, M. R., Kroner, . ., Hustedt, H., and Schutte, H., Ann.
NY Acad. Sci. 1981, 369, 341.
20.
Schutte, H., Kroner, . ., Hummel, W., Kula, M. R., Ann. NY
Acad. Sci. 1983, 413, 270.
21.
Hustedt, ., Kroner, K. H., Stach, W., Kula, M. R., Biotech.
Bioeng. 1978, 20, 1989.
22.
Andersson, ., Johansson, A-C., Hahn-Hagerdal, ., Enzyme and
Microb. Tech. 1985, 7, July, 333-338.
23. Veide, ., Smeds, A. L., Enfors, S. O., Biotech. Bioeng. 1983,
25, 1789.
24.
Kroner, . H., Hustedt, H., Kula, M. R., Proc. Biochem. 1984,
19, 170.
25.
Craig, L. C., Craig, D., "Techniques of Organic Chemistry";
ed. A. Weissberger; Interscience Publishers: New York; 1956, vol.3.
26.
Higgins, J. J., Lewis, D. J., Daly, W. H., Mosqueira, F. G.,
Dunnill, P., Lilly, M. D., Biotech. Bioeng. 1978, 20, 159.
27. Bradford, ., Analytical Biochemistry 1976, 72, 248-254.
28.
Coan, M. H., Brockway, W. J., Eguizabel, H., Krieg, T.,
Fournel, ., Vox Sang. 1985, 48, 333.
29. Burton, K., Biochem. J. 1956, 62, 315.
-30. Giles, K. W. and Myers, ., Nature, London 1965, 206, 93.
31. Dove, G. and Naab, P., personal communications.
32. Albertsson, P.., Advances in Protein Chemistry 1970, 24, 309.
33.
Bronsted, J. ., Z. phys. Chem., A. (Bodenstein-Festband)
1931, 257; from ref. 2.
34.
Bronsted, J. ., Warming, ., Z. phys. Chem., A. 1931, 155,
343; from ref. 2.

9
Modeling of Precipitation Phenomena in Protein
Recovery

C. E. Glatz and R. R. Fisher

Department of Chemical Engineering, Iowa State University, Ames, IA 50011
A review of efforts to experimentally characterize and

model the phenomena important in protein precipitation
shows that, despite successes, continued work is
necessary to produce accurate mechanistic descriptions
of this method of protein recovery.
The formation and growth of the primary particle in
acid precipitation has been described in terms of the
protein supersaturation. Aggregate growth by collision
results in a size-dependent rate expression. Aggregate
breakage, by shear or collision, remains to be
adequately described in light of recent work.
Population balances serve to model the combined
phenomena.
Recent work identifies mixing during precipitant
addition as a determinant of aggregate physical
properties; such effects are described with a
floc-strength model.
R e a p i n g t h e b e n e f i t s o f t h e new b i o l o g y a n d e v e n t h e c o n t i n u e d
development o f t r a d i t i o n a l biotechnology poses problems i n s e v e r a l
areas.
Two o f t h e s e , s y n t h e s i s o f t h e d e s i r e d p r o d u c t a n d i t s e n d
u s e , h a v e b e e n a n d w i l l c o n t i n u e t o b e t h e f o c u s o f much r e s e a r c h .
R e l a t i v e l y n e g l e c t e d has been the r e c o v e r y and p u r i f i c a t i o n o f these
b i o l o g i c a l p r o d u c t s , the i n t e r m e d i a t e s t e p s t h a t c o n s t i t u t e the a r e a
o f "downstream p r o c e s s i n g . "
I t i s t h i s l a s t area that i s proving t o
r e q u i r e the g r e a t e s t e f f o r t i n p r a c t i c e and t h a t h a s the p o o r e s t base
o f f u n d a m e n t a l e n g i n e e r i n g u n d e r s t a n d i n g on w h i c h t o draw.
The t o p i c o f t h i s p a p e r i s t h e m o d e l i n g o f e v e n t s o c c u r r i n g i n
the r e c o v e r y o f p r o t e i n s and i nthe c o n d i t i o n i n g o f the product
streams f o r f u r t h e r p u r i f i c a t i o n u s i n g p r e c i p i t a t i o n .
The t y p i c a l
g o a l o f downstream p r o c e s s i n g i s the r e c o v e r y o f a d e s i r e d p r o d u c t
from a very d i l u t e stream w h i l e m i n i m i z i n g the l o s s o f the m a t e r i a l
i n what i s u s u a l l y a m u l t i - s t e p s e p a r a t i o n p r o c e s s .
Precipitation
enables an e a r l y c o n c e n t r a t i o n o f the p r o d u c t and c a n s i m u l t a n e o u s l y
s e r v e t o remove c o n t a m i n a n t s t h a t would i n t e r f e r e w i t h s u b s e q u e n t
purification steps.
F u r t h e r , the wide v a r i e t y o f p o t e n t i a l
0097-6156/86/0314-0109$06.00/0

110
p r e c i p i t a t i n g a g e n t s t h a t e x i s t s p e r m i t s s e l e c t i o n o f the p a r t i c u l a r
agent c a p a b l e o f r e c o v e r i n g the t a r g e t s p e c i e s under c o n d i t i o n s where
a c t i v i t y i s retained.
The t a r g e t s p e c i e s c o n s i d e r e d here a r e p r o t e i n s , and the
p r i n c i p l e s d e v e l o p e d may be a p p l i e d t o any p r o t e i n - c o n t a i n i n g aqueous
stream, i n c l u d i n g f e r m e n t a t i o n b r o t h s , p l a n t e x t r a c t s , and waste
streams, whether the m a t e r i a l i s d e s t i n e d f o r f o o d , p h a r m a c e u t i c a l ,
or c h e m i c a l a p p l i c a t i o n .

Protein
Precipitation
The s t a b i l i t y o f p r o t e i n s i n s o l u t i o n i s d e t e r m i n e d by a number o f
f a c t o r s t h a t govern p r o t e i n - p r o t e i n , p r o t e i n - s o l v e n t , and
solvent-solvent interactions.
S u f f i c i e n t a l t e r a t i o n o f any o f t h e s e
i n t e r a c t i o n s can r e s u l t i n d r a m a t i c r e d u c t i o n s i n s o l u b i l i t y .
Hence,
p r o t e i n s can be p r e c i p i t a t e d by a v a r i e t y o f agents i n c l u d i n g o r g a n i c
s o l v e n t s , d i v a l e n t c a t i o n s , h e a t , a c i d s / b a s e s (pH a d j u s t m e n t ) , s a l t s ,
n o n i o n i c polymers ( e g . p o l y e t h y l e n e g l y c o l ) , and p o l y e l e c t r o l y t e s .
These means o f a l t e r i n g s o l u b i l i t y have been known and used f o r some
time.
What had been l a c k i n g was a d e s c r i p t i o n o f the mechanism o f
f o r m a t i o n o f the p a r t i c u l a t e phase, the e n v i r o n m e n t a l d e t e r m i n a n t s o f
the c h a r a c t e r i s t i c s o f t h i s phase, and the c o n n e c t i o n between t h e s e
c h a r a c t e r i s t i c s , p a r t i c u l a t e b e h a v i o r i n the subsequent p u r i f i c a t i o n
s t e p s , and r e t e n t i o n o f f u n c t i o n a l a c t i v i t y .
There was, t h e r e f o r e ,
l i t t l e knowledge on which t o base d e s i g n o f the p r e c i p i t a t i o n s t a g e
so t h a t the p r e c i p i t a t e would be e a s i l y r e c o v e r e d , the maximum amount
of p r o t e i n would be i n i t s n a t i v e or a c t i v e s t a t e , and as many
c o n t a m i n a n t s as p o s s i b l e would be removed.
Recent r e s e a r c h , the b u l k o f which has been g a t h e r e d from s t u d y
o f the i s o e l e c t r i c p r e c i p i t a t i o n o f soy p r o t e i n , has p r o v i d e d a good
deal of t h i s missing i n f o r m a t i o n .
Grabenbauer and G l a t z (l_) and V i r k a r e t a l . (2) have shown t h a t
p r e c i p i t a t i o n p r o c e e d s by an i n i t i a l r a p i d f o r m a t i o n o f submicron
p r i m a r y p a r t i c l e s f o l l o w e d by c o l l i s i o n - c o n t r o l l e d a g g r e g a t i o n of
these p r i m a r y p a r t i c l e s .
The l a t t e r growth phase i s c o m p l i c a t e d by
the s i m u l t a n e o u s s h e a r - c o n t r o l l e d breakup o f the a g g r e g a t e s .
We w i l l
examine each s t e p i n t u r n , i n c l u d i n g m o d e l i n g approaches f o r each.
Primary P a r t i c l e Formation.
The i n i t i a l s t a g e o f p r i m a r y p a r t i c l e
f o r m a t i o n had been o b s e r v e d by P a r k e r and D a l g l e i s h (3) f o r
enzymatically destabilized casein.
They used l i g h t s c a t t e r i n g and
t u r b i d i t y measurements to f o l l o w the weight-average m o l e c u l a r weight
(M ) o f the a s s o c i a t i n g c a s e i n p a r t i c l e s .
A f t e r an i n i t i a l p e r i o d o f
a c c e l e r a t i n g r a t e , the k i n e t i c b e h a v i o r c o u l d be d e s c r i b e d by von
Smoluchowski s t h e o r y o f p e r i k i n e t i c c o a g u l a t i o n . The r e s u l t i n
terms o f
was
w
1^ = M
+ 2 wkt
(1)
where M
i s the m o l e c u l e weight o f the i n d i v i d u a l p r o t e i n s , k i s the
c o a g u l a t i o n r a t e c o n s t a n t , w i s the c o n c e n t r a t i o n (weight b a s i s ) o f
p r o t e i n , and t i s t i m e .
N e l s o n and G l a t z (4)
examined the r o l e o f e n v i r o n m e n t a l
c o n d i t i o n s i n d e t e r m i n i n g the s i z e and number o f these p r i m a r y
particles.
They found s i z e t o depend on the p r e c i p i t a t i n g agent
Q

9.
111
Modeling of Precipitation Phenomena
GLATZ A N D FISHER
(HCl, H 2 S O 4 , or C a ) and t o t a l protein concentration, but not on

mixing conditions. Their conclusion was that primary p a r t i c l e growth
i s governed by supersaturation-controlled nucleation/growth
phenomena
This mechanism f o r the formation of primary p a r t i c l e s can be
described using Nielsen's (5) expressions f o r homogeneous nucleation
with d i f f u s i o n - c o n t r o l l e d growth i n p r e c i p i t a t i o n . In h i s
discussion, the nucleation rate, J ( c ) , i s expressed as a power-law
function of supersaturation, c,
2+

J(c) =
(2)
where 1^ i s the nucleation rate constant and m i s the nucleation

power constant.
The number of primary p a r t i c l e s per unit volume, N>|,
formed i n a batch p r e c i p i t a t i o n can be calculated as
CO
CO
N-, = '/ J(c) dt = k

0
/c
dt
(3)
Supersaturation, c, may be expressed as a function of i n i t i a l

supersaturation, c ,
Q
c = (1 - a ) c
(4)
where a i s the f r a c t i o n of supersaturated protein that has been

precipitated. Combining a d i f f u s i o n - c o n t r o l l e d growth-rate
expression with the assumption of spherical primary p a r t i c l e s gives
the volumetric growth rate of formed p a r t i c l e s as f i r s t order i n
supersaturation
-3
dt
= 4 rev
m
, \
(5)
c
where V i s the p a r t i c l e volume, D i s the protein d i f f u s i v i t y , i s

the protein molecular volume, and r i s the molecular radius. Solving
Equations 3-5 together with an overall mass balance, Nielsen obtained
the approximate r e s u l t
m
where C i s a weak function of m, approximately equal to one.

Hence th stronger the dependence of nucleation on supersaturation,
the greater w i l l be the increase i n number of primary p a r t i c l e s as
i n i t i a l supersaturation increases. For (3m-l)/5 > 1 ( i . e . m > 2),
the size of those p a r t i c l e s w i l l decrease with i n i t i a l
supersaturation. No dependence on mixing conditions appears; the
concentration dependence f o r soy protein p r e c i p i t a t e s (via
hydrochloric acid addition) was found (4) to be
m
= 2.67 x 1 0
1 1
c - 4
o
(7)
requiring m = 1.7. Over the range of concentrations studied (0.15 -

112

30 kg/m3) the size of primary p a r t i c l e s increased from 0.16 to

0.27 Um.
Aggregate growth and breakup. The primary p a r t i c l e s (produced as

described above) form the starting material f o r the aggregation
stage. This stage p a r a l l e l s what occurs i n other
aggregation/coagulation/flocculation systems. I t d i f f e r s from many
of these however, i n that the aggregates are p a r t i c u l a r l y prone to
breakup and their size i s smaller than the Kolmogorov microscale of
turbulence, subjecting them to d i f f e r e n t c o n t r o l l i n g f l u i d forces
during growth and breakup (6^). Since p a r t i c l e size i s one of the
determinants of the e f f i c i e n c y of s o l i d - l i q u i d separations
( f i l t r a t i o n rate and s e t t l i n g v e l o c i t y are both proportional to the
square of p a r t i c l e diameter (7)), the modeling and characterization
of the p a r t i c l e size d i s t r i b u t i o n s i s important.
Population balances were combined with the proposed mechanisms
to model the size d i s t r i b u t i o n s from continuous s t i r r e d
p r e c i p i t a t o r s . The postulated f a i l u r e of c o l l i s i o n s between larger
aggregates to form lasting agglomerates reduces the growth process to
one where only primary p a r t i c l e s and small aggregates can serve as
growth units, though larger sizes may serve as c o l l e c t o r s . Modelled
in t h i s way, growth becomes a continuous process. P a r t i c l e size
d i s t r i b u t i o n s have been successfully modelled over a wide range of
conditions f o r continuous stirred-tank precipitators ( 1_, 6^ 8).
Models of the p a r t i c l e size d i s t r i b u t i o n
Asssumptions. The mathematical models based on the population
balance incorporate the following physical features and simplifying
assumptions :
1.
Protein comes out of solution very quickly and therefore an

accounting i s needed only f o r the s o l i d material. This i s
supported by tubular reactor studies (2, 9) where p r e c i p i t a t i o n ,
in terms of removal of soluble protein, i s completed within 1 s
for the protein concentrations above 2 kg/m^.
2.
Growth of an aggregate occurs by c o l l i s i o n with primary p a r t i c l e s

and smaller aggregates. However, c o l l i s i o n s between larger
aggregates are i n e f f e c t i v e i n forming lasting aggregates.
Gregory (10) has shown that c o l l i s i o n e f f i c i e n c y decreases
considerably with increasing size of equal-sized c o l l i d i n g
species. In addition, aggregate-aggregate attachments would be
r e l a t i v e l y weak as the result of the lower bond densities at
these points compared to bond densities within aggregates. A
r a t i o of 10 to 1 has been reported as t y p i c a l (11) f o r the r a t i o
of contacts within an established aggregate to contacts between
two such aggregates. Growth i s therefore viewed as the
incremental addition of small units to the growing aggregates.
3.
The effectiveness of these c o l l i s i o n s of small p a r t i c l e s with

growing aggregates i s independent of the size of the growing
species.

113
9. G L A T Z A N D FISHER
Population Balances.
Three d i f f e r e n t models based on two
a p p r o x i m a t i o n s r e g a r d i n g t h e mode o f b r e a k a g e a n d t w o a p p r o x i m a t i o n s
r e g a r d i n g t h e s i z e d e p e n d e n c e o f g r o w t h r a t e h a v e b e e n e x a m i n e d . The
d i f f e r e n t i a l e q u a t i o n s f o r m o d e l i n g the s i z e d i s t r i b u t i o n are based
on a p o p u l a t i o n b a l a n c e o n a g g r e g a t e s o f s i z e L w h i c h , f o r a CSTR a t
s t e a d y s t a t e , mean r e s i d e n c e t i m e , a n d w i t h n o p a r t i c l e s i n t h e
feed, reduces t o
^ i

= 0
+ ^ + D - B
at
(8)
w h e r e i s t h e n u m b e r d e n s i t y o f p a r t i c l e s , i s t h e r e a c t o r mean
r e s i d e n c e t i m e , a n d w h e r e G, D, a n d a r e t h e r a t e s f o r d i f f e r e n t i a l
growth, v o l u m e t r i c death by breakup, andv o l u m e t r i c b i r t h by breakage
o f l a r g e r p a r t i c l e s , r e s p e c t i v e l y ; n , G, D, a n d may b e f u n c t i o n s o f
L.
The f i r s t m o d e l i n c o r p o r a t e s e x p r e s s i o n s f o r e a c h o f t h e s e t e r m s
(6), i n which growth i s approximated as l i n e a r w i t h aggregate s i z e ;
G = A<j>,,v L = K L
g
(9)
where A i s a c o n s t a n t i n c o r p o r a t i n g c o l l i s i o n e f f e c t i v e n e s s , ^ i s
the volume f r a c t i o n o f p r i m a r y p a r t i c l e s , V g i s t h e r o o t - m e a n - s q u a r e
v e l o c i t y g r a d i e n t , andK i s the product o f these t h r e e , c a l l e d the
growth rate c o n s t a n t .
Breakage i s d e s c r i b e d b y
Q
D = k'V ( ^ g ) n= k L n
(10)
tfya
where k a n d k a r e d e a t h - r a t e c o n s t a n t s , y i s the s o l u t i o n v i s c o s i t y ,
o
i s the a g g r e g a t e y i e l d s t r e s s , a n d 6 a n d a r e b r e a k a g e power
constants.
Breakup r e q u i r e s terras a c c o u n t i n g f o r the sudden d i s a p p e a r a n c e
(death) o f parent aggregates and c o r r e s p o n d i n g appearance ( b i r t h ) o f
daughter fragments.
The f i r s t a n d s e c o n d m o d e l s assume t h a t
a g g r e g a t e s b r e a k up t o f o r m a s m a l l n u m b e r o f d a u g h t e r f r a g m e n t s o f
s i g n i f i c a n t mass.
The number o f d a u g h t e r f r a g m e n t s w o u l d t e n d t o be
greater f o r l a r g e r parent aggregates; t h i s i s approximated as an
a v e r a g e f r a g m e n t n u m b e r , f , d e p e n d e n t o n t h e mean s i z e o f t h e
distribution.
Daughter f r a g m e n t s from a g i v e n p a r e n t a r e assumed t o
be o f e q u a l v o l u m e .
This gives
v a
= fD(f /3L)
The
resulting
dn
(11)
e q u a t i o n r e l a t i n g number d e n s i t y
B - l
/ 3 > + l
l / 3
to size i s
S u m m a r i z i n g , t h e m o d e l p a r a m e t e r s a r e K , k, 3, a n d f . T h e
d e a t h and b i r t h e x p r e s s i o n s assume b r e a k a g e i n t o e q u a l - s i z e d
Q

1 2
114
fragments such t h a t t o t a l p a r t i c l e volume i s c o n s e r v e d and t h a t a

power law d e s c r i b e s the i n c r e a s e d s u s c e p t i b i l i t y o f a g g r e g a t e s t o
breakup as s i z e i n c r e a s e s .
The growth term assumes t h a t c o l l i s i o n
due t o the s p a t i a l v a r i a t i o n o f t u r b u l e n c e i s the predominant f a c t o r ,
as has been demonstated ( e . g . ( 1 2 ) ) .
The second model c o n s i d e r e d here (1_) r e s u l t s from assuming
t h a t the growth r a t e , G , i s independent o f s i z e , g i v i n g
m
dn
.K_ P
L
( f
(e/3)+l
l / 3

The boundary c o n d i t i o n used f o r E q u a t i o n s 12 and 13 i s t h a t t h e

c a l c u l a t e d t o t a l volume o f a g g r e g a t e s e q u a l s the measured aggregate
volume.
Parameters.
P a s t work has s u g g e s t e d t h a t f o r a g i v e n p r o t e i n
c o n c e n t r a t i o n , 3 and f c o u l d be f i x e d a t r e a s o n a b l e v a l u e s , l e a v i n g
o n l y two f u l l y v a r i a b l e p a r a m e t e r s .
The method o f s o l u t i o n i s
d i s c u s s e d elsewhere ().
F i g u r e 1, from d a t a o f Brown and G l a t z
(13),
shows t h a t breakup o f l a r g e a g g r e g a t e s r e s u l t s i n two o r more
main fragments and a number o f s m a l l fragments c o m p r i s i n g r e l a t i v e l y
l i t t l e mass. The l a t t e r can be n e g l e c t e d i n the b a l a n c e , a l t h o u g h
t h e y w i l l s e r v e t o i n c r e a s e -j as t h e y a r e c o n s i d e r e d c a p a b l e o f
b e i n g growth u n i t s .
The l a r g e r a g g r e g a t e s a r e e x p e c t e d t o form the
g r e a t e r number o f daughter f r a g m e n t s .
However, Pandya and S p i e l m a n
(13) f o u n d t h a t a l l o w i n g f o r t h i s w i t h i n a g i v e n d i s t r i b u t i o n o f
kaolin-Fe(OH)-* f l o e s was no b e t t e r than u s i n g a c o n s t a n t f = 2.5.
The model based on a growth r a t e i n d e p e n d e n t o f a g g r e g a t e s i z e ,
E q u a t i o n 13, gave r e a s o n a b l e f i t s by r e s i d u a l sum o f s q u a r e s c r i t e r i a
except a t high p r o t e i n c o n c e n t r a t i o n s .
However, a t a l l p r o t e i n
c o n c e n t r a t i o n s s t u d i e d , the p r e d i c t e d c u r v e s were b i a s e d i n t h e
manner i n which t h e y d e v i a t e d from e x p e r i m e n t a l o b s e r v a t i o n .
This
was p a r t i c u l a r l y e v i d e n t a t s m a l l s i z e s where the l o c a l
minimum/maximum t r a i t s were l a r g e l y l o s t .
The f i r s t model, based on
a growth r a t e l i n e a r i n a g g r e g a t e s i z e , E q u a t i o n 12, gave
s a t i s f a c t o r y f i t s o f the p a r t i c l e s i z e d a t a ( i n f a c t , f o r most runs
the model f i t the e x p e r i m e n t a l p o i n t s more c l o s e l y t h a n d i d the
s i x - p a r a m e t e r Chebyshev p o l y n o m i a l on which the model f i t t i n g was
a c t u a l l y based) as w e l l as s u c c e s s f u l l y d e s c r i b i n g the l o c a l
minimum/maximum t r a i t s .
The c u r v e s p r e s e n t e d i n F i g u r e 2 a r e based
on t h i s model, u s i n g the d a t a o f G l a t z e t . a l . (0) who d i s c u s s the
b e h a v i o r o f model parameters k and K a t d i f f e r e n t r e a c t o r
conditions.
The t h i r d and f i n a l p a r t i c l e s i z e d i s t r i b u t i o n model assumes
t h a t growth i s l i n e a r as i n the f i r s t b u t t h a t breakup r e s u l t s i n
p r e d o m i n a n t l y s m a l l p a r t i c l e s ( t h o r o u g h breakage) which a r e t o o s m a l l
to measure by the e l e c t r o n i c p a r t i c l e c o u n t e r s used t o c h a r a c t e r i z e
the s u s p e n s i o n . P e t e n a t e and G l a t z (6) have p r o v i d e d a n a l y t i c a l
s o l u t i o n s f o r t h i s model.
The f o c u s o f the above m o d e l i n g has been on c o n t i n u o u s
stirred-tank reactors.
The g e n e r a l p r i n c i p l e s have been extended t o
i n t e r p r e t r e s u l t s from b a t c h and t u b u l a r r e a c t o r s , as w e l l , though
d e t a i l e d m o d e l i n g has n o t y e t been attempted
(8).
Q


9.
115
G L A T Z A N D FISHER
AGGREGATE SIZE, ym
F i g u r e 1. P l o t o f c h a n g e i n a g g r e g a t e v o l u m e v s . a g g r e g a t e s i z e
f o r given time i n t e r v a l during breakup o f i s o e l e c t r i c a l l y p r e c i p
i t a t e d soy p r o t e i n .
P a r t i c l e volume f r a c t i o n , 0.00531.
Shear
r a t e , 1010 s " .
1
71
0
10
15
20
I
25
SIZE,
F i g u r e 2. P a r t i c l e ( n u m b e r ) s i z e d i s t r i b u t i o n s f o r i s o e l e c t r i
c a l l y p r e c i p i t a t e d soy p r o t e i n showing t h e e f f e c t s o f shear r a t e
and p r o t e i n c o n c e n t r a t i o n .
Points are experimental data; curves
a r e t h e m o d e l f i t u s i n g E q u a t i o n 12.
S h e a r r a t e s : , 417 - l ;
V , 108 s " ; , 8 5 s " .
s

116

P r e v i o u s a t t e m p t s h a v e b e e n made t o m o d e l s i z e d i s t r i b u t i o n s
a l l o w i n g f o r growth by c o l l i s i o n s o fa l l aggregate-aggregate
combinations.
S u c h m o d e l s (2, 14) p r e d i c t e d much h i g h e r g r o w t h r a t e s
t h a n were o b s e r v e d , o f f e r i n g f u r t h e r e v i d e n c e f o r t h e i n e f f e c t i v e n e s s
of a g g r e g a t e - a g g r e g a t e c o l l i s i o n s .
The m o d e l i n g e q u a t i o n s , when a l l p a r t i c l e - p a r t i c l e c o l l i s i o n s
are assumed e f f e c t i v e , a r e r e p r e s e n t e d ( i n a b a t c h r e a c t o r ) b y
(14)
w h e r e N, t h e n u m b e r c o n c e n t r a t i o n o f p a r t i c l e s , a n d r , t h e p a r t i c l e
r a d i i , a r e s p e c i f i e d f o r p a r t i c l e s i z e s i , j , and k such t h a t r ^=
k
j T h i s e q u a t i o n r e p l a c e s E q u a t i o n 8, b u t d o e s n o t e x p l i c i t l y
account f o r aggregate breakage.
V i r k a r (2) i n t r o d u c e d b r e a k a g e t o
t h i s balance b y n o t a l l o w i n g c o l l i s i o n s t h a t would r e s u l t i n a
p a r t i c l e l a r g e r t h a n a f i x e d maximum s i z e .
Breakage Models.
We a r e c o n t i n u i n g o u r s t u d y o f s t i r r e d t a n k
behavior o f i s o e l e c t r i c p r e c i p i t a t e s by examining the breakup
p h e n o m e n a a n d t h e m o d e l i n g e q u a t i o n f o r b r e a k u p i n more d e t a i l .
Data
are i n t e r p r e t e d i n t h e l i g h t o f t h r e e p r o p o s e d t r e a t m e n t s o f b r e a k u p
(15) Two a r e b a s e d o n b r e a k u p u n d e r f l u i d s h e a r , u s i n g t h e c o n c e p t s
o f a maximum s t a b l e s i z e ( 1 6 - 1 8 ) a n d s i m i l a r i t y (19-20).
The t h i r d
i s based on c o l l i s i o n a l breakage which has been d i s c u s s e d b u t n o t
o b s e r v e d b y Glasgow and Luecke (21), and t h o u g h t t o o c c u r w i t h
p r o t e i n a g g r e g a t e s i n l a m i n a r s h e a r (22).
O t h e r Prcipitants. E x t e n s i o n a n d m o d i f i c a t i o n o f t h e s e m o d e l i n g
e f f o r t s w i l l be r e q u i r e d f o r t h e i r a p p l i c a t i o n t o p r e c i p i t a t i o n s
other than i s o e l e c t r i c .
O t h e r l o w m o l e c u l a r w e i g h t prcipitants h a v e
b e e n s h o w n t o r e s u l t i n t h e same s o r t o f a g g r e g a t e m o r p h o l o g y
(Ca ;
(4)) a n d g r o w t h k i n e t i c s ( e t h a n o l , C a , ( N H ^ ^ S O ^ ; 0 9 ) ) . F o r t h e s e
prcipitants n o m o d i f i c a t i o n s h o u l d b e n e c e s s a r y , a l t h o u g h t h e
dependence o f aggregate s t r e n g t h on t h e p h y s i c o c h e m i c a l c o n d i t i o n s
w i l l change and w i t h i t t h e s t r e n g t h - d e p e n d e n t model p a r a m e t e r s .
B a s e d o n v e r y p r e l i m i n a r y r e s u l t s (23) t h e b e h a v i o r o f p o l y e t h y l e n e
g l y c o l i s a l s o e x p e c t e d t o be q u a n t i t a t i v e l y s i m i l a r .
2 +
2 +
Precipitate
Behavior
Beyond d e s c r i b i n g what i s o c c u r r i n g i n t h e p r e c i p i t a t i o n s t e p i t s e l f ,
work h a s b e e n done i n r e l a t i n g t h e c h a r a c t e r i s t i c s o f t h e m a t e r i a l
l e a v i n g the p r e c i p i t a t o r t o i t s b e h a v i o r i n subsequent o p e r a t i o n s .
The i m p o r t a n c e o f a g g r e g a t e " a g i n g " t o c o n d i t i o n t h e a g g r e g a t e s t o
r e s i s t b r e a k u p d u r i n g s h e a r e n c o u n t e r e d i n pumps a n d c e n t r i f u g e s i s
d o c u m e n t e d (24-25) The i n f l u e n c e o f p r e p a r a t i o n c o n d i t i o n s o n


9.
117
c e n t r i f u g e c a p a c i t y a n d s l u d g e r h e o l o g y h a s b e e n o b s e r v e d (26), a n
e x p l a n a t i o n f o r a g g r e g a t e s t r e n g t h and r h e o l o g i c a l p r o p e r t i e s
p r o p o s e d (4) i n t e r m s o f a n e l a s t i c f l o e m o d e l ( 1 1 ) , a n d t h e
p r e p a r a t i o n - ( 2 7 ) a n d a g i n g - (14) d e p e n d e n t s e t t l i n g b e h a v i o r
reported.
The w o r k o f F i s h e r e t a l . ( 2 7 ) i l l u s t r a t e s some d e s i g n
c o n s i d e r a t i o n s beyond t h a t o f the p a r t i c l e s i z e a t the p r e c i p i t a t i o n
outlet.
These w o r k e r s were c o n c e r n e d w i t h the m i x i n g c o n d i t i o n s
d u r i n g the a d d i t i o n o f a c i d t o the p r o t e i n s o l u t i o n . L o c a l extremes
i n pH a r e known t o c a u s e i r r e v e r s i b l e d e n a t u r a t i o n o f p r o t e i n s
(28)
which w i l l a l t e r t h e i r p r e c i p i t a t i o n behavior.
F u r t h e r , H o a r e (297
has r e p o r t e d t h a t the p r e c i p i t a t e p r o p e r t i e s d i f f e r w i t h e x t r e m e s i n
operating conditions.
One e x t r e m e e x p l o i t e d i n i n o r g a n i c
p r e c i p i t a t i o n s i s h o m o g e n e o u s p r e c i p i t a t i o n (30), w h i c h i n v o l v e s t h e
homogeneous p r o d u c t i o n o f t h e p r e c i p i t a n t , u s u a l l y b y a c o n t r o l l e d
c h e m i c a l r e a c t i o n , w i t h i n the s o l u t i o n . Advantages o f t h i s i n c l u d e
the p r o d u c t i o n o f denser p r e c i p i t a t e and reduced c o p r e c i p i t a t i o n .
F i s h e r e t a l . (27) a p p r o x i m a t e d homogeneous i s o e l e c t r i c p r e c i p i t a t i o n
by the a d d i t i o n o f a c i d from a d i a l y s i s b a g suspended i n the p r o t e i n
s o l u t i o n on a r o t a t i n g s h a f t .
The d e g r e e o f f r a c t i o n a t i o n o f t w o
p r o t e i n s ( g l y c i n i n , p i = 6.0, a n d 3 - c o n g l y c i n i n , p i = 4$) from a
t o t a l soy e x t r a c t and the p h y s i c a l c h a r a c t e r i s t i c s o f the
p r e c i p i t a t e s w e r e c o n t r a s t e d w i t h t h e same p r o p e r t i e s o f p r e c i p a t e s
f o r m e d d u r i n g r a p i d a c i d a d d i t i o n . P r e c i p i t a t e f r a c t i o n s were t a k e n
a t p H 6.0 a n d
4.8.
The c o m p o s i t i o n s o f t h e f r a c t i o n s , T a b l e I , i n d i c a t e t h a t
s e p a r a t i o n o f the g l y c i n i n and 3 - c o n g l y c i n i n d i d occur,
with
s u b s t a n t i a l e n r i c h m e n t o f t h e g l y c i n i n p h a s e i n t h e p H 6.0 f r a c t i o n .
T a b l e I a l s o shows t h a t no d i f f e r e n c e s i n the f r a c t i o n a t i o n o f
g l y c i n i n o r 3 - c o n g l y c i n i n can be a t t r i b u t e d t o t h e m i x i n g d u r i n g a c i d
addition.
I n h i n d e r e d s e t t l i n g t e s t s o n t h e pH 4 8 p r o d u c t t h e a g g r e g a t e
prepared by slow a c i d a d d i t i o n c l e a r l y s e t t l e d f a s t e r than that
prepared by rapid a c i d a d d i t i o n .
Since aggregate s i z e and d e n s i t y ,
as measured p r i o r t o s e t t l i n g , c o u l d n o t a c c o u n t f o r the d i f f e r e n t
s e t t l i n g r a t e s , a n o t h e r e x p l a n a t i o n was s o u g h t .
I t was c o n c l u d e d
t h a t the c o n t r o l l i n g c h a r a c t e r i s t i c o f the hindered s e t t l i n g i s the
a g g r e g a t e ' s a b i l i t y t o a g g r e g a t e f u r t h e r a n d become l a r g e e n o u g h t o
settle.
The c h a r a c t e r i s t i c s o f t h e i s o e l e c t r i c a l l y p r e c i p i t a t e d
a g g r e g a t e s were i n t e r p r e t e d i n l i g h t o f a model o f f l o e s t e n g t h a s
d e v e l o p e d b y F i r t h a n d H u n t e r (11) a n d a p p l i e d b y N e l s o n a n d G l a t z
(4)
T h i s model h o l d s t h a t the s t r e n g t h , a , o f an a g g r e g a t e o f
p r i m a r y p a r t i c l e s i s a p r o d u c t o f the number o f b o n d s p e r a r e a a n d
the a t t r a c t i v e f o r c e per bond.
They f u r t h e r showed t h a t
y a
ya
Q<f>
dT
(15)
w h e r e Q i s a n i n t e r a c t i o n p o t e n t i a l f u n c t i o n t h e sum o f
c h a r g e - c h a r g e r e p u l s i v e a n d van der Waals a t t r a c t i v e
c o n t r i b u t i o n s a n d w h e r e d>j i s t h e d i a m e t e r o f t h e p r i m a r y p a r t i c l e .
The a p p l i c a t i o n o f t h i s m o d e l h e l p e d t o i d e n t i f y o r i e n t a t i o n o f t h e
p r o t e i n d u r i n g i t s i n c o r p o r a t i o n i n t o the p r i m a r y p a r t i c l e a s a
d e t e r m i n i n g s t e p i n the subsequent s t r e n g t h o f the a g g r e g a t e .

118
T a b l e I . P h y s i c a l and C o m p o s i t i o n a l P r o p e r t i e s o f t h e P r o t e i n
E x t r a c t and t h e R a p i d and Slow P r o t e i n P r e c i p i t a t e s
Fraction

Property
protein
a
extract
pH
Total
% glycinin
in fraction
27.6
%3-conglycinin
in fraction^
16.7
Slow
8.1
4.0
4.1
27.4
21.8
0.13
0.19
hindered s e t t l i n g
time (min)f
Determined
by b i u r e t
Slow
10.0
(ym)
5 Q
4.8
Rapid
52.-6
49.3
(ym)
d
Rapid
pH
6.0
colorimetric
0.51
0.36
11.31
7.88
17
assay.
Îmmunologically a c t i v e p r o t e i n expressed as percent of

t o t a l p r o t e i n , determined by r o c k e t - g e l
immunoelectrophoresis.
P r i m a r y p a r t i c l e d i a m e t e r , measured from
micrographs of the aggregate.
scanning
electron
^Mean a g g r e g a t e d i a m e t e r ( o n a v o l u m e b a s i s ) , d e t e r m i n e d
particle size distribution.
from
T h e pH 6.0 a g g r e g a t e s
size distribution.
from
Time f o r i n t e r f a c e t o drop
T h e pH 6.0 a g g r e g a t e s
tling test.
w e r e t o o weak t o b e c h a r a c t e r i z e d
t o 30% of i n i t i a l
slurry height.
d i dnot s e t t l e i n the hindered

set-
9.
119
Conclusion

Models f o r continuous, s t i r r e d p r e c i p i t a t i o n behavior are a t a

r e a s o n a b l y s u c c e s s f u l s t a g e , t h o u g h t h e e x p r e s s i o n f o r b r e a k u p c a n be
improved i n t h e l i g h t o f r e c e n t s t u d i e s .
Batch and t u b u l a r r e a c t o r
models must a d d i t i o n a l l y i n c l u d e an e x p l i c i t a c c o u n t i n g f o r p r i m a r y
p a r t i c l e formation and behavior o f small p a r t i c l e s .
Some o f t h e d a t a
n e c e s s a r y t o do s o h a v e b e e n c o l l e c t e d , b u t n u m b e r d e n s i t y d a t a a t
the s m a l l s i z e s i s s t i l l l a c k i n g .
F i n a l l y i t r e m a i n s t o b e s e e n how
s u c c e s s f u l t h e m o d e l s d e v e l o p e d f o r i s o e l e c t r i c p r e c i p i t a t i o n w i l l be
i n d e s c r i b i n g p r e c i p i t a t i o n w i t h o t h e r c l a s s e s o f prcipitants.
Acknowledgments
T h i s work was s u p p o r t e d b y t h e E n g i n e e r i n g R e s e a r c h I n s t i t u t e o f Iowa
S t a t e U n i v e r s i t y t h r o u g h N a t i o n a l S c i e n c e F o u n d a t i o n G r a n t No.
CPE-8120568.
Literature Cited
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Grabenbauer, G. C.; Glatz, C. E. Chem. Eng. Comm. 1981, 12, 203219.

Virkar, P. D.; Chan, M. Y. Y.; Hoare, M.; Dunnill, P. Biotechnol.
Bioeng. 1982, 24, 871-887.
Parker, T. G.; Dalgleish, D. G. Biopolymers 1977, 16, 2533-2547.
Nelson, C. D.; Glatz, C. E. Biotechnol. Bioeng. 1985, 27, 14341444.
Nielson, A. E. "Kinetics of Precipitation"; Macmillan Co.:
New York, 1964, Chap. 7.
Petenate, A. M.; Glatz, C. E. Biotechnol. Bioeng. 1983, 25,
3059-3078.
McCabe, W. L.; Smith, J. C. "Unit Operations of Chemical Engineering, 3rd Edition"; McGraw-Hill, Inc.:New York, 1976; p. 938971.
Glatz, C. E.; Hoare, M.; Landa-Vertiz, J. AIChE J. 1986, in
press.
Chan, M. Y. Y.; Hoare, M.; Dunnill, P. Biotechnol. Bioeng., 1985,
accepted for publication.
Gregory, J. Chem. Eng. Sci. 1981, 36, 1789-1794.
Firth, . .; Hunter, R. J. J. Colloid Interface Sci., 1976, 57,
266-275.
Yuu, S. AIChE J. 1984, 30, 802-807.
Pandya, J. D.; Spielman, L. A. J. Colloid Interface Sci. 1982,
90, 517-532.
Hoare, M. Trans. Inst. Chem. Eng. 1982, 60, 157-163.
Brown, D. L.; Glatz, C. E. Paper presented at the AIChE Annual
Meeting 1985, Chicago.
Tomi, D. T.; Bagster, D. F. Trans. Inst. Chem. Eng. 1978, 56,
1-8.
Parker, D. S.; Kaufman, W. J.; Jenkins, D. J. J. Sanit Eng. Div.
ASCE 1982, 98, 79-99.
Tambo, N.; Hozumi, H. Water Res. 1979, 13, 421-427.
Ramkrishna, D. Chem. Eng. Sci. 1974, 29, 987-992.

120
20.
21.
22.

23.
24.
25.
26.
27.
28.
29.
30.
Narsimhan, G.; Ramkrishna, D.; Gupta, J. P. AIChE J. 1980, 26,

991-1000.
Glasgow, L. .; Luecke, R. H. Ind. Eng. Chem. Fundam. 1980, 19,
148-156.
Twineham, M.; Hoare, M.; Bell, D. J. Chem. Eng. Sci. 1984, 39,
509-513.
Glatz, C. ., unpublished data.
Bell, D. J., Dunnill, P. Biotechnol. Bioeng. 1982, 24, 1271-1285.
Hoare, M.; Narendranathan, T. J.; Flint, J. R.; Heywood
-Waddington, D.; Bell, D. J.; Dunnill, P. Ind. Eng. Chem. Fundam.
1982, 21, 402-406.
Bell, D. J.; Dunnill, P. Biotechnol. Bioeng. 1982, 24, 23192336.
Fisher, R. F.; Glatz, C. E.; Murphy, P. A. Biotechnol. Bioeng.
1985, accepted for publication.
Salt, D. J.; Leslie, R. B.; Lillford, P. J.; Dunnill, P. Eur. J.
Appl. Microbiol. Biotechnol. 1982, 14, 144-148.
Hoare, M. Trans. Inst. Chem. Eng. 1982, 60, 79-87.
Berg, E. W. "Physical and Chemical Methods of Separation";
McGraw-Hill, Inc.:New York, 1963; pp. 279-284.

10
Process Considerations for Scale-up of Liquid
Chromatography and Electrophoresis

S. R. Rudge and M . R. Ladisch

Laboratory of Renewable Resources Engineering, School of Chemical Engineering, and
Department of Agricultural Engineering, Purdue University, West Lafayette, IN 47907
Chromatography is an important preparative and industrial

process. Scale-up of chromatographic processes requires computation of mass transfer characteristics as a function of
column area, support particle size and feed volume. In this
context, an analytical solution for longitudinal diffusion in
packed beds, developed by Lapidus and Amundson, is used to
demonstrate the characteristics of a typical size exclusion
separation of proteins, including estimation of maximum sample size as allowed by support properties. Electrophoresis is
also a powerful fractionation technique for proteins, but is subject to many microscopic effects. These include electric double
layers, hydrodynamic drag, and electrical relaxation. In addition, macroscopic effects, such as electroosmosis and thermal
gradients, also impact separation efficiency. These effects are
discussed in relation to elution processes using selected examples. The combination of an electric field with a chromatographic process has recently been proposed to extend the
power of electrophoresis separations. Analysis of such a process, referred to as electrochromatography, is also presented.
Mixtures can be separated by taking advantage of a physical property which
varies among the mixture's components. Such properties include boiling
points, equilibria with other substances, solubilities, isoelectric points, size,
and density. These physical properties can be exploited by manipulating thermodynamic variables, which include temperature, pressure, ionic strength, p H ,
and electric potential. While all of these variables have been used to separate
compounds, there has recently been a great deal of interest in the effect of
electric potential on separations, particularly in biological systems, as well as
application of process chromatography to a variety of molecules. This
chapter discusses both the key factors which impact the practical
0097-6156/86/0314-0122$08.50/0


10.
123
Scale- Dp of LC and Electrophoresis
RUDGE AND LADISCH
implementation of chromatography, and the current thinking on its combina

tion with electric potential to improve resolution.
Many industrial or preparative separation processes are elution
processes. In elution processes a pulse of sample followed by eluent is con
tinuously fed, and another stream is continuously recovered. However, pro
duct leaves the system in pulses, making this process a batch process. This is
different from a continuous process, in which feed and product streams are
continuously fed and recovered, respectively, and are at steady state. Semicontinuous processes alternate between continuous product and regeneration
cycles. Simple fixed bed adsorption is an example of a semi-continuous pro
cess, while distillation is an example of a continuous process. Chromatogra
phy is typically an elution process although continuous forms of this separa
tion have been reported (1-4).
Elution processes allow introduction of feed and withdrawal of product
without irreversible changes in stationary media. Elution of products is usu
ally driven by the flow of a fluid, although other driving forces are conceiv
able. Fluid driven elution is readily controlled on a process scale, especially
in the case of incompressible fluids. Pressure drop is always a consideration
when a fluid is driven, and becomes more important with increasing equip
ment length.
The Capacity Factor in Chromatography
One challenge in process chromatography applied to biologically active or
derived molecules is that these molecules exist with many other co-products,
particularly if the desired product molecule is fermentation derived (5). In a
separation scheme, a target molecule can be separated from other components
based on differences in shape or size, ionic character, hydrophobic character,
and/or bio-affinity (6). The resin's capacity to interact with the molecule is a
key parameter, and is described by the capacity factor:
[1]
where n\ is the moles of solute / in the stationary phase, c', is the moles of
solute i in the mobile phase; is the distribution coefficient; v is the fluid
volume displaced by the stationary phase and v is the fluid volume of the
s
mobile phase.
Since V = v
T
+ v , we can define = - r r - and v as (l - a)

s
Vj
V . Then
T
[2]
a

124
If a pore fraction, e, can be determined for a particular support, then these

equations may be further refined:
.,l-.
iL^.
[3]
Ki is described differently from K'i and reflects only the volume available to
y

fluid within the support. K'i = Ki e, where e =
res
The difference between
capacity factors of the desired solute and other products reflects the extent of
separation which can be expected. When the capacity factor is large (k', l),
the support takes on characteristics of an adsorbent. In this case, a solute will
adsorb onto the support at the conditions of sample loading, and desorb only
when conditions in the eluent, such as salt concentration, p H , polarity, or
temperature, are changed. This fits the category for adsorption/desorption
for which quantitative descriptions are available (7-10). This chapter primarily addresses cases where k'i is small, i.e., chromatographic separations.
Bio-Affinity. A n affinity support is an example of a support with a high
capacity factor. This support has a spacer arm attached to a ligand with a
highly specific affinity for a solute (11). The ligand acts here as an adsorbent
in which a change of conditions in the bulk fluid surrounding the support is
required to wash off the adsorbed solute. While quite elegant in theory, many
times the product will be present in small concentrations, and nonbinding
impurities will be present at much higher concentrations. If the ratio of product to other solutes is low (for example, 1 to 1000), even slight adsorption of
nonbinding solute onto the matrix or ligand can translate into significant loss
in selectivity. Effective use of an affinity support may therefore require sample clean up steps upstream as well as the appropriate selection of a matrix
which is inert with respect to the solutes. Given current technology and the
potentially high cost of affinity supports, such an approach suggests bioaffinity might be best considered in the context of a product polishing step
(6).
Ion Exchange Resins As Chromatographic Supports. A versatile type of support is one which can exhibit differential ion exclusion, size exclusion, ion
complexing, and hydrophobic interaction characteristics with respect to a
variety of molecules. In fact, a generic support of this type exists, and is
based on a copolymer of sulfonated styrene-divinylbenzene (DVB). Sulfonation impacts the ability of the support to complex counter-ions which, in turn,
can complex with the solute or exclude an ionic species. The degree of resin
cross-linking is proportional to D V B content and determines effective porosity
of the resin and size exclusion characteristics.
The separation shown in Figure 1 illustrates the versatility of such a
resin (12,13) packed in a 6 mm i.d. by 60 cm long jacketed column, maintained at 80 C . In this case, separation of oligosaccharides G to G
7


RUDGE A N D LADISCH
Scale-Up of LCand
Electrophoresis
GLUCOSE
XYLOSE
MANNOSE
ARABINOSE
METHA NO L
ETHANOL
600
1200
ION
EXCLUSION I
Figure 1.
1800
2400
TIME (sec.)
.HYDROPHOBIC
INTERACTION
SIZE
_
EXCLUSION
SIZE
and
COUNTER
ION
COMPLEX
Ion exchange separation of oligosaccharides, sugars, and
alcohols, conditions as given in text.

126
(molecular weight of 1152 to 342), monosaccharides (molecular weights of 180

and 150), and alcohols is shown. As indicated in Figure 1, ion exclusion
(NaCl), size exclusion ( G through glucose, hexoses and pentoses), ion complexing (glucose/mannose and xylose/arabinose), and hydrophobic interac
tions (alcohols elute in order of increasing size and hydrophobic character) all
contribute to the observed separation. The conditions for this separation were
(12,13):

eluent:
eluent velocity:
resin:
sample size:
detector:
particle size:
water
2.1 cm/min (based on cross-sectional area of empty column)
Aminex 50WX4, Ca
20
Waters differential refractometer
20 to 30 microns
++
If scale-up can be achieved using larger particle size ranges (250 to 1000
micron), a significant potential for this type of support is indicated particu
larly when price ($100 to $500/cubic foot), availability of commercial quanti
ties of cation ion exchange resins, and their history of use in the food and
pharmaceutical industries are considered.
Scale-Up Considerations
Once a support having appropriate surface area, pore size, particle size, and
surface characteristics is identified, engineering input for its use on a large
scale is required. Analytical applications are characterized by a small sample
size (less than 0.1% of column volume), low solute concentration (usually less
than 1%), and use of small particle size (5 to 30 microns). In comparison,
process-scale separations will probably be characterized by large sample size
(up to 20% of column volume), high solute concentration (up to 30%); and
relatively large particle size chromatographic supports (100 to 1000 micron).
Thus, it may be more difficult to obtain clear resolution of the solutes on a
process scale than it would be on an analytical scale. By careful control of
process conditions, however, a simple column system (Figure 2) with a modest
plate count (100 per meter based on glucose) can give satisfactory separation
for relatively large sample volumes (Figure 3, reproduced from reference 14).
Experience in our laboratory on a variety of column sizes ranging from 2 to
160 mm in diameter and 10 cm to 600 cm in length has shown that published
semi-empirical correlations are useful in obtaining a first estimate of column
performance (5,14,15).
Column Area. The desired sample volume to be applied to a column can be
expressed in terms of column void volumes:
= a
[4]

Scale- Up ofLC

RUDGE AND LADISCH
and Electrophoresis
On line
Analysis/Detection/
I
Control
-
^ - J Input to Turn Valve
Comp.
Comp. A
Figure 2. Chromatographic apparatus for process system.
1.0
lOV
0.8
0.6
0.4
0.2
0
0.2
0.4
0.6
0.
2.0
2.2
2.4
2.6
2.8
3.0
_V Total Vol. of Effluent

V,.
Column Void Vol.
Figure 3. Ion exclusion separation of glucose and sulfuric acid.

Sample size of 0.1 v , = 55 C .
m

128
where v is sample volume; and a is dependent on support characteristics.

For a column packed with support of a fixed particle size, operated at a fixed
temperature and eluent linear velocity, the column cross-sectional area should
increase in proportion to the sample size. However, proper column operation
also depends on the ability to distribute the feed as a plug onto the column
with as little back mixing as possible (5). As column diameter increases, this
condition becomes more difficult to approximate due to practical problems in
evenly distributing a liquid at velocities approaching those associated with
creep flow. Hence, the length to diameter ratio, L / D , also becomes a factor.
In general, an L / D of 10 or greater is desirable. A n increase in length with
an increase in column diameter may, however, be limited by the mechanical
stability of the support and pressure drop. Special attention is then required
for the liquid distribution systems and experimental trial and error becomes
essential for scale-up.

Column Length. Column length causes an increase in dispersion of a solute

due to both diffusion and backmixing caused by motion of the eluent. This
results in an increase in the volume over which the sample will elute, relative
to the volume of sample placed on the column. This phenomena is reflected
by an increase in peak standard deviation () where the width of a Gaussian
peak is 4. In process chromatography, particularly in the case of sample
overload, the peak may be skewed (16). A standard deviation can still be
defined leading to a value of plate height, although moment analysis of the
eluting peak must be used to obtain values of (5,17).
The concept of theoretical plates in chromatography is described else
where, and is not repeated here (18,19). Pieri et al. have described the appli
cation of the theoretical plate concept to column scale-up and demonstrated
its utility in scale-up of pheromone separation over silica gel (15). We have
found this same approach to be useful in separations over ion exchange resins
used as chromatographic supports. In essence, this approach gives an esti
mate of column length for a given separation if the particle size is changed.
This is based on empirical correlation of the number of theoretical plates, N :
(
where D is a coefficient which reflects dispersion, L is column length, d is

particle diameter, is the eluent linear velocity, and m is an empirical con
stant. In this approach, the molecules separated are assumed to be chemically
and physically similar. Therefore, D, can also be assumed to be similar for
these molecules as a first approximation (15). For the same linear velocity
and plate count, column length will change with particle size as the 1.5 power:
t
[6]
d
P.A

10.
Scale- Up of LC and Electrophoresis
RUDGE A N D LADISCH
129
Consequently, a 1 foot long column with a 50 micron support is equivalent to

an 8 foot long column with a 200 micron particle size of the same support.
For a binary system, resolution can be defined (19) by
(-1) N*
[7]
k'
\+k'

where
N, + N
[8]
*'l+*'2
P]
/.
'
k
,^,
where k >
2
[10]
Under process conditions, equations (5) to (10) are approximations when sam
ple volumes and concentrations are larger than those usually associated with
analytical chromatography. If support chemistry and physical characteristics
are maintained through scale-up, and if the support is packed in a properly
designed column, constant resolution is assumed to reflect a constant plate
count. This is the basis for estimating column length by equation (6).
Although approximate, this approach is useful for preliminary sizing in the
absence of detailed data.
Theoretical Considerations In Size Exclusion Chromatography
In order to better understand chromatography it is important to study the
underlying mass transfer operations which are occurring. These mass transfer
phenomena are well studied (20-27), and analytical solutions exist to most
limiting cases. In general, a mass balance for one component in a packed
column is given by:
-L =
d
2
i<L + i + J _ i i L
dx
dt
a dt
where
D Mr
dx
= dispersive flux (
m o l s
f
time vol

130
Table I. Summary of Selected Initial and Boundary Conditions Applicable

to Chromatography
Initial or Boundary Condition
1. Constant Inlet Concentration

c = c at =
a
0, / = 0
= o a t * = 0 , / >0
dt
or
4f = at = 0 0<t
at
<t
= 0 at
* = L, /
>0
dx
3. Initial concentration in column is 0

c =
0 at / = 0,
>0
Ref.
When solutes are being

eluted by an eluent of
constant concentra
tion, such as in linear
chromatography, or
when sample is being
injected as a finite
pulse.
21.25
Used when D and

are very similar inside
and outside the
column and when
accumulation is small
compared to disper
sion and convection.
20,22,
24
feed
2. No adsorption past column outlet
Application
Used in nearly all

cases. Applicable to
all boundary condi
tions listed.
w=0ati=0, * > 0
4. Danckwert's Condition
dc
D dx
vc
vc
at = L, / >0
Allows for backmixing

at the column outlet
by adding a diffusive
flux term. Lets
material diffuse into or
out of the column if a
gradient exists.

22-24,
26
10.
Scale-Up of LCand
RUDGE A N D LADISCH
dc
convective flux ( .
131
Electrophoresis
,)
time vol

dc_
dt
J_ dn_
a dt
accumulation of solute in mobile phase (
m o l s
,)
time vol
accumulation of solute on stationary phase (

J
,)
time vol'
There are a variety of boundary and initial conditions which are useful for
different situations. Some examples of these boundary conditions and their
applications are given in Table I. A relationship between (adsorbed species)
and c (mobile species) must be found. These relationships may either be
equilibrium or kinetic relationships (mass transfer rates). Some examples of
equilibrium and mass transfer relationships may be found in Tables II and III,
respectively. As pointed out by Lapidus and Amundson (25), equilibrium
relationships in themselves are useful in cases where mass transfer rates are
not limiting. In any case, the equilibrium characteristics of the support and
solute have a direct bearing on column performance.
Table II. Selected Equilibrium Expressions
Application
Relationship
1. Linear
Ref.
When concentrations are very low,

or when linear adsorption occurs.
25
Popular isotherm in chromato

graphic and adsorption systems.
This isotherm is asymptotically
linear when c is very small or very
large.
27
Empirical isotherm which can be

made to fit most equilibrium data,
but has no asymptotic limits.
27
kc
2. Langmuir
k c
a
"~
\+k c
b
3. Freundlich
n=k
X l k b
a C
Table III. Selected Mass Transfer Expressions

Application
Relationship
1.
dn_
dt
kma C ~kmb
2.
dn
dt
^ma ( " Ceq)
3.
dn
dt
dr
dr
Ref.
Mass transfer is finite.
25
Mass transfer is finite and

approaches equilibrium: Special
case of previous relationship.
25
Mass transfer is finite, and solute

diffuses in and out of a sphere.
Additional boundary conditions
reflecting particle environment are
required here.
21

132

Prediction of Elution Profiles {Linear Equilibrium). For the case of local

linear equilibrium (infinite rate of mass transfer), Lapidus and Amundson (25)
derived equations for computing concentration distributions in a packed
column. With concentrations at the inlet of the column, and initial conditions
throughout the column known, concentration profiles at a specific distance
from the column inlet can be computed. The derivation was based on a
semi-infinite column, which differs mathematically from a finite column, in
that effects of the mobile phase leaving the stationary phase are not modeled.
Nonetheless, the solution obtained is useful for giving a qualitative picture of
important parameters in column performance. The equation is:
c(*,0 = [fi (*,0 + / 2 ( * , 0 ] p ( - g - -
where
and
V
c (5)exp[^
0
s_
y
ds
AD{t-s)*
[14]
(,- )3/2
5
where
= 1+
* L =
a
1 +
distribution coefficient
_
VM
init.{s) = initial concentration profile in the column

c (s)
0
= inlet concentration profile for the column
Note that D , the dispersion coefficient, is not the molecular diffusivity, but a
measure of combined dispersive effects inherent in packed bed operations, of
which molecular diffusivity is a minor component. As pointed out by Kramers and Alberda (24), eddy diffusivity involves fluctuations of a statistical
nature, and should not be applied to macroscopic effects, such as by-passing
and mixing. This equation is important because it allows the modeling of
chromatographic results using the dispersion coefficient as a free parameter.

10.
RUDGE A N D LADISCH
133
Chromatographic zone broadening, however, is also influenced by finite

mass transfer rates, or porous diffusion. Selection of the proper relationship
between and c is therefore needed.
The functions / , (*,/) and f (x,t) allow solutions to be derived for spe
cial cases of inlet and initial conditions. Note that 5 is a dummy variable of
integration. In the case where the column has been washed with eluent and a
sample has not been introduced, initial condition 3 from Table I applies, and
fx (x,t) = 0. After a sample has been introduced and washed into the column
by eluent, boundary condition 1 from Table I with c = 0 applies and fi{xj) =
0. The eluent volume is Q = Avta. The two limiting cases mentioned above
may be superimposed, offset from one another by A a t' = Q' V where
Q' is the feed volume. This superimposition is applicable only in the case of
linear equilibrium, which yields symmetric solutions. Figure 4 shows the
results of these equations graphically for selected values. These values may be
particularly applicable to proteins in size exclusion supports.
In size exclusion chromatography, components are excluded from the
resin on the basis of size. By definition, solutes are not absorbed. These sup
ports have a linear equilibrium, since a constant volume within the resin is
available for a given size solute. When the support comes to equilibrium with
the mobile phase, the volume in the pores available to the solute will have the
same molar concentrations as the surrounding mobile phase.
2

ci _
[15]
ni
e V
where K is the fraction of the pore volume available to the solute, and is
independent of molar concentrations. For this special case, the familiar
expression
D
can be derived. Hence K

defined previously where K
the only partitioning factor.
0, ki = 0 and 7 = 1 . For a
D
U L = _L a n d = .
7
ni_
is analogous to Ki, the distribution coefficient as

addresses the special case for size exclusion being
For a solute which is completely excluded, K =
solute which is completely included, K = 1, ki =
e V
L
-L.
a
Estimating Maximum Sample Volume. The maximum

volume which may be introduced into the column at any
defined for size exclusion supports. Since these supports
tion, their capacity for a solute may range from 0 (totally
[16]
lXs_
amount of sample
one time is readily
exhibit no adsorp
excluded solute) to
(, the ratio of support pore volume to mobile phase volume). A solute
moves through the column in a time equal to the total volume available to

134
that solute within the column, divided by the volumetric flow rate of eluent.
Since all solutes in the column experience the same volumetric flow rate, it is
the difference in column volumes available to the different solutes which
defines the separation. For a binary system, the two elution volumes are:
V
Ex
K'i e V
=V +
m
(for component 1 which eluted first)

and
v
El
=v
K *v
2
(for component 2 which elutes second).

Since the difference in elution volumes reflects the extent of separation,
the smaller elution volume, plus the volume of the feed, can be no larger than
the elution volume of the second peak, v
E
v +v <v
Ei
El
Otherwise the two peaks will partially overlap each other.

said that V < v
F
El
- V
E{
Hence, it can be
= V . The difference in elution volumes is further

E
given by:
V
Fmax
= AV
= K' eV -Kle
= (K -K[)e
and since, for size exclusion,

0 < K'< 1
the largest difference between elution volumes is ^'.

V
EmSLX
= eV
Similarly, this can be related to capacity factors for size exclusion chromatog
raphy:
(K - K\) e V =(k' -k[)
2

10.
Scale-Up of LC and Electrophoresis
RUDGE A N D LADISCH
135
where:
which gives
[(K -K[)e

[20]
Vs
since K = 1, K\ = 0 for k' = e V / V and A:' = 0, respectively. This result

would imply that increasing support pore volume may allow greater feed
volumes. This is true for columns of identical length. Increasing length alone
does not, in general, increase throughput, since concomitant increases in elution time, and thus, processing time will also occur for constant eluent velocity, v. Column throughput may be increased by increasing column radius
within the constraints mentioned previously (see section on Column Area).
2
Electrophoresis
Background. There have been many applications of an electric potential to
separation processes. Electrophoresis is a separations process of great resolving power, capable of 10 theoretical plates per meter, according to Jorgenson
& Lukacs (28). Applications of electric potential to different flow systems
have accelerated in recent years. It is important, in the development and
analysis of these systems, to understand the nature of an electrolyte system,
and the events which occur when such a system is placed under an electric
field.
6
Factors Affecting Electrophoretic Mobilities. The rate of migration of a

solute through a conducting liquid in an electric field is based primarily upon
three physical phenomena. These phenomena are: the presence of a charge
on the particle, hydrodynamic drag around the solute, and relaxation forces in
the ionic atmosphere of the solute. These forces are discussed by Overbeek
and Bijsterbosch (29) in an excellent review. There are also three macroscopic
affects which may occur in electric environments which affect the net migration of a solute. These are: electro-osmosis, (30), natural convection due to
Joule heating, and forced convection, in the case of an elution process.
A l l charged particles or solutes in free solution attract ions of the opposite sign. These attracted ions cluster around the outer surface of the solute,
creating an ionic environment which is equal and opposite to the particle's
charge. These charged layers are known as the electric double layer, and have
been studied in the context of electrophoresis by many investigators (31, 32,
33). Under the influence of an electric potential, charged species are attracted
in one direction at magnitudes relative to their zeta potentials. The zeta
potential is the electric potential of the solute at the surface of shear, the
boundary between the moving particle's associated counterion layer and the


136
bulk. The zeta potential, in general, is of the same sign but of a smaller mag
nitude than the solute charge found by titration (29). The dimension of the
double layer is important in the theoretical analysis of electro-induced separa
tions, because it is a measure of how closely the counterions surround the
solute. Several developments of the impact of the double layer dimension on
electrophoretic mobility have been published for a variety of geometries, and
have been reviewed elsewhere (29).
Hydrodynamic drag causes a resistance to the flow of a particle at its
outer surface. In general, particles and large biological molecules obey some
form of Stokes law for drag in creep flow, although modifications for
geometry are sometimes required. The critical dimension in all cases is the
distance from the particle center into the double layer at which flow and
hence, drag, are actually occurring. This is referred to as the "slipping plane"
or "surface of shear" (29). Although the exact location of the "slipping plane"
is not explicitly known, it is in the region of the "Inner Helmholz Plane" or
"Stern Layer". The Helmholtz plane distinguishes between counterions that
are physically absorbed to the particle, compared to those surrounding it in a
boundary layer extending into the bulk fluid.
All charged particles migrate under the influence of electric potentials.
This applies to the counterions directly surrounding the solute. Since these
ions are opposite in charge to the solute, they migrate in the opposite direc
tion, exerting an extra drag on the particle which is not predicted merely by
hydrodynamic drag. This effect is greatest when the counterion layer thick
ness is large compared to the characteristic dimension of the solute.
When a particle migrates in a direction opposite to its counterion layer
(i.e. when a potential is applied), a distortion of the ionic boundary layer of
the particle takes place. The particle will no longer be in the center of its
double layer, but rather, towards the edge to which it is migrating. This
polarization results in the build-up of opposite charge in the opposite direc
tion of the applied potentials. This segregation of charge relative to the solute
results in a small attraction in the direction opposite to the applied potential,
thereby reducing mobility (Figure 5). This is known as a relaxation effect and
may reduce mobilities by 10-50% (29). This phenomenon is greatest when the
dimension of the counterlayer is on the order of the dimension of the solute.
Several microscopic effects impact the electrophoretic mobility, , of a
solute. The electrophoretic mobility is defined as the velocity with which a
particle moves in a field divided by the strength of the field, and has units of
length squared per time per voltage. It can be seen, by the complexity of
these factors, that the electrophoretic mobility is dependent on solute size and
charge, as well as medium viscosity, ionic strength and temperature.
Electroosmosis. Electroosmosis occurs in systems with applied potentials and
results from preferential adsorption of charges at a fixed surface, such as a
column wall. This ionic adsorption results in the build-up of a charged
"counter" layer at the surface, which migrates electrophoretically. This flux
along the wall induces a convective flux in the bulk due to viscous shear. If

RUDGE A N D LADISCH
0.080
0.060
g 0.050
iz
1.5
LU
^ 0.040
r
= 2.l
2 0.020
or
0.010
10
20
-LA
30
40
50
ELUENT VOLUME
60
70
Figure 4. Calculated elution profile to simulate a protein separa

tion on Sephadex G-75 using equation (12). This simulation is
based on parameter values for a 30 1.5 cm column with a
flowrate of 1.77 ml/min and a longitudinal diffusivity of 0.01
cm /min. The ratio of mobile phase volume to pore volume was
0.9, and the sample volume was 0.17 ml. Capacity factors for each
of the solutes are 0, 0.5, and 1.1, respectively.
2
r= relaxation displacement
Figure 5. Schematic representation of the relaxation effect for a

charged solute experiencing an electric potential.


138
the system is closed, and the fluid is incompressible, conservation of mass

requires that the net flow across any plane be zero (30). Therefore, a circulating flow occurs, with flow in the center of the cell occurring in a direction
opposite to the flow at the walls. Obviously, if a sample is introduced uniformly across a cell, the shape of the sample band will be distorted by the
electroosmotic flux. Therefore, either resolution is decreased, or the amount
of the channel which may be used is limited to portions in which the fluid
velocity is nearly equal. Electroosmosis has been extensively reviewed (30).
There have been three methods used to minimize electro-osmosis, or its
effect on separation. The first is to use an inert coating at the cell walls which
will not adsorb ions. One such coating is methylcellulose (30). The problem
with coatings of this nature, however, is that they are difficult to attach to
glass. Several coatings have been successfully employed in solving this problem including one used in tests during an Apollo-Soyuz flight (34), and more
recently, on the Space Shuttle (35).
Lowering the surface to volume ratio of a cell is another method of
reducing the impact of electroosmosis on separations.
Widening the
apparatus will accomplish this. It has been shown (30,34) that rectangular
channels are much more suitable for this kind of manipulation than circular
channels. The disadvantage of widening a channel lies in reduction of the surface area/volume ratio, and hence, reduced heat dissipation. At the same
time, power requirements are increased, due to the cell's wider cross section.
The last method of reducing the impact of electro-osmosis is with
porous media. Resins and gels dampen out non-ideal flow effects and can
minimize a nonuniform flow profile. The smaller the resin, or tighter the
cross-linking, the more effective the dampening. These types of supports may
have other properties, such as size exclusion properties, or the ability to
adsorb charges, which are not always desirable.
Joule Heating, Viscosity and Buoyancy. When an electrical current is passed
through a medium, energy is converted due to the resistivity of that medium,
and is given off as Joule heat. When the current is large, the heating is great.
Since most cells exchange heat at one or more boundaries, a temperature gradient arises in the cell. A thinner cell minimizes this effect. The effects of
gravity on bodies of different density produce a naturally convective (and
macroscopic) flow. Hot, lighter bodies (usually in the center of the cell) rise,
while other bodies sink. It is the elimination of this effect which makes electrophoresis in microgravity so appealing. Different temperatures also create
different liquid viscosities. Since the viscosity of the liquid impacts hydrodynamic drag, temperature gradients can alter electrophoretic mobilities,
independent of gravitational forces. This viscosity effect is estimated to
change mobilities by about 2% per degree centigrade (28).
Electrophoresis with Controlled Convection.
The study of how electrophoresis within controlled convection can be applied efficiently to flow systems in preparative and industrial scale separations is an area for further


10.
RUDGE A N D LADISCH
139
development. The potential for such separations is great, based on the

number and diversity of systems which have been proposed over the last fif
teen years (34-50).
A n electrochromatography system which is receiving attention is that of
O'Farrell (40). This system (Figure 6) consists of a layered bed of size exclu
sion gels, in which a less porous support rests upon a more porous gel. Since
the more porous gel has more pore volume available to solute, a solute's velo
city in this layer is slower than in the less porous layer. O'Farrell found that
an electric field could be selected, which would focus one solute at the inter
face of the layered gels. The solute must concentrate in one of the two layers
near the interface. The solute has been shown to concentrate in the lower gel,
because the local increase in electrolyte concentration decreases the local resis
tance, and therefore decreases the local potential. In regions other than the
concentrated solute region, the net flux of solute is always toward the concen
trated solute region. This system operates in batch mode for the processing of
one solute, with the remaining components being eluted in the bulk.
Very recently, Gassmann, Kuo, and Zare (44) demonstrated the "electrokinetic" separation of racemic mixtures of amino acids. Their system used
a potential applied along the axis of a capillary column, with open electrode
reservoirs. Their results show a chromatographic type separation of dansyl
derivalized L and D amino acids. The flow in this system is due to electro
osmosis, and is large enough in magnitude to elute all species (positive, nega
tive, and neutral) from the cathodic end of the column. Since the electrode
reservoirs are open, buffer flows freely through the capillary, and none of the
distortion associated with electroosmosis on closed systems is observed. The
investigators employed a specially designed laser fluorescence detector to
quantify extremely small quantities (i.e. 10 M ) of solute. Although the
column was 75 cm in length, analysis time was reportedly 6 to 10 minutes.
As a result of experiments originally conducted on Apollo 16 (34), com
mercial electrophoretic efforts have been undertaken on space shuttle flights
(45,46). The apparatus involved employs free flow electrophoresis of a pro
tein mixture for the purification and recovery of a very high valued product.
The process itself is proprietary. However, data and models are available
from earlier flights and other experiments (34,47,48).
15
Isoelectric Focusing. Isoelectric focusing has also been scaled up to take

advantage of its equilibrium resolution. By equilibrium resolution, it is meant
that the system depends upon solutes arriving at an equilibrium position, and
that neither duration of processing, nor length of equipment, will disrupt this
position. Isoelectric focusing takes advantage of the fact that the zeta poten
tial of a protein changes according to the H of the environment. When a
protein reaches the point, in a H gradient, where its zeta potential reaches
zero (its "isoelectric point"), it ends its migration and "focuses". The focusing
is due to the fact that, if the protein randomly migrates in either direction
from its isoelectric point, it acquires a charge and migrates back. Bier et. al
(49,50) have exploited this phenomena in a preparative recycle instrument,

a) Sample flow in column (Absence of potential)
Convective velocity
LESS
POROUS
Electrophoretic
velocity
i>

Legend
= Charged species I
<--I)
= Other species 2 , 3
A,D
< . <,>
2
MORE
POROUS
b) Sample flow in column (Application of potential)
+
LESS
POROUS
MORE
POROUS
=0
= '
Figure 6. Schematic diagram of electrochromatographic apparatus

described by O'Farrell (40).

10.
RUDGE AND LADISCH
141
which we estimate to be capable of processing samples at the gram level or

higher. This instrument appears adaptable for a variety of capacities.

Plate Height and Resolution Concepts in Electrophoresis

Theoretical plates can be used to evaluate electrophoretic and electrochromatographic performance, in a manner analogous to chromatographic perfor
mance. These type of numerical evaluations are useful to compare results,
both between different applications of the same apparatus, and between simi
lar applications of different apparatus.
Following the definitions and derivation of rate of generation of vari
ance with apparatus length (dal/dX) set forth by Giddings (51), Jorgenson
and Lukacs (28) defined a\ as
= 2Dt
It should be noted here that D is most properly referred to as longitudinal

dispersion, which may be attributed to a number of effects, the least of which
is usually molecular diffusion. Other dispersive effects are usually lumped
into "eddy diffusivity". The development of Jorgenson and Lukacs was
applied to a system in which molecular diffusivity was the major cause of
dispersion, but their approach appears to be applicable to other systems as
r 2
well. Substituting in analysis time, t =
we obtain:
al = 2DL*/V
where
= electrophoretic mobility
Volt min
so
LL == L
N- =
2D
2
1
[22]
In this case, the number of theoretical plates, N, is independent of length of

the apparatus. Since is a molecular parameter, and D is related to the
apparatus and the solute, the easiest variable to manipulate is the applied vol
tage, V. can therefore be increased by increasing the applied voltage. Since
analysis time

142
is inversely proportional to voltage, separating efficiency (N/t) will be maxim

ized at high voltage and short apparatus lengths. This has been shown experi
mentally (28).
The plate height ( H E T P ) is then shown to be

[24]
2DL
which is a direct function of length. This is in direct contrast to chromatogra

phy, where is a function of length, and is independent of length. A n
interesting variation may be seen by applying Ohm's law (V = IR) to the equa
tions for and H.
JdK
2D
[25]
[26]
_ 2DL_
H
~ /*
2
Joule heating is proportional to W I R. Hence, a continuous phase having

a high R would serve to increase both applied voltage and heat generation at
constant current. Assuming good control of heat removal a low ionic
strength buffer would thus be most appropriate for high separation efficiency.
Jorgenson and Lukacs also suggest that the sample be of an ionic strength less
than that of the buffer, in order to negate adsorption and interaction affects
within the sample that may lead to zone broadening.
Jorgenson and Lukacs also described resolution in electrophoresis sys
tems as
2D
77
or
V
R =0.177 ( - ) ( ^ - )
)
s
We see here that high voltage also enhances resolution. If another flux, for
example, a convective flux, is imposed upon the system, we speculate that this
equation could be empirically rewritten as:

10.
Scale-Up of LCand
RUDGE A N D LADISCH
^=0.177(,- )
7777
143
Electrophoresis
[27]
( + vL)D
where
= convective flux

and
+
;
As pointed out by Jorgenson and Lukacs, this allows another variable for
optimization of resolution. If F + vL is made very small, i.e. - v L , reso
lution may be greatly increased. In order for both solutes to elute from the
same end of the apparatus, however, -vL < . The convective flux must be
large enough to push both solutes through the apparatus. Thus, for example,
if a solute / were to migrate in a direction opposite to flow due to its inherent
electrophoretic mobility, the flux given by vL would have to be greater in
magnitude than the electrophoretic flux, . Hence, the condition of
-vL < is proposed if all solutes are to elute in the direction of the flow.
Analysis time is given by:
2
, =
2 8
+ vL
Since the variables and V may be varied independently, it is seen that length
of apparatus is not necessarily a constraining factor, as it is in liquid chroma
tography. Hence separation optima may be discerned which are independent
of length.
Theoretical Considerations in Electrokinetic Separations. Models have been
proposed for electrophoretic (52) and electrochromatographic systems. Simi
lar to developments in chromatography, we may write a model equation for
these systems:
_
dc
dc .
D
dx
where the new term M ^ j ^
l
dx
d(Ec) , be ,
- - - +
1
dx
dt
[29]
1 dn
dt
= flux due to electric potential.
This equation
may be solved with the same boundary conditions which are applied to
chromatographic processes. A similar equilibrium or mass transfer equation
may be used as well. We find, however, that another relation becomes neces
sary for in terms of c. If we assume that is independent of

144
concentration (53) or that concentration in the column is nearly constant, we

speculate that the solution of Lapidus and Amundson (25) may be adapted,
where is replaced everywhere by + . Results from the substitution are
shown in Figure 7, which shows a numerical solution for equations (12) to
(15) modified by replacing with + .
This calculation shows how resolution might be obtained for a three
component mixture, where two components have the same liquid chromato
graphic capacity factors (, = o.5, / = 2, 3) with slightly different electrophoretic
fluxes ( = -2.7; = -2.2).
The assumption that the electric field is homogeneous throughout the
system is only a first approximation. Many electrophoretic systems, such as
isotachophoresis, rely upon potentials varying with the local ionic strength to
affect a separation. In modeling these systems, one must find a function
which describes the local potential in terms of total concentration. The most
commonly used form is Poisson's Law:

dx
where
= dialectric constant of electrolyte solution
= permittivity of free space
F = Faraday's constant
= valence
This relationship was used effectively by Coxon and Binder (54), and Lim and
Franses (55) to solve electrophoretic systems in which flow was not applied.
Lim and Franses used ionic reactions at the electrodes as boundary conditions
to model apparently decreasing electrophoretic mobilities in an electrophoretic
mass transport analysis. They showed that increasing ionic strength at one
electrode decreased the local potential, thus decreasing mobility. Coxon and
Binder specified concentrations at electrodes to arrive at a model for ionic
interfaces in isotachophoretic processes. Each team investigated different sys
tems, thus resulting in different boundary conditions.
These equations show that the final elution profiles will be affected by
several parameters, such as dispersivity, field strength, eluent velocity, as well
as time, distance, and ionic strength distribution within the column.
There have been numerous advances in both the understanding and
applications of electric potentials to separations in recent years. Electro
phoresis has been met with skepticism as an industrial unit operation (56,57),
but recent applications would suggest a "rebirth" of interest in the potential of
these systems, especially in biological applications.

RUDGE A N D LADISCH
0.030

(J
0.025 h
I
P
<
or
0.020 h
-0.13
'
O
Q
UJ
0.010
0.005
or
o
^-0.22
0.000
10
20
il
30
ELUENT
40
11
50
60
70
VOLUME
Figure 7. Calculated elution profile to simulate a protein separa

tion on Sephadex G-75 using a modified version of equation (12).
This simulation is based on parameter values for a 30 1.5 cm.
column with a flow rate of 1.77 ml/min, a longitudinal diffusivity
of 0.01 cm /min, and an electric field strength of -25 volts/cm. The
ratio of mobile phase volume to pore volume is 0.9, and the sample
volume is 0.17 ml. Capacity factors for each of the solutes are 0,
0.5 and 0.5. The electric potential results in an electric flux term of
-0.13, -0.22, and -0.27 cm/min for each solute respectively, in order
of elution. These values correspond to electrophoretic mobilities of
8.6 , 14.7 * and 18.0 10" cm / Vs, respectively.
2

146

Summary
The practical use of chromatographic and electrophoretic separations in the
biotechnology industry will be aided by correlations capable of relating bench
scale results to process scale conditions. Published physical and chemical property data on chromatographic systems for engineering calculation purposes
currently appears to be limited. Hence, an effort was made in this chapter to
present equations which can be used with physical parameters for which
values have been reported or which are readily determined on a bench scale.
Since size exclusion chromatography is important in fractionation of proteins,
the rationale and equations were presented for estimating:
1.
2.
3.
maximum sample volume (equations (19) and (20) );

elution profiles for non-interfering components (equation (12) to (14) )
with results for an example given in Figure 4; and
changes in column length if particle size of a chromatographic support is
changed upon scale-up (equation (6) ).
These calculations require knowledge of column void fraction (); fractional

pore volume of the stationary phase (e); capacity factors (kf) or distribution
coefficients (A:,); dispersitivity or diffusivity of the solute (>); and initial and
inlet column conditions. The determination of k, is described in reference 5.
Knowledge of limits for sample volume, elution volume, and column length
for a given target of product purity and recovery allows a "best-case" estimate
of throughput, eluent volumes, and support costs using correlations described
previously (5).
Electrophoresis gives fantastic resolution of small protein and peptide
samples on a bench scale, and has prompted numerous experimental
approaches for enhancing this technique. Recent developments include the
systems of O'Farrell (40), Gassmann et.al. (44), and the Space Shuttle (45,46).
Scale-up of a preparative recycle instrument was recently reported by Bier
(50).
Key parameters for describing electrophoresis are plate count (equation
(22) ) and resolution (equation (27) ). Unlike chromatography, the number of
plates is a function of applied voltage, V (equation (22) ) rather than length.
The combination of size exclusion chromatography with an electrophoretic
driving force is proposed for continuous elution and separation of a multicomponent mixture. A n estimate of profiles based on an unsteady material
balance (equation (29) ) and equations ( (12) to (14) ) suggests such a separation is theoretically possible (Figure 7). If so, a compact and scalable unit
operation combining convective and electrical effects to give rapid and
extraordinary resolution of proteins in existing size exclusion chromatographic systems should be possible.
:

10.
RUDGE A N D LADISCH
147
Acknowledgments
The material in this work was supported by N S F Grant C P E 8351916 and
Kraft, Inc. Support for activities in process chromatography and scale-up
from Artisan Industries is also acknowledged. We also thank M r . K. Ruettimann for helpful comments on electrophoretic effects.

Nomenclature
= empirical constant
= column cross sectional area (cm )
= concentration in mobile phase (moles /1)
= concentration constant (moles /1)
= concentration in mobile phase if mobile phase is

in equilibrium with stationary phase (moles / /)
eq
= solute concentration in mobile phase (moles /1)
ci
= moles of solute in mobile phase (moles)
init
initial conditions for column as a function

of distance, or a constant (moles /1)
= concentration at column inlet as a function

of time, or a constant (moles /1)
Dj
= dispersivity associated with solute /
= diffusivity inside particle (-^r-)
(^^)
mm
pA
= support particle diameter

original scale particle diameter (/w)
px
= scaled up support particle diameter ^ m )
= electric field strength (Volts/cm)
= Faraday's constant

Library
1155 16th St., N.W.
In Separation, Recovery, and
Purification inD.C
Biotechnology;
Washington,
20036 Asenjo, J., et al.;
1 4 8
= height equivalent to a theoretical plate (cm)
= applied current (amps)
k,k k
ai
equilibrium constants
= fraction of pore volume available to a particular solute
k\
= capacity factor (<n2kL)

moles
average of two capacity factors
Ki
= pore volume distribution coefficient (. / m^L)
= support volume distribution coefficient (iSSfeL / jnokL^
kma, k
mb
k'
= mass transfer constants (-r)

time
= length of apparatus (cm)
= original scale apparatus length (cm)
= scale up apparatus length (cm)
= empirical constant
= concentration in stationary phase (moles / /)
m o
= solute concentration in the stationary phase ( ^
ni
= moles of solute in stationary phase (moles)
= average of two plate counts,
Ni
= number of theoretical plates based on solute /
= volume of flow (ml)
Q'
feed volume (ml)
= distance along the support particle radius ()
e s
Ni +

10.
RUDGE A N D LADISCH
continuous phase resistance (ohms)
Rs
resolution
dummy variable of integration
time
t'
= time
of feed duration
mobile phase linear velocity (cm / min)

applied voltage (volts)

elution volume of solute / (ml)
difference in elution volumes of two solutes (ml)
^ VEmax
= maximum difference
VF
feed volume (ml)
VFmax
y
r
pores
V
= maximum
in elution volumes (ml)
sample volume (ml)
mobile phase volume (ml)
volume of support pores (ml)
stationary phase volume (ml)
= total
power (watt volt-amps)
longitudinal distance from column inlet (cm)
column volume (ml)

y
valence of solute /
Greek
= ratio of mobile phase volume to stationary phase pore volume (
= column void fraction (

150

y
= pore volume fraction (
= dielectric constant of media
= relative permittivity

7 = 1 +
p o r e
k'i
= electrophoretic mobility of solute i ( /7min ^
= average of two electrophoretic mobilities,
7
= ratio of capacity factors for two solutes,
= standard deviation
VQ
ol variance
,
= zeta potential of solute /
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39. Nerenberg, S.T.; Pogojeff, G. Am. J. of Clin. Path. 1969, 51(6), 728.
40. O'Farrell, P.H. Science. 1985, 227, 1586.
41. Oren, Y.; Soffer, A. J. Electrochem. Soc. 1978, 125(6), 869.
42. Reis, J.F.G.; Ramkrishna, D.; Lightfoot, E.N. AIChE Journal. 1978,
24(4), 679.
43. Salik, J.; Roch, P. J. Chromatogr. 1972, 71, 459.
44. Gassmann, E.; Kuo, J.E.; Zare, R.N. Science, 1985 230, 813.


152
S E P A R A T I O N , R E C O V E R Y , A N D P U R I F I C A T I O N IN B I O T E C H N O L O G Y
45. Walters, S., Mech. Eng., 1982, 104, 46.

46. Burlis, N.W., "Agony and Ecstasy of EOS" 187th National Meeting of
the American Chemical Society, 1985.
47. Materials Processing in the Reduced Gravity Environment of Outer
Space; Proceedings of the Annual Meeting, 1981.
48. Electrophoresis '82; Proceedings of the Fourth International Conference,
1982.
49. Bier, M.; Egen, N.B.; Cellgyes, T.T.; Twitty, G.E.; Mosher, R.A. Pepthides; Structure and Biological Functions, Pierce Chemical Company,
1979, 79.
50. Bier, M. "Recycling Isoelectric Focusing" 187th National Meeting of the
American Chemical Society. 1985.
51. Giddings, J.C. Sep. Sci., 1969, 4, 181.
52. Bier, M.; Palusinski, O.A.; Mosher, R.A.; Saville, P.A. Science. 1983,
219, 1281.
53. Reis, J.F.G.; Lightfoot, E.N.; Lee, H-L. AIChE Journal. 1974, 20(2),
362.
54. Coxon, M.; Binder, M.J. J. Chromatogr. 1974, 95, 133.
55. Lim, K.H.; Franses, E.I. Chem. Eng. Commun. 1983, 22, 181.
56. Graff, G.M. Chem. Eng. 1983, June 13, 22.
57. Webber, D. C & EN. 1984, April 16, 11.
Received April 16, 1986

11
Mathematical Modeling of Bioproduct Adsorption Using
Immobilized Affinity Adsorbents
Somesh C. Nigam and Henry Y. Wang

Department of Chemical Engineering, The University of Michigan, Ann Arbor,
MI 48109-2136
The
use of small affinity
adsorbent
particles
immobilized in hydrogel beads has been investigated for
whole broth processing (1). The adsorbent particles can
contain biospecific ligands covalently attached to a
porous solid
support.
A mathematical model was
developed to study bioproduct adsorption using
immobilized affinity adsorbent beads in batch operation.
The performance of immobilized and freely suspended
affinity adsorbents was compared by calculating
adsorption rates and selectivities for four different
bead geometries. Simulation results indicate that the
performance
of finely ground adsorbent particles
immobilized in hydrogel beads is superior compared to
freely suspended adsorbents. The mathematical model was
further used for simulation studies to investigate the
effect of bead design parameters on product adsorption.
A f f i n i t y a d s o r p t i o n , due t o i t s h i g h degree o f s e l e c t i v i t y , o f f e r s a
viable
alternative
to conventional
crude
bio-product
separation
schemes.
However, t h e r e a r e s e v e r a l problems a s s o c i a t e d w i t h u s i n g
f r e e l y suspended a f f i n i t y adsorbent
p a r t i c l e s i n t h e whole b r o t h .
L a r g e adsorbent p a r t i c l e s i z e i s r e q u i r e d t o ensure easy h a n d l i n g i n
the b r o t h .
But t h i s l e a d s t o h i g h i n t e r n a l mass t r a n s f e r r e s i s t a n c e
w h i c h s i g n i f i c a n t l y r e d u c e s the a d s o r p t i o n r a t e .
The p r e s e n c e o f
v a r i o u s o r g a n i c macromolecules i n t h e b r o t h c a n l e a d t o r a p i d f o u l i n g
of t h e adsorbent p a r t i c l e s .
A l s o , t h e b r o t h may c o n t a i n b y - p r o d u c t s
i n s u b s t a n t i a l c o n c e n t r a t i o n which may compete w i t h
the d e s i r e d
product f o r the l i g a n d .
The
use o f s m a l l a f f i n i t y adsorbent
p a r t i c l e s immobilized i n
h y d r o g e l beads has been p r o p o s e d t o circumvent some o f these problems
(1).
The h y d r o g e l
matrix
c a n be p r o v i d e d
by
Ca-Alginate,
K-Carrageenan o r any o t h e r r e v e r s i b l e g e l . P r e v i o u s r e s e a r c h i n our
l a b o r a t o r y has i n d i c a t e d t h a t s i g n i f i c a n t l y h i g h e r a d s o r p t i o n r a t e s
and
overall
adsorption
capacities
c a n be
achieved
by
using
i m m o b i l i z e d a f f i n i t y adsorbent beads i n t h e whole b r o t h .
These beads
p r o v i d e low o v e r a l l i n t e r n a l mass t r a n s f e r r e s i s t a n c e due t o the
0097-6156/ 86/ 0314-0153506.00/ 0



154
small adsorbent p a r t i c l e s i z e .
A r e l a t i v e l y l a r g e bead s i z e (1-3
mm)
ensures easy r e c o v e r y from the whole b r o t h at the end o f a d s o r p t i o n
process.
Polymerization
of
these
hydrogels
can
be
reversed
by
manipulating
the c o n c e n t r a t i o n o f exogenous c a t i o n s and
inducing
temperature s h i f t s .
Adsorbent p a r t i c l e s w i t h bound p r o d u c t can be
e a s i l y recovered
by
d i s s o l v i n g away the
hydrogel
matrix.
Large
macromolecules p r e s e n t
i n the whole b r o t h are e x c l u d e d
from
the
h y d r o g e l because of pore s i z e r e s t r i c t i o n .
U n d e s i r e d macromolecules
t h a t do p e n e t r a t e
f o u l the o u t e r h y d r o g e l
l a y e r f i r s t . T h i s saves
most
of
the
ligand distributed
i n s i d e the
bead.
Many of
the
available
biospecific
ligands
used
for bioseparation
are
more
e x p e n s i v e compared t o the p r o d u c t i t s e l f .
R e t r i e v i n g and r e u s i n g the
l i g a n d s a f t e r b i o s e p a r a t i o n i s c r u c i a l t o the economic s u c c e s s o f an
a f f i n i t y bioseparation process.
C o v a l e n t attachment of the l i g a n d t o
an i n s o l u b l e support was used t o m i n i m i z e l e a k a g e .
Therefore,
the
l i g a n d can be r e - u s e d f o r subsequent b i o s e p a r a t i o n s .
The
purpose o f t h i s a r t i c l e
i s to formulate
a model w h i c h
considers
s i m u l t a n e o u s d i f f u s i o n and b i n d i n g r e a c t i o n w i t h i n
the
i m m o b i l i z e d adsorbent p a r t i c l e s .
The model has been d e v e l o p e d f o r
batch adsorption processes.
I t can however be e a s i l y m o d i f i e d t o
p r e d i c t product a d s o r p t i o n i n other r e a c t o r c o n f i g u r a t i o n s .
Theory
A f f i n i t y a d s o r p t i o n i s a s e p a r a t i o n t e c h n i q u e based on s p e c i f i c
and
r e v e r s i b l e b i n d i n g o f two b i o l o g i c a l l y a c t i v e compounds.
Numerous
b i o l o g i c a l compounds r e c o g n i z e and b i n d t o s p e c i f i c compounds.
For
example enzymes form complexes w i t h s u b s t r a t e s i n the course of t h e i r
normal c a t a l y t i c mechanisms.
S i m i l a r l y , a n t i b o d i e s form v e r y s t r o n g
complexes w i t h
their
respective
antigens.
Various
proteins
also
i n t e r a c t s e l e c t i v e l y with other macromolecules.
Graves and Wu
have d e v e l o p e d a simple
e q u i l i b r i u m model f o r
describing
a f f i n i t y binding reactions (2).
The
binding reaction
between a p r o d u c t and an a f f i n i t y l i g a n d c o v a l e n t l y a t t a c h e d t o a
s o l i d support can be r e p r e s e n t e d as:
+ L
P.L
(1)
-1
In the s i m p l e s t
w r i t t e n as:
ads =
des = *-iIP.L*l
case
the
rates
of
adsorption
and
desorption
can
be
( 2 )
(3)
where [P] i s the p r o d u c t c o n c e n t r a t i o n , [L*] i s the c o n c e n t r a t i o n o f

the
bound
ligand
and
[P.L ]
is
the
concentration
of
the
p r o d u c t - l i g a n d complex.
T h i s y i e l d s an e q u i l i b r i u m
constant:

11.
NIG A M AND WANG
F
K
155
s
(4)
= *-3 i = - i
[P.L ]

Mathematical Modeling of Bioproduct Adsorption
fA
In t h i s approach i t i s assumed t h a t t h e p r o d u c t m o l e c u l e b i n d s t o a
s i n g l e b i n d i n g s i t e on t h e l i g a n d t h r o u g h monovalent i n t e r a c t i o n . F o r
t h i s mechanism, t h e r a t e o f a d s o r p t i o n c a n be e x p r e s s e d by a r e l a t i o n
which
is first
order w i t h respect t o both, product
and l i g a n d
concentration
( E q u a t i o n 2 ) . However, t h e r e may be c i r c u m s t a n c e s
where t h e p r o d u c t m o l e c u l e c o n t a i n s more than one b i n d i n g s i t e t h a t
i s r e c o g n i z e d by t h e l i g a n d .
Such a m u l t i v a l e n t i n t e r a c t i o n r e q u i r e s
a more complex a n a l y s i s (3.). Most o f t h e a f f i n i t y b i n d i n g r e a c t i o n s
are c h a r a c t e r i z e d by v e r y s n a i l e q u i l i b r i u m b i n d i n g c o n s t a n t s .
We
w i l l assume t h e r a t e o f a d s o r p t i o n ( r ^ ) t o be much h i g h e r compared
to the r a t e o f d e s o r p t i o n (
) so t n a t the a f f i n i t y b i n d i n g c a n be
c o n s i d e r e d as e s s e n t i a l l y i r r e v e r s i b l e .
F i g u r e 1 shows a schematic diagram o f an i m m o b i l i z e d
affinity
adsorbent bead.
H y d r o g e l , by v i r t u e o f i t s e x t r e m e l y h i g h water
content 0 9 0 % ) , o f f e r s l i m i t e d d i f f u s i o n a l r e s i s t a n c e t o the d e s i r e d
product.
I t i s t h e r e f o r e used
as an i n e r t
matrix
t o support
relatively
small
adsorbent
particles
which
otherwise
cannot be
r e a d i l y r e c o v e r e d from a h i g h l y heterogenous whole b r o t h . The reduced
adsorbent p a r t i c l e s i z e leads t o s i g n i f i c a n t d e c l i n e i n
internal
diffusional
resistance
which
offsets
any m a r g i n a l
increase i n
r e s i s t a n c e due t o t h e h y d r o g e l m a t r i x i t s e l f .
Several
assumptions
a r e made
to mathematically
model the
immobilized adsorbent.
The s m a l l a d s o r b e n t p a r t i c l e s a r e assumed t o
be d i s t r i b u t e d u n i f o r m l y i n s i d e t h e h y d r o g e l bead. The e x t e r n a l mass
t r a n s f e r r e s i s t a n c e due t o t h e boundary l a y e r i s assumed t o be
n e g l i g i b l e i f the bulk s o l u t i o n i s w e l l s t i r r e d .
T h i s assumption i s
s u p p o r t e d by t h e e x p e r i m e n t a l o b s e r v a t i o n s o f Tanaka e t a l . who
studied d i f f u s i o n of several
s u b s t r a t e s from w e l l
stirred
batch
s o l u t i o n s i n t o C a - a l g i n a t e g e l beads ( 4 ) . However, t h e boundary
c o n d i t i o n s c a n be e a s i l y m o d i f i e d t o i n c o r p o r a t e e x t e r n a l d i f f u s i o n
effects
i f needed.
Furthermore
product
diffusion
i n both the
h y d r o g e l and t h e porous adsorbent i s c o n s i d e r e d t o f o l l o w F i c k i a n
laws and i t s d i f f u s i v i t y i n each r e g i o n i s assumed t o be c o n s t a n t .
r
d e s
The unsteady s t a t e p r o d u c t and l i g a n d m a t e r i a l b a l a n c e s i n the

d i f f e r e n t r e g i o n s c a n be e x p r e s s e d as f o l l o w s .
The p r o d u c t mass b a l a n c e i n t h e h y d r o g e l c a n be r e p r e s e n t e d a s :
2.L.
2 3R
<
R 2
* J>
3R
( 3
"
1 >
3
ac.
i ~
at
9 C
Ai
3r
(5)
r=r
The p r o d u c t mass b a l a n c e i n t h e a d s o r b e n t p a r t i c l e s u s i n g a f i r s t
o r d e r b i n d i n g r e a c t i o n w i t h r e s p e c t t o b o t h p r o d u c t and l i g a n d i s
g i v e n by:
I
3r
*
OT
" " *.C C- Ai 1
<6>
A|
3t


156
Adsorbent matrix
Figure 1.
bead.
Schematic diagram o f an i m m o b i l i z e d a f f i n i t y
adsorbent

11.
N I G A M A N D WANG
Product
by:
d e p l e t i o n i n the b u l k s o l u t i o n o f the b a t c h a d s o r b e r
9C .
bi
3t
V
-47tnR D.

V
dC.
dR
157
i s given
(7)
R=R
The
l i g a n d b a l a n c e w i t h i n the a d s o r b e n t
aC
-K.C..CJ i L l
a
1 .
ot

particle i s :
(8)
In the case o f n e g l i g i b l e e x t e r n a l d i f f u s i o n r e s i s t a n c e , the

and boundary c o n d i t i o n s f o r e q u a t i o n s 5-8 can be w r i t t e n as:
Initial
C
t=0):
C o n d i t i o n s (at
= C
= 0;
A i
(zero i n i t i a l
= ^lo*
initial
product
l o a d i n g on the bead)
(uniform l i g a n d c o n c e n t r a t i o n )
S)i'
(uniform bulk concentration)
Boundary C o n d i t i o n s
3C.
R = 0:
- j g = 0;
RQ:
9 C
r = 0:
r
0
Ai
=
(radial
symmetry o f h y d r o g e l bead)
C^;
(concentration continuity
solution interface)
0;
(radial
at
hydrogel-bulk
symmetry o f a d s o r b e n t
particle)
C^;
(concentration continuity
hydrogel-adsorbent i n t e r f a c e )
at
The mass b a l a n c e e q u a t i o n s g i v e n above can be r e p r e s e n t e d i n

non-dimensional
form
by
employing
the
following
dimensionless
v a r i a b l e s and p a r a m e t e r s :
= r /
^1 "
lo:
5 = R/R ;
%i
t =
i o lo
t/R^
= %i'<Zi-> C
2
=
r e f
= c X - ;
2
/ D
Ai
a o ref
2
/ R
ref
A 1
2
D
Ai
*i
"
A i
/C^
2
A fCbi
e
i ;
refs
/ D

158
. = 4nnR D.R
/VD
ref
ref
r
,/R
J),
A. = 3ND .r
/D.R ; . = R D
g o ref
ref

r=l
B.3C.
3F
(9)

(10)
an
(11)
R=l
(12)
i=l
In the p a s t , s i m i l a r b i d i s p e r s e d systems have been i n v e s t i g a t e d
and modeled i n the c a t a l y s t
d e a c t i v a t i o n area
(5-7).
However,
modeling of immobilized
a f f i n i t y adsorbent beads i s more complex
due
to
the
non-linearity
introduced
by
the
rapid
ligand
b i n d i n g r e a c t i o n w h i c h i s dependent on the p r o d u c t c o n c e n t r a t i o n .
The
mathematical
model
described
above
involves
non-linear,
coupled, p a r t i a l d i f f e r e n t i a l equations.
The e q u a t i o n s were s o l v e d
using
a F i n i t e - D i f f e r e n c e method.
D e t a i l s of t h i s
mathematical
technique
have been d e s c r i b e d elsewhere i n the l i t e r a t u r e
(8,9).
F i g u r e 2 shows a f l o w s h e e t f o r the n u m e r i c a l s o l u t i o n o f these model
equations.
Simulation
Studies
S e v e r a l s i m u l a t i o n r u n s were c a r r i e d out t o g a i n i n s i g h t i n t o the

e f f e c t o f bead d e s i g n parameters on the a d s o r p t i o n c h a r a c t e r i s t i c s o f
i m m o b i l i z e d a d s o r b e n t beads.
The p h y s i c a l parameters ( r a t e c o n s t a n t ,
d i f f u s i v i t y e t c . ) f o r the s i m u l a t i o n s t u d i e s were determined from
e x p e r i m e n t a l data on the a d s o r p t i o n o f c y c l o h e x i m i d e , a low m o l e c u l a r
weight
antibiotic,
onto XAD-4 n o n - i o n i c
polymeric
resin
(10,11)
(Table
I).
The
f i t between
the
model
and
the
experimentally
determined a d s o r p t i o n c u r v e s i s q u i t e good ( F i g u r e 3 ) .
S i n g l e component d i f f u s i o n and b i n d i n g .
F i g u r e 4 shows f o u r c a s e s
w h i c h were s i m u l a t e d t o observe the e f f e c t s o f i m m o b i l i z a t i o n i n
hydrogel
and
reduction
of
adsorbent
particle
size.
Case
(a)
r e p r e s e n t s a f r e e l y suspended a d s o r b e n t p a r t i c l e of r a d i u s 1.1
mm.
Case (b) r e p r e s e n t s the same s i z e p a r t i c l e i m m o b i l i z e d i n a h y d r o g e l
bead o f 2.8 mm.
In case ( c ) , the same a d s o r b e n t p a r t i c l e as i n cases
(a) and (b) was assumed t o be c r u s h e d t o 80 s m a l l e r p a r t i c l e s which
were i m m o b i l i z e d w i t h i n a h y d r o g e l bead o f r a d i u s 2.8 mm.
Case (d)
r e p r e s e n t s the extreme s i t u a t i o n i n which the a d s o r b e n t p a r t i c l e was
c r u s h e d t o f i n e powder such t h a t the t o t a l number of p a r t i c l e s w i t h i n
the i m m o b i l i z e d
bead may
be r e g a r d e d
as i n f i n i t e .
This i s also

11.
159

Initial Conditions
t +At
I
Use eqn.(9) to calculate Cj
from R = 0 to 1
Use eqn.(10) to calculate C j

A
from R = 0 to 1
=0
Compute
to 1
dC j
A
r = 1
from R = 0 to 1
Use eqn.(l 1) to calculate
Use eqn.(12) to calculate

fromR= 0 to 1
r = 0 to 1
No
F i g u r e 2 . Flowsheet
model e q u a t i o n s .
of basic
s t e p s i n the n u m e r i c a l s o l u t i o n o f

160

' J
.2
.4
.6
.8
TIME dimensionless
F i g u r e 3.
Concentration p r o f i l e of cycloheximide
in a
a d s o r b e r employing
i m m o b i l i z e d adsorbent beads (see T a b l e
experimental c o n d i t i o n s ) .
(a) Free adsorbent
(b) Immobilized adsorbent

N=1
batch
I for
(c) Immobilized adsorbent

N=80
(d) Immobilized adsorbent

= oo
F i g u r e 4.
Diagrammatic r e p r e s e n t a t i o n o f f o u r c a s e s
the s i m u l a t i o n s t u d i e s .
employed i n

11.
Table
I.
P h y s i c a l Parameters used f o r S i m u l a t i o n
161
Studies
Adsorber parameters:
V = 50 ml
= 107
= 81
R

= R
r
= 2.8 mm
= 0.25 mm
1.0 g m / l i t e r
bi
ref
= 0.513
D i f f u s i o n and R e a c t i o n
0.95
8
parameters:
3
K. = 7.05x10*" s e c "
D. = D
= 5.8xl0" cm /sec
ref
6 2
D.. = 1.1x10 cm / s e c
Ai
= 0.13 gm cycloheximide/gm
adsorbent
equivalent t o d i s p e r s i n g the l i g a n d i n the hydrogel without

another
immobilization matrix.
F i g u r e 5 shows n u m e r i c a l l y g e n e r a t e d p l o t s o f a d s o r p t i o n r a t e as
a f u n c t i o n o f time f o r t h e above mentioned c a s e s .
The a d s o r p t i o n
r a t e was d e f i n e d as t h e amount o f l i g a n d consumed p e r u n i t time u s i n g
d i m e n s i o n l e s s u n i t s . As e x p e c t e d , a d d i t i o n o f the h y d r o g e l l a y e r on
the f r e e l y suspended a d s o r b e n t p a r t i c l e i n case (b) causes t h e mass
t r a n s f e r r e s i s t a n c e t o go up which r e d u c e s t h e a d s o r p t i o n
rate
compared t o case
( a ) . As shown i n F i g u r e 4, t h e i n t e r n a l mass
t r a n s f e r r e s i s t a n c e i n ( c ) i s reduced because t h e a d s o r b e n t p a r t i c l e s
are s m a l l e r .
T h i s d e c r e a s e i n mass t r a n s f e r r e s i s t a n c e more t h a n
overcomes
the e f f e c t
of a d d i t i o n a l hydrogel
resistance.
The
a d s o r p t i o n r a t e f o r ( c ) t h e r e f o r e shows a sharp i n c r e a s e over t h a t
f o r f r e e l y suspended a d s o r b e n t p a r t i c l e s .
T h i s i l l u s t r a t e s one o f
the advantages o f u s i n g
immobilized
a d s o r b e n t beads over t h a t o f
f r e e l y suspended adsorbent p a r t i c l e s .
A f t e r c r u s h i n g the adsorbent
i n t o an i n f i n i t e number o f p a r t i c l e s and d i s p e r s i n g i t w i t h i n t h e
h y d r o g e l bead (case d ) , o n l y a m a r g i n a l i n c r e a s e i n the a d s o r p t i o n
r a t e over case ( c ) i s o b s e r v e d . T h i s happens because below a c e r t a i n
s i z e the i n t e r n a l mass t r a n s f e r r e s i s t a n c e w i t h i n t h e a d s o r b e n t
p a r t i c l e becomes low enough t h a t i t does n o t c o n t r o l t h e o v e r a l l
a d s o r p t i o n r a t e . Based on these r e s u l t s i t can be c o n c l u d e d t h a t the
a d s o r p t i o n r a t e increases m o n o t o n i c a l l y with r e d u c t i o n i n adsorbent
p a r t i c l e s i z e w i t h i n t h e h y d r o g e l bead.
However, below a c e r t a i n
s i z e the a d s o r p t i o n r a t e does not i n c r e a s e a p p r e c i a b l y . As d i s c u s s e d
e a r l i e r , t h e r e may be added d i f f i c u l t i e s
i n recovering very
fine
a d s o r b e n t p a r t i c l e s from t h e bead
a f t e r d i s s o l v i n g the hydrogel.
Thus, o p t i m i z a t i o n o f t h e adsorbent p a r t i c l e s i z e s h o u l d take i n t o
account the a d d i t i o n a l c o s t a s s o c i a t e d w i t h t h e l o s s o f a d s o r b e n t
during
recovery
compared
t o the advantages
o f i n c r e a s i n g the
adsorption rates.

162

0.8
0.
0.2
0.4
0.6
0.8
TIME cHmnsionless
Figure
5.
Adsorption
simulated cases.
rate
as
function
of
time
for

four

11.
NIGAM AND WANG
163
The
diffusivity
of the d e s i r e d product
i n the hydrogel
will
depend on t h e g e l m a t e r i a l , t h e g e l c o n c e n t r a t i o n and t h e d e g r e e o f
c r o s s - l i n k i n g by m u l t i v a l e n t c a t i o n s .
The d i f f u s i v i t y o f t h e p r o d u c t
i n t h e a d s o r b e n t p a r t i c l e s c a n a l s o v a r y d e p e n d i n g on t h e c h o i c e o f
t h e a d s o r b e n t m a t r i x u s e d f o r l i g a n d i m m o b i l i z a t i o n . The c h o i c e o f
t h e h y d r o g e l and t h e a d s o r b e n t m a t r i x w i l l u s u a l l y depend on s e v e r a l
f a c t o r s such as the s t a b i l i t y o f the b e a d a g a i n s t s h e a r f o r c e s , the
s u s c e p t i b i l i t y t o f o u l i n g by v a r i o u s a g e n t s , and
the presence
of
competing by-products.
F o r e f f i c i e n t b e a d d e s i g n one w i l l t h e r e f o r e
need
t o know t h e
effect
of
diffusivity
on
product
adsorption.
F i g u r e s 6a a n d 6b show t h e e f f e c t o f v a r y i n g t h e p r o d u c t d i f f u s i v i t y
i n t h e h y d r o g e l and i n t h e a d s o r b e n t m a t r i x r e s p e c t i v e l y .
I t was
found
that
i n both
cases,
l i g a n d s are
consumed
faster
as
d i f f u s i v i t i e s are increased.
However, s i m i l a r t o e a r l i e r runs
the
ligand
consumption p r o f i l e
approaches a l i m i t
as t h e r e s p e c t i v e
d i f f u s i o n a l r e s i s t a n c e s become s m a l l e r .
Two
component
diffusion
and
binding.
There
is
a
frequent
possibility
of
having
one
or
more
oompounds
present
in
the
f e r m e n t a t i o n b r o t h w h i c h may c o m p e t e f o r t h e a v a i l a b l e l i g a n d s i n t h e
adsorbent
particles.
The
o b j e c t i v e here
i s t o o p t i m i z e the bead
d e s i g n so as t o m a x i m i z e t h e p u r i t y o f t h e d e s i r e d p r o d u c t
adsorbed
onto the adsorbent p a r t i c l e s .
I n order t o n u m e r i c a l l y s i m u l a t e such
a s i t u a t i o n i t was a s s u m e d t h a t two c o m p o u n d s a r e b e i n g a d s o r b e d
onto
t h e i m m o b i l i z e d a d s o r b e n t s : a d e s i r e d p r o d u c t '1' a n d a n u n d e s i r e d
by-product
'2'.
The
adsorption rate
constant
f o r the
desired
product,
i s a s s u m e d t o be 10 t i m e s t h a t o f t h e u n d e s i r e d p r o d u c t ,
The d i f f u s i v i t i e s f o r b o t h o f t h e s e p r o d u c t s a r e a s s u m e d t o be
similar.
Two a d d i t i o n a l p a r a m e t e r s a r e d e f i n e d t o s t u d y t h e d y n a m i c
b e h a v i o r of such
systems.
,
^. . ^
Selectivity
0
Adsorption rate of d e s i r e d product

( S ) = 73
~
r
r
Adsorption rate of undesired product
A1 1
A V
S A2 1
A V
(
C
Product
purity
1 3 )
(Pu)
Amount o f p r o d u c t
Amount o f p r o d u c t ' 1' a d s o r b e d

' a d s o r b e d + A m o u n t o f p r o d u c t '2'
(\A)
adsorbed
F i g u r e 7 shows t h e v a r i a t i o n o f s e l e c t i v i t y w i t h r e s p e c t t o t i m e
f o r t h r e e t y p e s o f a f f i n i t y b e a d s ( C a s e s ( a ) , (b) and ( c ) ) . I n a l l
t h r e e c a s e s , s e l e c t i v i t y d e c r e a s e s f r o m t h e i n i t i a l maximum v a l u e a s
time p r o g r e s s e s .
Due
t o i d e n t i c a l d i f f u s i v i t i e s , t h e two
products
have v e r y
similar
concentration profiles
within
the
immobilized
adsorbent bead at i n i t i a l time.
Thus the i n i t i a l s e l e c t i v i t y i s j u s t
the r a t i o of t h e i r a d s o r p t i o n r a t e c o n s t a n t s . However, s i n c e p r o d u c t


164
0.2
0.4
8.6
0.8
TIME dimensionless
0.5J
0.
1 1 1 1 1"
0.2
0.4
0.6
0.8
TIME dimensionless
F i g u r e s 6 a , b . E f f e c t o f b i o p r o d u c t d i f f u s i v i t y i n h y d r o g e l (D)
and
i n adsorbent
matrix
(D^) o n l i g a n d
consumption
using
immobilized adsorbent beads.


11.
165
'1'
i s adsorbed
at a higher
rate,
(Figure
7,
right),
the
c o n c e n t r a t i o n o f p r o d u c t ' w i t h i n t h e bead g r a d u a l l y becomes lower
than t h a t o f p r o d u c t '2' due t o s i g n i f i c a n t d i f f u s i o n a l r e s i s t a n c e .
The b u l k c o n c e n t r a t i o n o f d e s i r e d p r o d u c t ' a l s o d e c l i n e s f a s t e r
than t h a t o f the u n d e s i r e d p r o d u c t . The combined e f f e c t o f these two
mechanisms l e a d s t o the i n i t i a l d e c r e a s e o f the s e l e c t i v i t y i n a l l
t h r e e c a s e s . D i f f u s i o n a l r e s i s t a n c e e f f e c t s d i m i n i s h as the l i g a n d
g e t s consumed and the c o n c e n t r a t i o n w i t h i n t h e bead becomes c l o s e r t o
the b u l k c o n c e n t r a t i o n . I n some c a s e s , t h i s l e a d s t o an i n c r e a s e i n
the s e l e c t i v i t y near the end o f the a d s o r p t i o n p r o c e s s .
I t was found t h a t the d e c l i n e i n s e l e c t i v i t y was l e a s t i n case
(c) because o f a s m a l l e r o v e r a l l d i f f u s i o n a l r e s i s t a n c e o f t h e bead.
F i g u r e 8 shows the v a r i a t i o n o f p r o d u c t p u r i t y (Pu) as a f u n c t i o n o f
time f o r these t h r e e c a s e s .
The p r o d u c t p u r i t y c u r v e s show t h e same
g e n e r a l t r e n d as the s e l e c t i v i t y c u r v e s .
F i n a l p r o d u c t p u r i t y was
a l s o found t o be h i g h e s t f o r case ( c ) . By v i r t u e o f t h e i r
lower
o v e r a l l mass t r a n s f e r r e s i s t a n c e case ( c ) i m m o b i l i z e d adsorbent beads
not o n l y d i s p l a y a h i g h e r a d s o r p t i o n r a t e but a l s o o f f e r a h i g h e r
s e l e c t i v i t y f o r the d e s i r e d product.
Conclusions
The use o f s m a l l adsorbent p a r t i c l e s i m m o b i l i z e d i n h y d r o g e l beads
f o r whole b r o t h p r o c e s s i n g r e p r e s e n t s a n o v e l approach t o i n c r e a s e
the o v e r a l l e x t r a c t i o n y i e l d o f b i o s y n t h e t i c a l l y d e r i v e d p r o d u c t s .
Immobilized
adsorbent
beads d i s p l a y major advantages over
freely
suspended
adsorbents
both
i n terms
of adsorption
r a t e and
selectivity.
Other
practical
advantages
o f these
immobilized
adsorbent
beads
are
easy
handling
and
reduced
fouling
characteristics.
A mathematical
model was d e v e l o p e d
and used t o
investigate
simultaneous
mass
transfer
and b i n d i n g
w i t h i n the
immobilized
adsorbent
beads.
Numerical
simulation of a
batch
a d s o r p t i o n p r o c e s s employing these i m m o b i l i z e d beads was found t o be
a u s e f u l way t o study t h e i r dynamic b e h a v i o r and o p t i m a l d e s i g n .
Acknowledgments
We would l i k e t o acknowledge t h e f i n a n c i a l
support
S c i e n c e F o u n d a t i o n which made t h i s work p o s s i b l e .
from
National
Legend o f Symbols
"Ai
lo
product
c o n c e n t r a t i o n i n adsorbent
product
c o n c e n t r a t i o n i n h y d r o g e l , gm/ml
ligand concentration ( f r a c t i o n of
s i t e s remaining)
i n i t i a l l i g a n d c o n c e n t r a t i o n (1.0)
bulk c o n c e n t r a t i o n o f the product,
bi
particle,
initial
gm/ml
original
binding
gm/ml
bulk c o n c e n t r a t i o n o f the product,
gm/ml
Legend o f Symbols c o n t i n u e d on p, 167


166
.2
8.4
.6
8.8
TIME dinrwnslonlMS
Figure
7.
(left)
Selectivity
as a function
o f time f o r
c o m p e t i t i v e a d s o r p t i o n o f two compounds, ( r i g h t )
Concentration
p r o f i l e s w i t h i n the immobilized
adsorbent bead and t h e b u l k
solution.
PROOUCTPURfTY-
PRODUCT 'ADSORBED
PRODUCT ADSORBED PRODUCT 7 ADSORBED
I
8.
1
8.5
1.5
HMEdtantlonlMS
F i g u r e 8.
adsorption
Product p u r i t y as a f u n c t i o n
o f two compounds.
o f time
f o r competitive

11.
c o n c e n t r a t i o n o f d e s i r e d p r o d u c t i n adsorbent p a r t i c l e ,
gm/ml
concentration
of undesired
product
i n adsorbent
p a r t i c l e , gm/ml
r a d i a l d i s t a n c e w i t h i n adsorbent p a r t i c l e , cm
r a d i u s o f adsorbent

radial
r e
r e
p a r t i c l e s , cm
d i s t a n c e i n h y d r o g e l bead, cm
r a d i u s o f h y d r o g e l bead, cm
a r b i t r a r y r e f e r e n c e d i s t a n c e f o r making
d i m e n s i o n l e s s , cm
time, sec
167
product
diffusivity
i n adsorbent
product
diffusivity
2
i n h y d r o g e l , cm / s e c
matrix,
arbitrary reference d i f f u s i v i t y
s c a l e d i m e n s i o n l e s s , cm / s e c
a d s o r p t i o n r a t e c o n s t a n t , 1/sec
the time
scale
2
cm / s e c
f o r making
number o f adsorbent
particles
h y d r o g e l bead
number o f beads i n a b a t c h
immobilized
NC
number o f a d s o r b i n g components i n t h e b r o t h
&
p o r o s i t y o f adsorbent
p o r o s i t y of hydrogel
t h e time
within
matrix
AV
volume element i n s i d e adsorbent
particle
u l t i m a t e l o a d i n g c a p a c i t y , gm/unit
ligand
Subscripts :
i
r e p r e s e n t s i ' t h a d s o r b i n g component
i n the b r o t h
Superscripts :
represents variable
i n dimensionless
form.
Literature Cited
1.
2.
3.
4.
5.
Wang, . Y. Annals of the New York Academy of Sciences,

Biochemical Engineering III, 1984, 413, 313.
Graves, D. J.; Wu, Y. T. Methods Enzymol. 1974, 34, 140.
Chase, H. A. Chem. Eng. Sci., 1984, 39, 1099.
Tanaka, H.; Matsumura, M.; Veliky, I. A. Biotech. Bioeng., 1984,
26, 053.
Ors, .; Dogu, R. AIChE J., 1979, 25, 723.

168

6.
Kulkarni, B.D.; Jayaraman, V. K.; Doraiswamy, L. K. Chem. Eng.

Sci., 1981, 36, 943.
7. Maheshwari, J.; Nigam, S. C.; Kunzru, D. AlChE J., 1985, 31,
1393.
8. Carnahan, B.; Luther, . .; Wilkes, J. O. 'Applied Numerical
Methods'; John Wiley Sons; New York, NY, 1969.
9. von Rosenberg, D. U. 'Methods for the Numerical Solution of
Partial Differential Equations'; American Elsevier Publishing
Co., Inc.; New York, 1969.
10. Wang, . Y.; Sobnosky, ., unpublished data, 1984.
11. Payne, G. F., Ph.D. Thesis, The University of Michigan, Michigan,
1984.

12
High-Resolution, High-Yield Continuous-Flow
Electrophoresis
William A. Gobie and Cornelius F. Ivory

Department of Chemical Engineering, University of Notre Dame, Notre Dame, IN 46556
Recycling effluent through a thin-film continuous

flow electrophoresis (CFE) chamber allows virtually complete separation of a binary feed with
negligible dilution of products and permits
throughput to be increased by 0(100-10,000) over
present thin-film CFE devices. An approximate
model of recycle CFE is developed for the high
Peclet number regime and solved analytically. The
solution is used to characterize the behavior of a
recycle CFE device.
V i r t u a l l y a l l b i o l o g i c a l l y derived materials including proteins,
n u c l e i c a c i d s and c e l l s , can be c h a r a c t e r i z e d by one or more of
the p o p u l a r bench-top e l e c t r o p h o r e t i c t e c h n i q u e s . O f t e n the
suspending medium r e s i d e s i n an i n e r t m a t r i x which a c t s not o n l y
t o s t a b i l i z e the s o l v e n t a g a i n s t n a t u r a l c o n v e c t i o n but may a l s o
c o n t r i b u t e c o n s i d e r a b l y to m o l e c u l a r c l a s s i f i c a t i o n through g e l
' s i e v i n g ' or f i l t r a t i o n e f f e c t s .
In f r e e s o l u t i o n e l e c t r o p h o r e s i s r e t a i n s the a b i l i t y to r e s o l v e homologous s p e c i e s and,
because the s t r e s s e s a s s o c i a t e d w i t h e l e c t r o p h o r e t i c p r o c e s s i n g
a r e r e l a t i v e l y s m a l l , b i o l o g i c a l a c t i v i t y l o s s e s due to mechanic a l denaturation are g e n e r a l l y n e g l i g i b l e .
However, i n f r e e
s o l u t i o n one must be e x t r a o r d i n a r i l y c a r e f u l to a v o i d e x c e s s i v e
thermal e x c u r s i o n s which may d r i v e n a t u r a l c o n v e c t i o n and/or
i n a c t i v a t e product.
The f i r s t f r e e - f l o w e l e c t r o p h o r e s i s d e v i c e (I) was d e s i g n e d
s p e c i f i c a l l y f o r l a r g e s c a l e b i o l o g i c a l s p u r i f i c a t i o n s and,
a l t h o u g h t h a t u n i t never became f u l l y o p e r a t i o n a l , i t preceded a
p l e t h o r a of i n n o v a t i v e i n s t r u m e n t s which e i t h e r r e s o l v e d or e l i minated the drawbacks i n h e r e n t i n the e a r l i e s t d e v i c e .
For
i n s t a n c e , one a p p a r a t u s which has been c o m m e r c i a l l y a v a i l a b l e
s i n c e the e a r l y s i x t i e s , the ' t h i n - f i l m ' d e v i c e (2) i s s u c c e s s f u l l y o p e r a t e d a t both a n a l y t i c a l and p r e p a r a t i v e s c a l e s , e . g .
0097-6156/86/0314-0169$06.00/0


170
up to 5ml/hr o f a 2% p r o t e i n s o l u t i o n or about 0.1gm/hr.

Further
s c a l e - u p of t h i s a p p a r a t u s i s l i m i t e d by buoyancy-induced c o n v e c t i o n i n the f l u i d f i l m ( 3 , 4 ) which g r e a t l y reduces s o l u t e r e s o l u t i o n (7).
Two a l t e r n a t i v e d e s i g n s have succeeded i n s t a b i l i z i n g the
hydrodynamic f l o w and i n c r e a s i n g d e v i c e c a p a c i t y by, i n one c a s e ,
a p p l y i n g a shear s t r e s s a c r o s s a c y l i n d r i c a l annulus w i t h a
r a d i a l e l e c t r i c f i e l d and a x i a l f l o w ( 6 , 7 ) a n d , i n the second
c a s e , by o p e r a t i n g a ' t h i n - f i l m ' CFE i n the low g r a v i t y e n v i r o n ment a v a i l a b l e i n space ( 8 h
The former a p p a r a t u s i s c a p a b l e of
p r o c e s s i n g p r o t e i n s o l u t i o n s a t throughputs g r e a t e r than
2000ml/hr but w i t h s i g n i f i c a n t l y poorer r e s o l u t i o n than t h a t
o b t a i n a b l e i n the t h i n - f i l m d e s i g n . The l a t t e r column a l s o
a c h i e v e s throughputs o f t h i s magnitude but space s h u t t l e payload
expenses, n e a r l y $100/ounce, r e n d e r e x t r a t e r r e s t r i a l p r o c e s s i n g
e c o n o m i c a l l y p r o h i b i t i v e i n a l l but a handful of e x c e p t i o n a l
cases.
A m o d i f i e d v e r s i o n of the ' t h i n - f i l m ' chamber has been p r o posed (9^ which i s c a p a b l e of p r o c e s s i n g l a b i l e b i o m a t e r i a l s a t
e l e v a t e d throughputs w h i l e r e c o v e r i n g p r o d u c t a t a r b i t r a r i l y high
purity.
The proposed d e s i g n i s based on the ' t h i n - f i l m ' d e v i c e
b u t uses e f f l u e n t r e c y c l e w i t h p r e s c r i b e d b a c k s h i f t to a c h i e v e
e f f e c t i v e c o u n t e r c u r r e n c y i n the e l e c t r o p h o r e s i s column, thus
a l l o w i n g v i r t u a l l y complete s e p a r a t i o n of feed i n t o two p r o d u c t
streams.
In a d d i t i o n , r e p e a t e d c o n t a c t between s o l u t e and
e l e c t r i c f i e l d a l l o w s the power i n p u t to be reduced and the
t r a n s v e r s e t h i c k n e s s of the chamber to be i n c r e a s e d , y i e l d i n g a
p r o p o r t i o n a t e i n c r e a s e i n the d e v i c e c a p a c i t y .
In the f o l l o w i n g
paper the performance and c h a r a c t e r i s t i c s of the m o d i f i e d
e l e c t r o p h o r e s i s chamber are i n v e s t i g a t e d i n the l i m i t of
v a n i s h i n g l y small d i f f u s i o n c o e f f i c i e n t s by u s i n g an approximate
mathema t i c a l model.
Continuous Flow E l e c t r o p h o r e s i s
The c l a s s i c t h i n - f i l m CFE d e v i c e d e p i c t e d i n F i g u r e 1 c o n s i s t s of
a t h i n , broad chamber through which a l a m i n a r c u r t a i n of b u f f e r e d
f l u i d flows a x i a l l y .
Feedstock i s c o n t i n u o u s l y i n j e c t e d i n t o
t h i s flow near the chamber e n t r a n c e and an e l e c t r i c f i e l d imposed
a c r o s s the c u r t a i n causes charged s p e c i e s to m i g r a t e l a t e r a l l y as
they pass through the chamber. The s o l u t e s form d i s c r e t e bands
a c c o r d i n g to t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s and e l u t e a t the
bottom of the chamber where the i n d i v i d u a l bands are c o l l e c t e d
through a m u l t i p l i c i t y of p o r t s .
J o u l e heat generated by the i o n i c c u r r e n t i s removed through
c o o l i n g j a c k e t s mounted on the t r a n s v e r s e w a l l s . T h i s c r e a t e s a
t r a n s v e r s e temperature g r a d i e n t which d r i v e s a s t a b l e and u s u a l l y
d e l e t e r i o u s c o n v e c t i v e f l o w (3).
F u r t h e r m o r e , the a x i a l temp e r a t u r e g r a d i e n t i n the thermal e n t r a n c e r e g i o n (10,11) near the
t i p s of the e l e c t r o d e s may d r i v e a buoyancy i n s t a b i l i t y when the
thermal g r a d i e n t exceeds a c r i t i c a l v a l u e ( 4 , 1 2 ) .
A p a r t from the
p h y s i c a l p r o p e r t i e s o f the c a r r i e r f l u i d , the c r i t i c a l temperature


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171
g r a d i e n t r e q u i r e d to d r i v e t h i s i n s t a b i l i t y depends on the r a t e
o f power d i s s i p a t i o n i n the f l u i d and i n c r e a s e s w i t h the f i f t h
power of the t r a n s v e r s e chamber t h i c k n e s s . N o t i n g t h a t the power
i n p u t i s f i x e d by the r e q u i r e m e n t t h a t s e p a r a t i o n be a c c o m p l i s h e d
i n a chamber of moderate l e n g t h , t h i s s t r o n g dependence on the
t r a n s v e r s e t h i c k n e s s e n s u r e s t h a t t e r r e s t r i a l CFEs a r e g e n e r a l l y
r e s t r i c t e d to chamber t h i c k n e s s e s of 0(2mm). Thus the r e q u i r e
ment of s t a b l e , l a m i n a r flow d i r e c t l y l i m i t s throughput by
c o n s t r a i n i n g the t r a n s v e r s e t h i c k n e s s of the d e v i c e .
The l a t e r a l d e f l e c t i o n e x p e r i e n c e d by a p a r t i c l e p a s s i n g
through the chamber depends s t r o n g l y upon i t s t r a n s v e r s e p o s i t i o n
s i n c e the a x i a l v e l o c i t y p r o f i l e i s p a r a b o l i c . P a r t i c l e s n e a r e r
the t r a n s v e r s e w a l l s are exposed to the e l e c t r i c f i e l d l o n g e r
than those near the c e n t e r l i n e , a n d , i n the absence of
e l e c t r o o s m o s i s , m i g r a t e a g r e a t e r l a t e r a l d i s t a n c e a c r o s s the
chamber, d i s t o r t i n g the s o l u t e i n t o c r e s c e n t shaped bands.
E l e c t r o o s m o s i s , which i s the e l e c t r i c a l l y d r i v e n f l o w o f f l u i d i n
the double l a y e r a d j a c e n t to the charged t r a n s v e r s e w a l l s , s e t s
up a l a t e r a l c i r c u l a t i o n p a t t e r n , 0 ( y ) , which f l o w s p a r a l l e l to
the w a l l s , r e v e r s e s d i r e c t i o n near the e l e c t r o d e s and r e t u r n s
a l o n g the chamber c e n t e r l i n e ( 1 3 ) . The r e s u l t i n g p a r a b o l i c flow
s t r o n g l y a f f e c t s s o l u t e d i s p e r s i o n by a l t e r i n g the shape of the
' c r e s c e n t ' and, to some e x t e n t , the c r e s c e n t phenomenon may be
used to f o c u s s o l u t e s i n t o compact bands (14,15) by proper
a d j u s t m e n t of the - p o t e n t i a l of the t r a n s v e r s e s u r f a c e s .
Bands d i s t o r t e d by c r e s c e n t f o r m a t i o n w i l l tend to n e s t
i n s i d e one another c a u s i n g the s o l u t e e l u t i o n p r o f i l e s to
overlap.
As a r e s u l t , d e v i c e r e s o l u t i o n s u f f e r s and the y i e l d of
p u r i f i e d p r o d u c t d r o p s . To reduce c r e s c e n t f o r m a t i o n i n the
t h i n - f i l m CFE, the s o l u t e feed i s u s u a l l y r e s t r i c t e d to the p o r
t i o n of the f l o w f i e l d w i t h the l e a s t v a r i a t i o n i n a x i a l and
l a t e r a l v e l o c i t i e s . T h i s i s a c c o m p l i s h e d by c e n t e r i n g the f e e d
stream between the t r a n s v e r s e w a l l s and l i m i t i n g i t s d i a m e t e r to
no more than 30% of the chamber t h i c k n e s s ( 2 ) . T h e r e f o r e
c r e s c e n t f o r m a t i o n f u r t h e r reduces t h r o u g h p u t by l i m i t i n g the
p o r t i o n of the chamber through which s o l u t e may p a s s . Note t h a t
i f the feed were i n j e c t e d through a square p o r t spanning the
t r a n s v e r s e a x i s of the chamber, an o r d e r of magnitude i n c r e a s e i n
t h r o u g h p u t would immediately be r e a l i z e d . E l i m i n a t i n g or compen
s a t i n g f o r the d i s p e r s i v e i n f l u e n c e s a s s o c i a t e d w i t h c r e s c e n t
f o r m a t i o n would t h e r e f o r e y i e l d a s i g n i f i c a n t i n c r e a s e i n s c a l e
of the ' t h i n - f i l m ' a p p a r a t u s .
o s
R e c y c l e CFE (RCFE)
In the RCFE e f f l u e n t i s c o n t i n u o u s l y r e i n j e c t e d i n t o the chamber
v i a r e c y c l e streams as i n d i c a t e d i n F i g u r e 2 . Each r e c y c l e
stream r e i n j e c t i o n p o r t i s o f f s e t from i t s c o r r e s p o n d i n g e l u t i o n
p o r t by a s p e c i f i e d l a t e r a l d i s t a n c e , S, so t h a t upon r e c y c l e the
e f f l u e n t i s s h i f t e d back a g a i n s t the s o l u t e ' s e l e c t r o p h o r e t i c
m i g r a t i o n . When the s h i f t i s s m a l l s o l u t e m i g r a t e s i n the
p o s i t i v e z d i r e c t i o n but i f the s h i f t i s i n c r e a s e d s u f f i c i e n t l y


172
F i g u r e 1. Schematic o f the c l a s s i c CFE d e v i c e .

Solutes injected
near the e n t r a n c e s e p a r a t e l a t e r a l l y as they pass through the
chamber.
As d e p i c t e d here c r e s c e n t f o r m a t i o n causes the s o l u t e s '
e l u t i o n d i s t r i b u t i o n s to o v e r l a p .
mm
Buffer
W \ | V
W W
Recycle Streams
Low mobility
product
High mobility
product
F i g u r e 2. R e c y c l e CFE d e v i c e ,
a . Schematic of e l u t i o n p o r t to
i n l e t p o r t c o n n e c t i o n . E l u a n t i s c o n t i n u o u s l y r e i n j e c t e d a t the
chamber i n l e t . Feed i s i n j e c t e d i n t o one of the r e c y c l e s t r e a m s ,
b. Schematic o f complete RCFE d e v i c e .
R e c y c l e streams connect
the e l u t i o n and i n l e t p o r t s i n the r e c y c l e s e c t i o n .
Separated
p r o d u c t s are r e c o v e r e d through the e l u t i o n p o r t s f l a n k i n g the
r e c y c l e s e c t i o n , and an equal volume of b u f f e r i s fed through the
i n l e t p o r t s on e i t h e r s i d e of the r e c y c l e s e c t i o n .


12.
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High-Resolution,
High- Yield CFE
173
the s o l u t e w i l l begin to m i g r a t e i n the o p p o s i t e d i r e c t i o n .

The
v a l u e of the s h i f t a t which a s o l u t e begins to m i g r a t e i n the
o p p o s i t e d i r e c t i o n i s r e f e r r e d to below as i t s ' f l i p ' p o i n t .
By
a d j u s t i n g e i t h e r the s h i f t or the e l e c t r i c f i e l d s t r e n g t h , two
s o l u t e s of d i f f e r i n g e l e c t r o p h o r e t i c m o b i l i t y can be made to
migrate in opposite d i r e c t i o n s .
C r e s c e n t f o r m a t i o n o c c u r s i n the r e c y c l e chamber to the same
degree as i s found i n the c o n v e n t i o n a l CFE.
However, s i n c e the
e f f l u e n t i n c l u d e s s o l u t e g a t h e r e d from the t r a n s v e r s e w a l l
r e g i o n as w e l l as from the c e n t e r l i n e of the chamber and because
the r e c y c l e streams are t h o r o u g h l y mixed b e f o r e r e i n j e c t i o n ,
a f t e r a s u f f i c i e n t number of c y c l e s through the RCFE each s o l u t e
w i l l have sampled a l l p o s s i b l e t r a n s v e r s e p o s i t i o n s and w i l l thus
be d i s p l a c e d e n t i r e l y t o the l e f t or t o the r i g h t s i d e of the
chamber. T h i s e n s u r e s t h a t the d i s p e r s i o n a s s o c i a t e d w i t h c r e s c e n t
f o r m a t i o n i s e v e n t u a l l y c o u n t e r a c t e d by r e p e a t e d c o n t a c t w i t h the
electric field.
As noted above, when two components w i t h d i f f e r e n t m o b i l i t i e s
are i n t r o d u c e d i n t o the chamber, the s h i f t may be a d j u s t e d so
t h a t the s o l u t e s s e p a r a t e i n the r e c y c l e s e c t i o n of the chamber
and e l u t e on e i t h e r s i d e .
RCFE Model
A model of the RCFE has been developed to help c h a r a c t e r i z e
d e v i c e performance under v a r i o u s o p e r a t i n g c o n d i t i o n s . To f a c i l i t a t e s o l u t i o n v a r i o u s s i m p l i f i c a t i o n s have been i n t r o d u c e d i n t o
the model to a l l o w a n a l y t i c a l s o l u t i o n of the mass c o n s e r v a t i o n
e q u a t i o n w h i l e r e t a i n i n g the e s s e n t i a l o p e r a t i n g f e a t u r e s of the
device.
For example, i n the model p r e s e n t e d below the r e c y c l e
s e c t i o n extends i n f i n i t e l y a l o n g the l a t e r a l a x i s and i s assumed
to have i n f i n i t e s i m a l l y r e s o l v e d r e c y c l e p o r t s so t h a t the
g o v e r n i n g e q u a t i o n and boundary c o n d i t i o n s are c o n t i n u o u s .
F u r t h e r m o r e , the f e e d i s modeled as a l i n e source of u n i t
s t r e n g t h , e x t e n d i n g a c r o s s the t r a n s v e r s e dimension of the
chamber a t the i n l e t and no p r o v i s i o n i s made f o r p r o d u c t removal
a t the l e f t and r i g h t - h a n d s i d e s of the chamber.
S o l u t e t r a n s p o r t i n the i n t e r i o r of the e l e c t r o p h o r e s i s
chamber i s d e s c r i b e d by the c o n v e c t i v e - d i f f u s i o n e q u a t i o n ,
V {-DVC + UC} = 0
(1)
where D i s the m o l e c u l a r d i f f u s i o n c o e f f i c i e n t and

jJ = U ( y ) i + o s ( y ) k + u E k , i n c l u d i n g c o n v e c t i v e , e l e c t r o o s m o t i c and
e l e c t r o p h o r e t i c v e l o c T t y components.
The r e c y c l e boundary c o n d i t i o n equates the s o l u t e f l u x a t the chamber i n l e t a t x=0 t o the
f l u x through the chamber o u t l e t a t x=L w h i l e t a k i n g i n t o a c c o u n t
the l a t e r a l b a c k s h i f t of e f f l u e n t , S, d u r i n g r e c y c l e .
Feed
i n j e c t i o n i s i n c l u d e d i n the boundary c o n d i t i o n as an impulse of
strength, J , located at (x,z)=(0,0).
x

174
u (y)
"ZbW
~B
'
J (L,y,z-S)dy + J 6(y,z)
x
(2)
In the low P e c l e t number l i m i t , Pe=<U >B/D<<l, an a n a l y t i c a l

s o l u t i o n of E q u a t i o n 1 has been d e r i v e d ( 1 6 , 9 ) and used to
i n v e s t i g a t e the performance c h a r a c t e r i s t i c s of the r e c y c l e
chamber.
S i m i l a r l y , f o r a packed column the d i f f u s i o n c o e f
f i c i e n t s are r e p l a c e d w i t h s e p a r a t e a x i a l and l a t e r a l d i s p e r s i o n
c o e f f i c i e n t s computed u s i n g T a y l o r - A r i s t h e o r y . The r e s u l t i n g
e q u a t i o n i s l i n e a r w i t h c o n s t a n t c o e f f i c i e n t s and i s a l s o ame
n a b l e to a n a l y t i c a l s o l u t i o n .
However, i n the high P e c l e t number
r e g i m e , the p a r a b o l i c dependence on the t r a n s v e r s e c o o r d i n a t e of
the c o n v e c t i v e and e l e c t r o o s m o t i c v e l o c i t i e s s e v e r e l y c o m p l i c a t e s
s o l u t i o n of the g o v e r n i n g e q u a t i o n s . In g e n e r a l , r e p o r t e d d i f f u s i v i t i e s of o l i g o p e p t i d e s are l e s s than or a p p r o x i m a t e l y equal
t o 1 0 - c m / s e c so t h a t P e c l e t numbers, Pe=<Ux>B/D, i n the CFE
chamber are g r e a t e r than about 1 0 , 0 0 0 . Because of t h i s d i f f u s i o n
c o n t r i b u t e s i n s i g n i f i c a n t l y to the d i s p e r s i o n and t h i s suggests
t h a t a c o n v e c t i v e d i s p e r s i o n model c o u l d be e f f e c t i v e l y used to
p r o v i d e a good q u a l i t a t i v e d e s c r i p t i o n of column performance.
To o b t a i n a model of p r a c t i c a l u t i l i t y which s t i l l emphasizes
the i m p o r t a n t c h a r a c t e r i s t i c s of the RCFE i n the high Pe l i m i t ,
i t i s d e s i r a b l e to s u b s t i t u t e a d i s p e r s i o n e q u a t i o n i n v o l v i n g the
t r a n s v e r s e average c o n c e n t r a t i o n f o r E q u a t i o n 1. The y - d e p e n d e n t
v e l o c i t i e s are r e p l a c e d by t h e i r t r a n s v e r s e averages and the c o n
v e c t i v e d i s p e r s i o n of s o l u t e a s s o c i a t e d w i t h the c r e s c e n t pheno
menon i s lumped i n t o a l a t e r a l d i s p e r s i o n c o e f f i c i e n t , K, which
s i m p l i f i e s the a n a l y s i s c o n s i d e r a b l y and a l l o w s a n a l y t i c a l s o l u
t i o n of the governing e q u a t i o n ,

(3)
T h i s model of c o n v e c t i v e d i s p e r s i o n i n the chamber y i e l d s

o n l y a f a i r a p p r o x i m a t i o n of the c o n c e n t r a t i o n p r o f i l e f o r a
s o l u t e which makes a s i n g l e pass through the chamber ( F i g u r e 3)
and, i n p a r t i c u l a r , i t n e g l e c t s the asymmetry i n the p r o f i l e
caused by the c r e s c e n t phenomenon.
However, u s i n g moments to
compute the d i s p e r s i o n c o e f f i c i e n t d i r e c t l y from the a n a l y t i c a l
s o l u t i o n f o r the f l u x i n a s i n g l e pass through the chamber a l l o w s
the approximate d i s p e r s i o n model to q u a l i t a t i v e l y p r e d i c t the
most i m p o r t a n t f e a t u r e s of the RCFE as w i l l be d i s c u s s e d below.
In o r d e r t h a t the d i s p e r s i o n model mimic the c o n v e c t i v e model,
i s a d j u s t e d so t h a t the s o l u t e d i s t r i b u t i o n p r e d i c t e d by each
model has i d e n t i c a l f i r s t and second moments. T h i s ensures t h a t
the s i m p l i f i e d d i s p e r s i o n model w i l l y i e l d a r e a s o n a b l e e s t i m a t e
o f the band s p r e a d i n g .
F i g u r e 4 , which i s a p l o t of the d i s p e r s i o n c o e f f i c i e n t as a
f u n c t i o n of the e l e c t r o p h o r e t i c m o b i l i t y f o r f i x e d e l e c t r i c f i e l d

12.
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GOBIE A N D IVORY
15-
10-

5^
-1
(cm)
F i g u r e 3 . Comparison o f s i n g l e - p a s s CFE models u s i n g parameter
v a l u e s g i v e n i n T a b l e I I . The long t a i l e x h i b i t e d by the z e r o
d i f f u s i o n model (sharp peak) i s caused by c r e s c e n t f o r m a t i o n .
The d i s p e r s i o n caused by c r e s c e n t f o r m a t i o n has been approximated
i n the d i s p e r s i o n model (normal curve) by a d j u s t i n g so t h a t
both d i s t r i b u t i o n s have i d e n t i c a l v a r i a n c e .
0.012-
0 -008-
0 .004H
0 .000-8
"
1 1 1 1 1 1
-6
1 1 1 1 1
1 '
-2
(nm/sec cm/v)
F i g u r e 4 . E f f e c t i v e d i s p e r s i o n c o e f f i c i e n t , K, v s . e l e c t r o
p h o r e t i c m o b i l i t y , IJ. A S approaches , where
=
2.15 - / ^ , d i s p e r s i o n caused by c r e s c e n t f o r m a t i o n
v a n i s h e s . See T a b l e I I f o r parameters used o t h e r than i n d i c a t e d
i n the f i g u r e .
0

176
s t r e n g t h and e l e c t r o o s m o t i c w a l l v e l o c i t y , demonstrates t h a t a
d i s p e r s i o n c o e f f i c i e n t computed i n t h i s f a s h i o n w i l l c o r r e c t l y
p r e d i c t f o c u s i n g of the s o l u t e band ( 1 4 , 1 5 ) , i . e . a minimum i n
the d i s p e r s i o n , when the e l e c t r o p h o r e t i c m o b i l i t y i s equal and
o p p o s i t e to the e l e c t o o s m o t i c w a l l m o b i l i t y .
Note t h a t t h e
e f f e c t i v e l a t e r a l d i s p e r s i o n c o e f f i c i e n t , K, i s t h r e e o r more
o r d e r s of magnitude g r e a t e r than the m o l e c u l a r d i f f u s i o n c o e f
f i c i e n t , D*10"6cm /s, so d i f f u s i o n may s a f e l y be n e g l e c t e d i n
t h i s model.
We do n o t mean t o imply t h a t E q u a t i o n 3 i s a p p r o p r i a t e as a
model o n l y because i t can be s o l v e d a n a l y t i c a l l y .
But i n f a c t ,
under c e r t a i n c o n d i t i o n s i t i s p o s s i b l e to o b t a i n a n a y t i c a l s o l u
t i o n s to the f i r s t o r d e r PDE which r e s u l t s when the d i f f u s i v e
terms a r e n e g l e c t e d i n E q u a t i o n 1. Under these c o n d t i o n s t h i s
model p r e d i c t s s i n g l e pass c o n c e n t r a t i o n p r o f i l e s a c c u r a t e l y a n d ,
when a p p l i e d to the RCFE i t y i e l d s v a l u e s f o r the ' f l i p * p o i n t ,
i . e . S=-L{yE/<U >}, which a r e i d e n t i c a l to those p r e d i c t e d i n the
low P e c l e t number l i m i t , s u g g e s t i n g t h a t t h i s c o n d i t i o n i s i n d e
pendent of both m o l e c u l a r d i f f u s i o n and the e l e c t r o o s m o t i c
f l o w r a t e f o r a l l v a l u e s o f the P e c l e t number. I t i s t h e r e f o r e
e x p e c t e d t h a t E q u a t i o n 3 w i l l y i e l d a c c u r a t e p r e d i c t i o n s o f both
the f l i p p o i n t and the f l u x p r o f i l e s i n the RCFE.
S i n c e t h i s model n e g l e c t s a x i a l d i s p e r s i o n and uses a t r a n s
v e r s e average c o n c e n t r a t i o n , the r e c y c l e c o n d i t i o n , E q u a t i o n 2 ,
reduces to

<C(0,z)> = <CL,z - S)>
<5(z).
(4)
The t w o - s i d e d L a p l a c e t r a n s f o r m ( 7 ) i n
f
()
f(z)e"
p z
dz
(5)
i s used to o b t a i n the s o l u t i o n t o E q u a t i o n 3 s i n c e the s h i f t r u l e

w i l l r e a d i l y handle the r e c y c l e b a c k s h i f t i n boundary c o n d i t i o n
4.
The model e q u a t i o n i s s o l v e d i n t r a n s f o r m space and i n v e r t e d
v i a e x p a n s i o n i n r e s i d u e s so the s o l u t i o n i s e x p r e s s e d as a
s e r i e s of e x p o n e n t i a l f u n c t i o n s .
T a b l e I.
Dimensionless
Parameters.
xDi/B
zDi/B
LDi/B
SDi/B
/ U
Di = K/B U

12.
177
GOBIE A N D IVORY
The c o n c e n t r a t i o n d i s t r i b u t i o n a t the chamber e x i t , i n terms of

the d i m e n s i o n l e s s parameters d e f i n e d i n T a b l e I, i s determined to
be
<0(,)> = s g n U ) e x p U / 2 D i ) \ e x p ( C [ D i r
n=0
2
- /40 ]
2
+ r c)/{R'(r )}
n
(6)

where the c h a r a c t e r i s t i c e q u a t i o n i s
R(r )
n
= 1 - expU[Di r
w i t h two r e a l
- e / 4 D i ] - a[e/2Di +r ])=0
2
(7)
zeros,
-
2Di
^Q j
2 +
2XDi
o f which the f i r s t can be used to determine the f a r - f i e l d f l u x

and the second used to determine the l e n g t h s c a l e over which the
l a t e r a l c o n c e n t r a t i o n g r a d i e n t decays to z e r o .
In a d d i t i o n t h e r e
i s a m u l t i t u d e of complex z e r o s o f E q u a t i o n 7 which moderate
s o l u t e b e h a v i o r c l o s e r t o z=0.
The term i n the summation i n E q u a t i o n 6 which i n v o l v e s the
f i r s t r e a l z e r o has no z dependence and r e p r e s e n t s the s o l u t e
c o n c e n t r a t i o n a t i n f i n i t e l a t e r a l d i s t a n c e from the f e e d .
This
i s termed the " f a r - f i e l d " f l u x i n t h i s paper and, i n terms of
dimensional q u a n t i t i e s , i t i s
J
f f
(z)
B/L sgn
(z)
!
(<; * )
The denominator r e p r e s e n t s the mean d i s p l a c e m e n t of the
s o l u t e per c y c l e and i t s s i g n i n d i c a t e s the l a t e r a l d i r e c t i o n ,
e i t h e r to the l e f t (-) or r i g h t (+), to which the s o l u t e m i g r a t e s
under the combined i n f l u e n c e of e l e c t r o p h o r e s i s and r e c y c l e .
In
a l l cases the c o n c e n t r a t i o n a t the o t h e r end of the chamber f a l l s
to zero.
Note t h a t the f a r f i e l d c o n c e n t r a t i o n may become
a r b i t r a r i l y l a r g e when the two terms a r e equal i n magnitude and
o p p o s i t e i n s i g n and t h i s i s the p o i n t a t which the f a r - f i e l d
f l u x ' f l i p s ' to the o p p o s i t e s i d e of the chamber.

178
Table I I .
Nominal V a l u e s of Parameters Used i n Sample C a l c u l a t i o n s

2B
S
=
=
=
=
0.375 cm
0 . 0 cm
2 . 5 -cm/v-sec
1.0x10*6 cm /sec
2
L
U
x
0
= 1 6 . 0 cm
=
0.70 cm/sec
= +2.15 - c m / v - s e c
=
1 . 6 8 x l 0 " cm /sec
3
Discussion
To a s s e s s the performance of the RCFE a t e l e v a t e d P e c l e t numbers
s e v e r a l sample c a l c u l a t i o n s were performed u s i n g the parameters
given i n Table I I .
R e c y c l i n g a l l o w s the e l e c t r i c f i e l d s t r e n g t h
t o be reduced from 70 v/cm i n a s i n g l e pass CFE to 41.25 v/cm
i n t h i s example a n d , as a consequence of the r e d u c t i o n i n J o u l e
h e a t i n g , the chamber t r a n s v e r s e t h i c k n e s s can be i n c r e a s e d by a
f a c t o r of 1 . 7 0 . Assuming square i n l e t p o r t s , the throughput can
be i n c r e a s e d by a f a c t o r of 2.88 over the s i n g l e p a s s , ' t h i n f i l m ' chamber.
The l a t e r a l w i d t h of the chamber i s d i c t a t e d by the s m a l l e s t
p o s i t i v e and n e g a t i v e (nonzero) arguments of the the e x p o n e n t i a l
f u n c t i o n s i n E q u a t i o n 6 s i n c e these terms determine the
l e n g t h s c a l e o v e r which the c o n c e n t r a t i o n g r a d i e n t s decay to z e r o .
In the neighborhood of the f e e d p o r t E q u a t i o n 6 converges s l o w l y
and dashed l i n e s have been used i n F i g u r e s 5-7 t o i n d i c a t e
approximate c o n c e n t r a t i o n s i n those r e g i o n s where adequate c o n
vergence c o u l d n o t be o b t a i n e d even w i t h the use of n u m e r i c a l
a c c e l e r a t i o n techniques (18).
F i g u r e 5 i l l u s t r a t e s the e f f e c t on a s i n g l e s o l u t e of v a r y i n g
the s h i f t , S , w h i l e h o l d i n g a l l o t h e r parameters c o n s t a n t . When
S=0 the e f f l u e n t i s r e c y c l e d d i r e c t l y overhead and i s d i s p l a c e d
t o the r i g h t on subsequent c y c l e s through the chamber. As the
magnitude of the b a c k s h i f t i s i n c r e a s e d , S<0, the r e l a x a t i o n
l e n g t h s c a l e i n c r e a s e s s l o w l y w h i l e the f a r - f i e l d c o n c e n t r a t i o n
i n c r e a s e s r a p i d l y a c c o r d i n g to E q u a t i o n 9 . When the s h i f t r e a c h e s
the ' f l i p ' p o i n t , the f a r - f i e l d c o n c e n t r a t i o n i s p r e d i c t e d to be
i n f i n i t e and to f i l l both s i d e s of the column. I f the s h i f t i s
f u r t h e r p e r t u r b e d , s o l u t e i s d i s p l a c e d to the l e f t - h a n d s i d e of
the chamber a n d , as the magnitude of the b a c k s h i f t i s i n c r e a s e d
s t i l l f u r t h e r , the f a r - f i e l d f l u x d e c r e a s e s but s o l u t e e l u t e s on
the l e f t - h a n d s i d e of the chamber.
In F i g u r e 6 the l a t e r a l d i s p e r s i o n c o e f f i c i e n t , K, i s v a r i e d
by an o r d e r of magnitude about the nominal value of 1 . 7 x l 0 " c m / s
o b t a i n e d u s i n g the parameters i n T a b l e I I .
For s m a l l an
o s c i l l a t i o n i n the f l u x appears near z=0 because the feedband,
m o d e l l e d i n our c a l c u l a t i o n s as a D i r a c d i s t r i b u t i o n , i s not
e n t i r e l y d i s p e r s e d u n t i l i t has passed through the chamber
s e v e r a l t i m e s . The t h r e e peaks e v i d e n t i n t h i s curve r e p r e s e n t
the s o l u t e on i t s f i r s t , second and t h i r d c y c l e s through the
chamber, b e f o r e the impulse has spread out over the l a t e r a l a x i s .
3

12.
GOBIE A N D IVORY
179
Effect
of shift
7
6

\
-0.1875 c r r N ^
1
0
S = - 0 . 3 7 5 cm
-8
0 cm
-4
(cm)
F i g u r e 5 . E f f e c t of v a r y i n g the s h i f t .
As the b a c k s h i f t i s
i n c r e a s e d the f a r - f i e l d f l u x i n c r e a s e s as does the l e n g t h of the
toe of the d i s t r i b u t i o n .
Once the s h i f t passes the " f l i p p o i n t " ,
the s o l u t e m i g r a t e s o p p o s i t e to the d i r e c t i o n of i t s
e l e c t r o p h o r e t i c motion.
See T a b l e II f o r parameters used o t h e r
than i n d i c a t e d i n the f i g u r e .
F i g u r e 6.
E f f e c t of v a r y i n g the l a t e r a l d i s p e r s i o n c o e f f i c i e n t .
D i s p e r s i o n has a marked e f f e c t on the r e l a x a t i o n l e n g t h s c a l e . See
T a b l e II f o r parameters used o t h e r than i n d i c a t e d i n the f i g u r e .


180
As the d i s p e r s i o n c o e f f i c i e n t i s i n c r e a s e d t h i s o s c i l l a t i o n
d i s a p p e a r s s i n c e the s o l u t e i s q u i c k l y smeared over the e n t r a n c e
region.
For l a r g e K, the r e l a x a t i o n l e n g t h s c a l e , which i s d i c
t a t e d p r i m a r i l y by the second exponent of E q u a t i o n 8 , i n c r e a s e s
r a p i d l y with i n c r e a s i n g d i s p e r s i o n c o e f f i c i e n t since that expo
nent decreases w i t h the square of the d i s p e r s i o n number, D i .
F i g u r e 7 p r e s e n t s the r e s u l t s of a s i m u l a t e d b i n a r y s e p a r a
t i o n of s o l u t e s which have m o b i l i t i e s of 2.5p-cm/v-s and
3 . 0 y - c m / v - s , r e s p e c t i v e l y , w i t h s h i f t , S=-0.375cm. The r e l a t i v e
f l u x e s shown i n d i c a t e t h a t the low m o b i l i t y component i s c o n
c e n t r a t e d about 6x above i t s feed v a l u e w h i l e the f a s t e r moving
component i s c o n c e n t r a t e d to about 4x i t s feed v a l u e .
Note t h a t
the s e p a r a t i o n i s e f f e c t e d i n a chamber about 16cm wide and t h a t
both s o l u t e s can be r e c o v e r e d a t a r b i t r a r i l y high p u r i t i e s by
e x t e n d i n g the b r e a d t h of the r e c y c l e s e c t i o n .
Typical
Table III.
E l e c t r o k i n e t i c Parameters of C o l l o i d a l
and B i o l o g i c a l M a t e r i a l s
(pm-cm/volt/sec)
Serum P r o t e i n s :
Average A x i a l V e l o c i t y :
(Hemoglobin) 0.12
0.074 cm/sec
(Albumin)
0.59
Red Blood C e l l s :
(Human RBC) 1.16

0.093 cm/sec
(Sheep RBC) 1.44
P o l y s t y r e n e Latex
Particles:
(.2ym d i a . )
6.5
0.235 cm/sec
(.8 d i a . )
9.2
E l e c t r i c F i e l d S t r e n g t h : 25 volts/cm
E l e c t r o d e L e n g t h : 16 cm
E l e c t r o p h o r e t i c Wall M o b i l i t y : - 1 . 0 ijm-cm/volt-sec
To help put these c a l c u l a t i o n s i n p e r s p e c t i v e T a b l e I I I c o n

t a i n s t y p i c a l v a l u e s of the e l e c t r o p h o r e t i c m o b i l i t i e s measured
f o r v a r i o u s m a t e r i a l s of w i d e l y v a r y i n g s i z e .
In g e n e r a l , one
s h o u l d e x p e c t lower m o l e c u l a r w e i g h t p a r t i c l e s to have m o b i l i t i e s
i n the range, 0 . 1 - 3 . 0 y m - c m / v o l t - s e c , depending on the c o m p o s i
t i o n of the c a r r i e r f l u i d w h i l e c e l l s and p a r t i c u l a t e s would have
m o b i l i t i e s i n the r a n g e , 0 . 5 - 1 0 . 0 - / t - s e c .
However, d i f
f e r e n c e s i n the r e l a t i v e magnitudes of the m o b i l i t i e s between the
v a r i o u s groups c i t e d above are r e a d i l y compensated by a d j u s t i n g


12.
GOBIE A N D IVORY
181
the c a r r i e r f l u i d f l o w - r a t e so t h a t p a i r s of s p e c i e s which have

low m o b i l i t i e s a r e exposed l o n g e r to the e l e c t r i c f i e l d .
R e c a l l t h a t i n the h y p o t h e t i c a l RCFE modelled i n t h i s paper,
s e v e r a l s i m p l i f y i n g assumptions have been invoked to f a c i l i t a t e
i n v e s t i g a t i o n of the o p e r a t i n g c h a r a c t e r i s t i c s of the d e v i c e and
one of these assumptions was t h a t no p r o v i s i o n be made i n the
model f o r p r o d u c t w i t h d r a w a l .
The a d d i t i o n of w i t h d r a w a l p o r t s
a t e i t h e r end of the RCFE w i l l cause the c o n c e n t r a t i o n s of p r o d u c t to drop below t h e i r feed c o n c e n t r a t i o n s , t h w a r t i n g a t t e m p t s
to concentrate s o l u t e .
However, t h i s problem i s c i r c u m v e n t e d by
adding " r e g e n e r a t o r s " on e i t h e r end of the column as d e s c r i b e d i n
Figure 8.
The r e g e n e r a t o r s a r e e s s e n t i a l l y a d d i t i o n a l r e c y c l e s e c t i o n s
appended to e i t h e r s i d e o f the main r e c y c l e s e c t i o n i n which the
s h i f t i s a l t e r e d by one or more p o r t w i d t h s . When the s h i f t i s
p r o p e r l y a d j u s t e d , each s o l u t e ' s net l a t e r a l v e l o c i t y i s r e v e r s e d
i n the r e g e n e r a t o r s and upon e n t e r i n g the r e g e n e r a t o r s o l u t e i s
f o r c e d to r e t u r n to the r e c y c l e s e c t i o n . S i n c e the s o l u t e can
e x i t the chamber only through the e l u t i o n p o r t s l o c a t e d between
the r e c y c l e s e c t i o n and r e g e n e r a t o r , the c o n c e n t r a t i o n i n the
p r o d u c t stream w i l l approach the o r i g i n a l c o n c e n t r a t i o n i n the
feed.
F u r t h e r c o n c e n t r a t i o n of p r o d u c t s u s i n g the RCFE w i t h
r e g e n e r a t o r s can be e f f e c t e d by u s i n g m u l t i p l e f e e d s , d e c r e a s i n g
the w i d t h of the o u t l e t p o r t s or by r e c y c l i n g a p o r t i o n of the
p r o d u c t stream back i n t o the chamber near the i n n e r edge of the
regenerator s e c t i o n .
Conclusion
As has been demonstrated i n t h i s paper i n the high P e c l e t number
l i m i t , continuous e f f l u e n t recycle with s h i f t allows binary
s e p a r a t i o n of s o l u t e s to a r b i t r a r i l y high p u r i t i e s w i t h e s s e n t i a l l y complete r e c o v e r y of p r o d u c t s .
In a d d i t i o n , p r o d u c t s can
be r e c o v e r e d a t or above t h e i r feed c o n c e n t r a t i o n s a n d , because
the e l e c t r i c f i e l d can be s i g n i f i c a n t l y reduced when s o l u t e s a r e
r e p e a t e d l y c y c l e d through the chamber, the t r a n s v e r s e t h i c k n e s s
o f the chamber can be i n c r e a s e d p r o p o r t i o n a t e l y .
S e v e r a l f a c t o r s can be taken advantage of to i n c r e a s e
throughput i n the RCFE over the t h i n - f i l m d e s i g n on which the
chamber i s based: F i r s t , u s i n g the e n t i r e t r a n s v e r s e t h i c k n e s s
o f the chamber and assuming square i n l e t p o r t s i n c r e a s e s
throughput by 0 ( 1 0 ) .
Reducing the e l e c t r i c f i e l d s t r e n g t h and
r e o r i e n t i n g the d e v i c e i n t o a more s t a b l e c o n f i g u r a t i o n (18)
a l l o w s a f u r t h e r 0(10-100) increase i n throughput.
A further
0(10) i n c r e a s e i n throughput i s made p o s s i b l e by u s i n g m u l t i p l e
f e e d s t r e a m s , thus an o v e r a l l i n c r e a s e i n the c a p a c i t y of t h i n f i l m type c o n t i n u o u s e l e c t r o p h o r e t i c s e p a r a t i o n s of 0 ( 1 0 0 - 1 0 , 0 0 0 )
can be a c h i e v e d by u t i l i z i n g r e c y c l e .

182
Binary Separation
S = -0.375 cm
urn cm
5
4
/
/
/
/
/
/

\
L
/xm cm
^
- r
2
I
1
0
-4
1
4
1
8
12
(cm)
Figure
short
varies
other
7. S e p a r a t i o n of s o l u t e s d i f f e r i n g by 20% i n m o b i l i t y .
A
d i s t a n c e from the feed p o i n t , the p u r i t y of both components
e x p o n e n t i a l l y w i t h z.
See T a b l e II f o r parameters used
than i n d i c a t e d i n the f i g u r e .
F i g u r e 8 . Schematic of RCFE w i t h r e g e n e r a t o r s .
The s h i f t i n the
a d d i t i o n a l r e c y c l e s e c t i o n s i s chosen to r e v e r s e the s o l u t e ' s d i
r e c t i o n of m i g r a t i o n , c o n s t r a i n i n g i t to e x i t the chamber through
the e l u t i o n p o r t s between the r e c y c l e s e c t i o n and the r e g e n e r a
t o r s . P r o d u c t s are r e c o v e r e d a t or near t h e i r feed c o n c e n t r a t i o n .

12.
GOBIE A N D IVORY
Acknowledgment
T h i s m a t e r i a l i s based upon work supported i n p a r t by the
N a t i o n a l S c i e n c e Foundation under Grants No. CPE-8211483 and
CBT-8414218.
Legend of

Symbols
H a l f t h i c k n e s s of e l e c t r o p h o r e s i s
Concentration.
chamber.
<C>
Average c o n c e n t r a t i o n .
Molecular d i f f u s i o n c o e f f i c i e n t .
Di
Dimensionless d i s p e r s i o n c o e f f i c i e n t .
E l e c t r i c f i e l d strength.
"
Feed source
strength.
Axial
jff
Far-field
Length of e l e c t r o p h o r e s i s
chamber.
Length of e l e c t r o p h o r e s i s
chamber.
Electroosmotic velocity
Roots of the c h a r a c t e r i s t i c e q u a t i o n .
R(r )
C h a r a c t e r i s t i c equation for
S h i f t distance.
L[
Velocity
O S
flux.
flux.
Axial
Average a x i a l
x,y,z
axial,
r .
n
vector.
<U >
profile.
velocity.
velocity.
transverse,
lateral
coordinates.
Dirac d i s t r i b u t i o n .
Dimensionless e l e c t r o p h o r e t i c
Dimensionless l a t e r a l
coordinate.
D i m e n s i o n l e s s chamber
length.
Electrophoretic
Electroosmotic wall
mobility.
coordinate.
y s
0
velocity.
mobility.
Dimensionless a x i a l
Dimensionless s h i f t d i s t a n c e .
Gradient
operator.

184
Literature Cited
1.
2.
3.
4.

5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Philpot, J. St. L. Trans. Farad. Soc 1940, 36, 38.

Hannig, K. In "Techniques of Biochemical and Biophysical
Morphology"; Glick, D.; Rosenbaum, R., Eds.; Wiley: New York,
1972; Vol. 1, pp. 191-232.
Ostrach, S. J. Chrom. 1977, 140, 187-195.
Saville, D. .; Ostrach, S. "Fluid Mechanics of Continuous
Flow Electrophoresis"; Final Report, Contract NAS-8-31349
Code 361, 1978.
Rhodes, P. H. "Sample Stream Distortion Modeled in Continuous
Flow Electrophoresis"; NASA TM-78178, 1979.
Philpot, J. St. L. In "Methodological Developments in
Biochemistry"; Reid, E., Ed.; 1966; Longmans: England; Vol.
2, pp. 81-85.
Mattock, P.; Aitchison, G. F.; Thomson, A. R. Sep. Pur. Meth.
1980, 9, 1.
Morrison, D. R.; Barlow, G. H.; Cleveland, C.; et al. Adv.
Space Res. 1984, 4, 67-76.
Gobie, W. .; Beckwith, J. B.; Ivory, C. F. Biotechnology
Prog. 1985, 1, 60.
Lynch, E. D.; Saville, D. A. Chem. Eng. Commun. 1981, 9,
201-211.
Naumann, R. J.; Rhodes, P. H. Sep. Sci. 1984, 19,51.
Saville, D. A. PhysicoChemical Hydro. 1980, 1, 297-307.
Nee, T. W. J. Chrom. 1975, 105, 231.
Strickler, A. Sep. Sci. 1967, 2, 335.
Strickler, .; Sacks, T. Ann. New York Acad. Sci. 1973, 209,
497.
Ivory, C. F.; Gobie, W. A.; Turk, R. S. Electrophoresis '83,
1984, p. 293.
van der Pol, .; H. Bremmer, "Operational Calculus Based on
the Two-Sided Laplace Integral"; Cambridge University Press;
1950.
Turk, R. S.; Ivory, C. F. Chem. Eng. Sci., 1984, 39, 851.

13
Scale-Up of Isoelectric Focusing
Milan Bier

Biophysics Technology Laboratory, University of Arizona, Tucson, AZ 85721
The paper describes some applications to large scale protein

fractionation using a recycling isoelectric focusing apparatus.
Separation is achieved in free solution without the use of
supporting media. Various alternatives for the formation of
the pH gradient are discussed and results of a computer
simulation are presented.
We are presently witnessing revolutionary developments i n applied

biology due t o the rapid advances i n genetic engineering through
recombinant DNA and hybridoma technologies. The progress i n these
areas has surpassed even the most o p t i m i s t i c projections o f j u s t a
few years ago.
The economic impact o f these technologies has been
amply covered i n the s c i e n t i f i c and l a y press, including reviews by
the O f f i c e o f Technology Assessment o f the U.S. Congress (1).
At present, these technologies have rendered possible the production o f v i r t u a l l y unlimited quantities o f important new biologies
which were previously a v a i l a b l e only i n minutest q u a n t i t i e s . Human
i n s u l i n , interferon, human growth hormone, foot and mouth disease
vaccine are but a few examples. For the purpose o f t h i s symposium i t
should be emphasized that these proteins are often f i r s t obtained i n
the form of crude extracts, heavily contaminated by extraneous matter,
derived from the host organism. The p u r i f i c a t i o n o f the desired end
product i s e s s e n t i a l i f i t i s t o be used as a pharmaceutical. The
magnitude o f the problem can best be comprehended i f one r e a l i z e s
that the host c e l l , i . e . the modified microorganism or the hybridoma
c e l l , may contain w e l l over 5,000 d i f f e r e n t proteins, only one o f
which may be the desired a c t i v e p r i n c i p l e .
Isoelectric
Focusing
I s o e l e c t r i c focusing (IEF) i s unique among separation processes

as i t r e s u l t s i n a stationary steady state d i s t r i b u t i o n o f f r a c t i o n s
along the column a x i s . The f i n a l d i s t r i b u t i o n o f f r a c t i o n s i s
independent o f t h e i r i n i t i a l d i s t r i b u t i o n . As such, IEF has no
analogue i n other electrophoretic o r chromatographic methods and w e l l
deserves i t s current popularity (2).
0097-6156/ 86/ 0314-0185$06.00/ 0


186
IEF i s applicable only to amphoteric compounds, p r i m a r i l y proteins

or larger peptides, i . e . , compounds which have a c i d i c and basic
dissociable groups. As a r e s u l t , such compounds acquire a p o s i t i v e
net charge i n a c i d i c media and a negative net charge i n basic media.
The point o f charge inversion, i . e . , where the compounds e x h i b i t zero
net charge, i s refered to as the i s o e l e c t r i c point. When such species
are exposed to a d.c. e l e c t r i c f i e l d o f proper p o l a r i t y i n a pH
gradient, they w i l l migrate e l e c t r o p h o r e t i c a l l y toward the pH corresponding to t h e i r i s o e l e c t r i c point, where they become v i r t u a l l y
immobilized as d i f f u s i o n i s balanced by electrophoretic focusing.
This f i n a l d i s t r i b u t i o n o f f r a c t i o n s i s independent o f t h e i r i n i t i a l
d i s t r i b u t i o n within the pH gradient.
I s o e l e c t r i c point and molecular weight are the two most characteri s t i c parameters o f a protein. This i s the reason a n a l y t i c a l IEF has
so r a p i d l y gained widespread usage i n research as w e l l as i n q u a l i t y
control o f product development. The sequential separation according
to IEF and molecular weight i n tWD-dimensional electrophoresis gives
the sharpest r e s o l u t i o n o f p r o t e i n mixtures and permits the recognit i o n o f several thousand i n d i v i d u a l protein species i n extracts o f
various b i o l o g i c a l tissues (3).
A n a l y t i c a l IEF i s routinely c a r r i e d out i n gels o f polyacrylamide
or agarose, the pH gradient being formed n a t u r a l l y , i . e . through
the e f f e c t o f the e l e c t r i c a l current i t s e l f . Special buffer mixtures
were developed f o r t h i s purpose, the f i r s t and best known being
a v a i l a b l e under the tradename Ampholine . I t comprises the products
of random polymerization o f a mixture o f polyamines and a c r y l i c a c i d ,
thus containing a large number o f molecular species. Each species
focuses t o i t s i s o e l e c t r i c point and establishes thereby the pH
gradient.
Our laboratory has undertaken the task o f adapting the p r i n c i p l e s
of IEF t o large scale preparative purposes. We have taken a two
pronged approach:
1.
Development o f a better t h e o r e t i c a l understanding o f a l l
electrophoretic processes, IEF included, through mathematical modeling
and computer simulation. Our collaborators, Drs. S a v i l l e and
Palusinski, have presented t h i s work i n a paper (#5e) i n the Symposium
on Novel Separation Techniques i n Biotechnology I a t the present
AlChE meeting. Some o f the r e s u l t s were previously reported (4,5).
2.
Development o f instrumentation f o r large scale preparative
IEF. Gels are unsuitable f o r t h i s purpose and separation i n free
solution i s e s s e n t i a l . This e n t a i l s the solution to the two problems
common t o a l l electrophoretic processes:
stabilization of f l u i d
against convection and d i s s i p a t i o n o f Joule heat. Our f i r s t apparatus,
dubbed RIEF (Recycling IEF) solves these two problems i n a unique
fashion, by using screen elements f o r streamlining the f l u i d flow and
d i s s i p a t i n g the Joule heat i n heat-exchangers external to the focusing
apparatus (6). The apparatus i s o f modular design and could be
applied, a t l e a s t i n p r i n c i p l e , t o i n d u s t r i a l scale processing. The
demand f o r large scale processing has been lagging, however, and the
increased use o f the RIEF f o r research scale separations has prompted
us t o develop other instruments with smaller throughputs (7).
1

13.
BIER
Scale- Up of Isoelectric Focusing
187

Recycling I s o e l e c t r i c Focusing Apparatus

In most electrophoretic methods constituents separate according
to differences i n t h e i r mobility, i . e . , the rate o f t h e i r migration.
Scaling to i n d u s t r i a l l y meaningful throughputs i s complicated by two
problems: the need to d i s s i p a t e the Joule heat generated by the
e l e c t r i c current and the need to s t a b i l i z e the f l u i d against convecti v e remixing. IEF, to the contrary, i s not based on differences i n
migration rates, but instead a steady state i s obtained, proteins
d i s t r i b u t i n g themselves within the pH gradient i n accordance to t h e i r
i s o e l e c t r i c points. We have taken advantage of t h i s unique property
of IEF to solve the dual problem of heat d i s s i p a t i o n and f l u i d s t a b i l i z a t i o n by p h y s i c a l l y separating these two functions.
The apparatus i s presented schematically i n Figure 1.
The
solution to be fractionated i s continuously recycled between a m u l t i channel focusing apparatus and a multichannel heat-exchange r e s e r v o i r .
F l u i d flow through the focusing apparatus i s streamlined by means of
a p a r a l l e l array of b a f f l e s . Monofilament nylon screens of f i n e
porosity have been found most suitable f o r t h i s purpose as they allow
free transport o f a l l constituents, while s t i l l acting as convective
b a r r i e r s . Joule heat i s dissipated within the heat-exchange r e s e r v o i r , which i s external to the focusing apparatus i t s e l f (6).
Key components are the focusing c e l l and the heat exchange
r e s e r v o i r s . F l u i d i s r e c i r c u l a t e d between the corresponding channels
of these two components by the multichannel pump. We have a l s o
designed monitors f o r intermittent r e g i s t r a t i o n of p r o t e i n concentra t i o n (through u l t r a v i o l e t adsorption) and pH of each channel. While
not e s s e n t i a l f o r separations, these monitors are under the c o n t r o l
o f a Hewlett-Packard desk top computer (not shown i n the diagram).
The computer provides f o r numerical s c a l i n g and c a l i b r a t i o n of the
multiplexed monitors, receives the raw data from the i n t e r f a c e a t
preset time i n t e r v a l s , converts these i n t o o p t i c a l density and pH
units, provides printout of data i n r e a l time, stores them on tape,
or can display them on a p l o t t e r . In addition, i t can be programmed
f o r a v a r i e t y of s t a t i s t i c a l analyses or feed-back control of the
separation process.
I l l u s t r a t i v e Fractionation Results
The RIEF system i s completely modular and i s not r e s t r i c t e d to
any preset number o f compartments, r e s e r v o i r capacity or cross-area
of the focusing c e l l . Nevertheless, most of our data were obtained
with a ten compartment assembly. The p a r t i t i o n i n g of the focusing
apparatus by the screen b a f f l e s imposes a step-changing pH gradient,
rather than a l i n e a r one. The resolution i s l a r g e l y d i c t a t e d by the
pH range of Amholine used. For highest resolution, very narrow pH
range Ampholine i s needed. This can be obtained i n a preliminary
f r a c t i o n a t i o n i n the RIEF, followed by the reprocessing of selected
f r a c t i o n s i n a second run.
Figure 2 i l l u s t r a t e s the f r a c t i o n a t i o n of a complex sample, an
extract from Bermuda grass, which i s a common allergen. Because of
the heterogeneity o f the sample, a broad range Ampholine was used, pH
3.5 - 10. The r e s u l t s of the f r a c t i o n a t i o n were assessed by a n a l y t i c a l polyacrylamide g e l i s o e l e c t r i c focusing. Shown are the patterns
of the o r i g i n a l mixture and o f the ten RIEF f r a c t i o n s . As can be


188
Figure 1. Schematic presentation of the r e c y c l i n g i s o e l e c t r i c

focusing apparatus (RIEF). Reproduced with permission from
Ref. 6. Copyright 1979, Pierce Chemical Company.
Figure 2. RIEF separation of an aqueous extract of Bermuda grass

allergens. Reproduced i s a photograph of an a n a l y t i c a l polyacrylamide g e l focusing pattern of c o l l e c t e d samples from the
ten RIEF channels.


13.
BIER
189
seen, the RIEF has separated the o r i g i n a l mixture i n t o f r a c t i o n s of

increasingly basic i s o e l e c t r i c points. The f r a c t i o n s were tested
c l i n i c a l l y and a l l found to be s i m i l a r l y a l l e r g e n i c .
In Figure 3 i s documented the r e s o l u t i o n of a polyclonal antibody
sample e x h i b i t i n g only a few c l o s e l y spaced i s o e l e c t r i c bands. The
antibodies were r a i s e d to the b a c t e r i a l carbohydrates derived from
Micrococcus lysodeiktikus i n a r a b b i t from a colony outbred f o r
s i m p l i c i t y of t h e i r clonotype patterns. A f t e r p u r i f i c a t i o n by a f f i n i t y chromatography, the antibodies exhibited three major bands, with
very close i s o e l e c t r i c points. To effectuate t h e i r separation,
mmercial Ampholine was subf ractionated i n the RIEF and a narrow
cut, pH range 7.5 to 8.5, was used f o r the f r a c t i o n a t i o n (8). This
i l l u s t r a t e s the r e s o l u t i o n achievable i n c r i t i c a l separations.
Formation of pH
Gradients
Ampholine and other s i m i l a r c a r r i e r ampholytes are generally

used f o r the formation of pH gradients. They contain, however,
chemically i l l - d e f i n e d components which may contaminate the p u r i f i e d
products.
The development of other means f o r the generation of pH
gradients would be highly desirable and was a prime objective of much
of our t h e o r e t i c a l modeling. Three a l t e r n a t i v e s were pursued:
1. Focused (static) pH gradients: Mixtures of w e l l characterized low molecular compounds can be used instead of the i l l - d e f i n e d
polymeric buffer systems. A stable pH gradient i s again generated
through the focusing of components to t h e i r i s o e l e c t r i c point. In
Figure 4 we i l l u s t r a t e t h i s approach with the computer simulation of
the focusing of a mixture of cacodylic a c i d , h i s t i d i n e and t r i s (hydroxymethyl)andnomethane ( t r i s ) , a weak a c i d , an ampholyte and a
weak base, respectively. Starting from a uniform d i s t r i b u t i o n of a l l
three components, the focusing process begins with the accumulation
of the a c i d a t the anode and of the base a t the cathode. This generates a pH gradient which propagates towards the center of the column,
where the amphoteric h i s t i d i n e accumulates. This approach can be
u t i l i z e d experimentally f o r the focusing of proteins (9), but i t
suffers from the paucity of suitable compounds. The main amphoteric
compounds a v a i l a b l e are amino acids and oligopeptides, and t h e i r
i s o e l e c t r i c points do not adequately cover the e n t i r e pH range.
2. Dynamically formed pH gradients: Rather than allowing the
focusing of components to t h e i r i s o e l e c t r i c point, i t i s possible to
generate pH gradients by maintaining a constant f l u x of b u f f e r i n g
e l e c t r o l y t e s across the column. This requires the maintenance of
constant boundary conditions a t the two ends of the column. In
p r i n c i p l e , t h i s could be accomplished by maintaining large e l e c t r o l y t e
volumes a t the column ends, with an a c i d i c buffer at the anode and a
basic buffer a t the cathode. In p r a c t i c e , a r e c o n s t i t u t i o n of the
composition of the two buffers seemed more appropriate and various
approaches were studied experimentally as well as through computer
simulation (10).
3. Preformed pH gradients: I t i s possible to preform i n free
solution a pH gradient, using simple buffer components, such as, f o r
example, the above mentioned cacodylic a c i d and t r i s . Such gradients
are unstable, gradually degrading through ion transport.
Nevertheless,
we have demonstrated that such gradients are usable i n the neutral pH
region, about pH 6 to 8, provided only two components are used (11).


190
Figure 3. Polyacrylamide g e l pattern of ten RIEF fractions of a

polyclonal r a b b i t antibody sample.
Figure 4. Three dimensional p l o t of computer simulation data

of the focusing process of a mixture of cacodylic acid, h i s t i d i n e
and T r i s . Reproduced with permission from Ref. 5. Copyright
1983, American Association f o r the Advancement o f Science.

13.
BIER
191
Deterioration of gradients could be avoided by p h y s i c a l immobilization

of the functional groups of the buffer. This has been accomplished
i n a n a l y t i c a l gels (12) through the use of Immobilines (Tradename of
LKB Produkter, A.B., Bromma, Sweden) but these have not yet found
application i n free solutions.

Discussion
In p r i n c i p l e , the modular design of the RIEF would permit the
s c a l i n g up of i t s capacity to i n d u s t r i a l l y meaningful volumes. At
present, volumes ranging from 300 to 10,000 ml are being processed,
t h i s usually r e q u i r i n g two to four hours. Apparatus cross-sections
of 10, 20 and 100 cm are u t i l i z e d . In other electrochemical i n s t r u ments of somewhat s i m i l a r type, such as e l e c t r o d i a l y s i s or forcedflow electrophoresis, much larger cross-sections are u t i l i z e d .
Corresponding extension of the RIEF i s quite f e a s i b l e .
The demand f o r large scale focusing has been lagging, however,
due to several f a c t o r s : the w e l l entrenched status of chromatography,
the lack of o f f - t h e - s h e l f large scale focusing equipment and the need
as yet to use Ampholine- l i k e buffers f o r generation of the pH gradients. Our equipment has found, however, increasing demand f o r
research applications on smaller scale. This has prompted us to
design a smaller apparatus, based on a somewhat d i f f e r e n t p r i n c i p l e ,
with a priming volume of only 40 ml subdivided i n t o 20 f r a c t i o n s (7).
The RIEF system has been u t l i z e d f o r the f r a c t i o n a t i o n of a
large number of samples, most of which were provided to us by researchers from industry or academia. These encompassed fermentation
products, such as recombinant i n t e r f e r o n , products of mammalian
tissue culture and a great v a r i e t y of other proteins, enzymes, synt h e t i c peptide hormones, e t c . In general, preparative IEF i s p a r t i c u l a r l y w e l l suited f o r the p u r i f i c a t i o n of products of genetic engineering, as they tend to be more homogeneous than natural proteins.
This i s due to the avoidance i n non-mammalian systems of glycosylation,
a process secondary to DNA t r a n s c r i p t i o n , which accounts f o r much of
the heterogeneity of natural products. The same i s true f o r monoc l o n a l antibodies, which are obviously more homogeneous than the
polyclonal ones.
Center f o r Separation
Science
Our laboratory has recently been selected by the National Aeronautics and Space Administration (NASA) as one of i t s two national
centers of excellence i n separation science. This has given us an
opportunity to broaden our e f f o r t s towards the advancement of a l l
modes of electrophoresis f o r large scale processing. Thus, our
Center has been chosen by European manufacturers as the demonstration
s i t e f o r the United States of two unique electrophoretic instruments.
CJB Developments Ltd of Portsmouth, England, has i n s t a l l e d a t
our Center i t s BIOSTREAM (TM) production scale electrophoresis system.
Developed a t the Harwell Atomic Energy Laboratory i n UK, t h i s apparatus i s capable of separating i n d u s t r i a l l y meaningful quantities of
proteins, having a throughput of up to 100 grams of p r o t e i n per hour.
Residence time i n the apparatus i s of the order of a few seconds
only, f l u i d being s t a b i l i z e d against convection through shear induced
by an ingenious r o t a t i n g electrode assembly.


192
Bender & Hobein Gmbh o f Munich, West Germany, has contributed to

the Center i t s ELPHOR Vap 21 free flow electrophoresis apparatus. Of
the type flown by McDonnell Douglas Corp. aboard the NASA space
shuttle, t h i s apparatus embodies the p r i n c i p l e s f i r s t developed by
Hannig (13). I t i s an innovative apparatus best suited f o r sepa
r a t i o n o f l i v i n g c e l l s and c e l l organelles.
In addition, our Center remains dedicated to the development o f
novel f r a c t i o n a t i o n methods based on the r e c y c l i n g p r i n c i p l e
embodied i n our RIEF apparatus. Several new prototypes are being
tested. Experiments aboard the space shuttle are also planned, as
part o f our continuing study o f the p o t e n t i a l advantages i n terms o f
throughput and r e s o l u t i o n derivable from operation i n the microgravity environment p r e v a i l i n g i n o r b i t i n g spacecraft.
Acknowledgments
Supported i n part by NASA grant NSG-7333 and by NASA contract
NAS8-32950.
Literature Cited
1.
Commercial Biotechnology: An International Analysis, Office of

Technology Assessment, U.S. Congress, Washington, D.C., 1984.
2. P.G. Righetti: Isoelectric Focusing: Theory, Methodology and
Applications, Elsevier Biomedical, New York, N.Y. 1983.
3. D.W. Sammons, L.D. Adams and E.E. Nishizawa, Electrophoresis
2, 135, 1981.
4. .. Palusinski, T.T. Allgyer, R.A. Mosher, M. Bier and
D.A. Saville, Biophys. Chem. 13, 193, 1981.
5. M. Bier, .. Palusinski, R.A. Mosher and D.A. Saville,
Science 219, 1281, 1983.
6. M. Bier, N.B. Egen, T.T. Allgyer, G.E. Twitty and R.A. Mosher
in "Peptides: Structure and Biological Function"; E. Gross and
J. Meienhofer, Eds., pp. 35-48, Pierce Chemical Co., Rockford,
IL, 1979.
7. N.B. Egen, W. Thormann, G.E. Twitty and M. Bier in
"Electrophoresis '83"; H. Hirai, Ed., pp. 547-550, de
Gruyter, New York, NY, 1984.
8. S.B. Binion, L.S. Rodkey, N.B. Egen and M. Bier, Electrophoresis
3, 284, 1982.
9. M. Bier, R.A. Mosher and .. Palusinski, J. Chromatogr.
211, 313, 1981.
10. A. Tsai: "Study of a Coupled System of Two Electrophoretic
Columns with Opposing Current Polarity", Master Thesis,
University of Arizona, Tucson, AZ 1984.
11. M. Bier, R.A. Mosher, W. Thormann and A. Graham in
"Electrophoresis '83', H. Hirai, Ed., pp. 99-108, de Gruyter,
New York, NY, 1984.
12. B. Bjellqvist, K. Ek, P.G. Righetti, E. Gianazza, A. Georg,
R. Westermeier and W. Postel, J. Biochem. Biophys. Methods
6, 317, 1982.
13. K. Hannig in "Electrophoresis"; Vol. II, M. Bier, Ed.,
pp. 423-472, Academic Press, New York, 1967.

14
Large-Scale Gel Chromatography

Assessment of Utility for Purification of Protein Products

from Microbial Sources
1,3
1,4
James J. Kelley , George Y. Wang , and Henry Y. Wang

1
Molecular Genetics, Inc., Minnetonka, MN 55343

Department of Chemical Engineering, The University of Michigan, Ann Arbor, MI 48109
The cost of l a r g e - s c a l e gel chromatography

fractionation was found to be on the order of $5/gm of
product protein, a cost which limits its applicability
to high price products, like human pharmaceuticals.
The high cost is due to inherent limitations of the gel
permeation phenomenon, namely, poor resolution,
restricted throughput and product d i l u t i o n . A
quantitative model for assessing the u t i l i t y of gel
chromatography for protein purification was developed.
This methodology may be used to determine the ability
of gel chromatography to perform a desired separation
at the process-scale, and perform design calculations
and cost projection analyses.
S i n c e i t s d i s c o v e r y 25 y e a r s ago (I),
g e l chromatography has been
a p p l i e d e x t e n s i v e l y a t t h e l a b o r a t o r y s c a l e f o r a n a l y t i c a l and
p r e p a r a t i v e work and i n d u s t r i a l l y f o r t h e p r o d u c t i o n o f p o l y m e r s and
proteins.
S e v e r a l f a c t o r s which make i t a t t r a c t i v e f o r l a r g e - s c a l e
production of proteins are:
1. ) I t i s t h e o n l y
method f o r p r o t e i n f r a c t i o n a t i o n based
s o l e l y on m o l e c u l a r s i z e ;
2. ) S o l u t e c o n c e n t r a t i o n i s l i m i t e d o n l y b y s o l u b i l i t y
and v i s c o s i t y c o n s i d e r a t i o n s ;
3. ) I t i s g e n t l e a n d t h u s h i g h r e c o v e r i e s a r e u s u a l l y
obtained;
4. ) O p e r a t i o n
i s i s o c r a t i c w i t h r e s p e c t t o pH, b u f f e r
c o m p o s i t i o n , and temperature.
Current address: The Stroh Brewery Company, Detroit, MI 48207

'Current address: Amoco Research Center, Standard Oil (Indiana), Naperville, IL 60566
0097-6156/ 86/0314-0193$06.00/ 0

194

The use o f g e l chromatography has remained h i g h o v e r the y e a r s .

We
s u r v e y e d the 1982 volume o f The J o u r n a l of B i o l o g i c a l C h e m i s t r y and
found
t h a t 60% o f t h e p u b l i s h e d l a b o r a t o r y - s c a l e p r o t e i n
p u r i f i c a t i o n schemes i n c l u d e d a t l e a s t one g e l chromatography s t e p .
T h i s i s e s s e n t i a l l y the same r e s u l t found by D u n n i l l and L i l l y i n
1968
(2).
While
there
are
examples
of
industrial
use
of
gel
chromatography f o r p r o t e i n p u r i f i c a t i o n and e v e n though i t c o n t i n u e s
to
e n j o y g r e a t p o p u l a r i t y among b i o c h e m i s t s f o r s m a l l - s c a l e
s e p a r a t i o n s , we p e r c e i v e a h e s i t a n c y t o a p p l y the method w i t h i n the
B i o t e c h n o l o g y i n d u s t r y , due to some i n h e r e n t problems.
These p r o b l e m s a r e :
1. )
Low
p r o d u c t i v i t i e s due
to l i m i t e d
f e e d and
flow
rate capabilities;
2. ) Low column e f f i c i e n c i e s ;
3. ) S o l u t e d i l u t i o n ;
4. ) L a c k
of
information
about
costs
involved
in
large-scale operations.
In o r d e r to examine the v a l i d i t y of t h e s e s u s p i c i o n s , we f i r s t
c a l c u l a t e d the v o l u m e t r i c p r o d u c t i v i t i e s of i n d u s t r i a l and l a r g e s c a l e g e l c h r o m a t o g r a p h y p r o t e i n f r a c t i o n a t i o n s p u b l i s h e d i n the
l i t e r a t u r e ( T a b l e I ) . O n l y a p p l i c a t i o n s i n v o l v i n g column volumes
g r e a t e r t h a n 4 l i t e r s were c o n s i d e r e d .
The f i r s t s e v e n e x a m p l e s
(3-8) used s o f t , c o m p r e s s i b l e g e l s , l i k e Sephadex G-200 and U l t r o g e l
AcA34.
P r o d u c t i v i t i e s v a r i e d from 0.0016 to 0.045 l i t e r f e e d / l i t e r
g e l / 2 0 h day ( 1 / 1 / d )
and a v e r a g e d a b o u t 0.025 1 / 1 / d .
The n e x t
t h r e e examples (9-11) used l e s s c o m p r e s s i b l e g e l s , such as Sepharose
4B, Spehadex G-75 and G-50.
The p r o d u c t i v i t i e s were on the o r d e r of
0.1 1 / 1 / d , a b o u t 4 - f o l d h i g h e r t h a n t h e c o m p r e s s i b l e g e l s .
The
p r o d u c t i v i t y o f a c o n t i n u o u s a n n u l a r chromatograph (12) was found t o
be a p p r o x i m a t e l y 0.37
1/1/d, 1 5 - f o l d h i g h e r than the c o m p r e s s i b l e
g e l s . F i n a l l y , when p r o d u c t i v i t i e s f o r h y p o t h e t i c a l HPLC p r o d u c t i o n
s y s t e m s were c a l c u l a t e d f o r B i o - S i l 250 (13) and L i c r o s o r b d i o l
(14),
they were found to be a p p r o x i m a t e l y 25- and 250- f o l d h i g h e r
than c o m p r e s s i b l e g e l s , r e s p e c t i v e l y .
We a l s o e s t i m a t e d t h e c o l u m n e f f i c i e n c y f r o m t h e e l u t i o n
p r o f i l e f o r the i n d u s t r i a l s e p a r a t i o n of i n s u l i n (10). I t was found
to be 3-4 f o l d l e s s than t h a t p r e d i c t e d by the G i d d i n g s p l a t e h e i g h t
e q u a t i o n w i t h r e a s o n a b l e assumptions f o r g e l chromatography (15,16).
Thus the r e s t r i c t e d p r o d u c t i v i t i e s o f t e n a s s o c i a t e d with g e l
c h r o m a t o g r a p h y a r e the r e s u l t of the predominant use of s o f t ,
c o m p r e s s i b l e g e l s and low column e f f i c i e n c i e s a r e p r o b a b l y due t o
l o s s e s o u t s i d e the columns. E x c e p t f o r one case (10), t h e r e was no
d i s c u s s i o n i n any o f these p u b l i c a t i o n s c o n c e r n i n g how s c a l e - u p was
a c h i e v e d , how
v a l u e s f o r d e s i g n v a r i a b l e s were chosen, o r e v e n why
g e l chromatography was used.
C u r r e n t D e s i g n Methods.
Two
methods h a v e been used f o r g e l
chromatography system d e s i g n ( F i g u r e 1). The f i r s t method, which we
h a v e c a l l e d c o n v e n t i o n a l s c a l e - u p , i s t h a t commonly u s e d
by
b i o c h e m i s t s f o r p r e p a r a t i v e work. The c o n d i t i o n s f o r an a c c e p t a b l e
s e p a r a t i o n a r e f i r s t worked out i n s m a l l columns i n the l a b o r a t o r y .

14.
K E L L E Y ET AL.
195
Utility of Large-Scale Gel Chromatography
TABLE I
Protein
Separation
Medium

E.
coli
Iso-leucyl-t-RNA
transferase
E. c o l i
Methionyl-t-RNA
transferase
Citrobacter
L-asparaginase
Rape Seed P r o t e i n s
T r a n s f e r r i n from
Cohn F r a c t i o n IV
Plasma A l k a l i n e
Phosphatase
Plasma
Chollnesterase
Thyroglobulins
Insulin
Whey P r o t e i n
Concentrate
Continuous, annular
Chromatograph
Hypothetical
Industrial
HPLC S e p a r a t i o n
Sephadex G-200
Bed Dimensions
(D L )
Productivity
(cm cm)
(1 f e e d /
1 gel/day)
21.5 80
Sephadex G-200
14 180
Sephadex G-200
4.2 L
0.026
0.0016
0.007
Sephadex G-200
Sephadex G-200
45 85
45 75
0.025
0.019
6
7
U l t r o g e l AcA 34
16 100
0.045
9.3 90
0.026
37 45
37 90
37 15
0.125
0.071
0.128
9
10
11
0.37
12
5.5 60
0.6
13
2.5 25
6.5
14
Ultrogel
A c A 34
Sepharose 4B
S e p h a d e x G-50
Sephadex G-75
Sephadex G-75
B i o - S i l 250
G3000 SW
Lichrosorb
Diol
CONVENTIONAL S C A L E - U P
1.
Determine a c c e p t i b l e c o n d i t i o n s at lab scale
2. I n c r e a s e b e d a r e a p r o p o r t i o n a l to the i n c r e a s e in f e e d
volume, keeping all other v a r i a b l e s c o n s t a n t
METHOD O F C H A R M E T A L .
1.
Reference
1 7
Determine a c c e p t i b l e c o n d i t i o n s at lab s c a l e
2. Maintain geometric a s p e c t ratio ( L / D ) , R e y n o l d ' s number,

and f e e d fraction c o n s t a n t
Figure
1.
S c a l e - u p Methods.


196
To s c a l e - u p , t h e c r o s s - s e c t i o n a l a r e a o f t h e b e d i s i n c r e a s e d i n
p r o p o r t i o n to the i n c r e a s e i n feed volume w h i l e maintaining a l l
o t h e r v a r i a b l e s t h e same. To o u r knowledge, however, no s y s t e m a t i c
methodology f o r d e f i n i n g an a c c e p t a b l e s e p a r a t i o n has e v e r been put
forth.
The second method, a t t r i b u t e d t o Charm e t a l . (17), a g a i n c a l l s
for the establishment of a c c e p t a b l e c o n d i t i o n s at the l a b o r a t o r y
scale.
To s c a l e - u p , g e o m e t r i c a s p e c t r a t i o (column l e n g t h / c o l u m n
d i a m e t e r ) , c o l u m n R e y n o l d s Number, a n d t h e f e e d l o a d i n g a r e h e l d
constant.
When we a p p l i e d t h i s method t o a one-hundred f o l d s c a l e up o f a t y p i c a l g e l c h r o m a t o g r a p h y s y s t e m , we f o u n d
that
e x c e p t i o n a l l y l o n g c o l u m n s , on t h e o r d e r o f 4-5 m e t e r s , a n d v e r y
s l o w f l o w r a t e s were p r e d i c t e d .
Model Development
B e s i d e s these l i m i t a t i o n s , we submit t h a t n e i t h e r method i s s u i t e d
to o p t i m i z a t i o n , they cannot be a p p l i e d w i t h c o n f i d e n c e t o a l l g e l
chromatography systems,
a n d t h e y r e v e a l no k n o w l e d g e o f
r e l a t i o n s h i p s among v a r i a b l e s important t o t h e b i o p r o c e s s e n g i n e e r .
Consequently,
we s o u g h t t o d e v e l o p
a systematic approach to
p r o d u c t i o n - t y p e g e l c h r o m a t o g r a p h y u n i t o p e r a t i o n d e s i g n b a s e d on
e s t a b l i s h e d chromatographic
t h e o r y and e m p i r i c a l knowledge o b t a i n e d
experimentally.
M o d e l V a r i a b l e s . Some o f t h e v a r i a b l e s u s e d i n t h i s s y s t e m a r e
d e f i n e d w i t h r e f e r e n c e t o F i g u r e 2.
Shown t h e r e i s a t y p i c a l
e l u t i o n p r o f i l e from a g e l chromatography o p e r a t i o n i n which the
e a r l i e r e l u t i n g key contaminant ( s u b s c r i p t 1) i s s e p a r a t e d from the
l a t e r e l u t i n g p r o d u c t ( s u b s c r i p t 2). P r o t e i n c o n c e n t r a t i o n i s
p l o t t e d a g a i n s t the e l u t i o n v o l u m e as a f r a c t i o n o f t h e column
volume.
The a r r o w d e n o t e s t h e i n i t i a t i o n o f an i n j e c t i o n c y c l e ,
which i s d i v i d e d i n t o three regimes, the feed regime, f , the run
regime,r,
a n d t h e wash r e g i m e , w.
The e l u t i o n p r o f i l e s a r e
t y p i c a l l y d e s c r i b e d by G a u s s i a n d i s t r i b u t i o n p r o f i l e s h a v i n g two
parameters.
One p a r a m e t e r i s t h e e l u t i o n v o l u m e ,
V , which i s
a n a l o g o u s t o t h e mode o f a G a u s s i a n d i s t r i b u t i o n .
T h e o t h e r , , i s
r e l a t e d t o t h e w i d t h o f t h e e l u t i o n peak and i s a n a l o g o u s t o t h e
standard d e v i a t i o n of a Gaussian d i s t r i b u t i o n .
The p r o d u c t i s
c o l l e c t e d i n t h e volume between t h e " c u t " v o l u m e s , ( V ) - ^ and ( V ) 2
A p r o d u c t i o n scheme f o r a h y p o t h e t i c a l p r o d u c t i o n - t y p e g e l
chromatography o p e r a t i o n i s shown i n F i g u r e 3. Each b a t c h
having a
v o l u m e o f V ^ , m u s t be p r o c e s s e d w i t h i n t h e b a t c h t i m e t ^ .
The
b a t c h time i s d i v i d e d i n t o s e v e r a l i n j e c t i o n s ( i n t h i s case 4) and
dead time d u r i n g which no p r o d u c t i o n o c c u r s .
As mentioned above,
each i n j e c t i o n may h a v e t h r e e d i s t i n c t f l o w regimes, shown here as
v a r i o u s l y shaded b a r s .
The column must be taken out o f p r o d u c t i o n
a f t e r b a t c h e s f o r c l e a n i n g and a f t e r c l e a n i n g c y c l e s f o r
repacking.
I n t h e n e x t f i v e f i g u r e s a r e shown b l o c k s f r o m w h i c h a s y s t e m
f o r g e l c h r o m a t o g r a p h y u n i t o p e r a t i o n c a n be b u i l t .
Each b l o c k
c o n s i s t s o f equations from chromatographic
theory or e m p i r i c a l
correlations.
E a c h l i n e r e p r e s e n t s e i t h e r a p r o c e s s v a r i a b l e , shown
e n t e r i n g from the l e f t , a d e s i g n v a r i a b l e , or a s t a t e v a r i a b l e ,
which c a r r i e s i n f o r m a t i o n w i t h i n t h e system.
In these g e n e r a l
e


KELLEY ETAL.
- cycles -
DC
Time
F i g u r e 3. Sequence o f O p e r a t i o n s f o r a P r o d u c t i o n - T y p e G e l
Chromatography S y s t e m .

198

r e p r e s e n t a t i o n s of the b l o c k s , arrow heads have been o m i t t e d from

the d e s i g n and s t a t e v a r i a b l e l i n e s because any s e t of the them can
be s o l v e d f o r once the a v a i l a b l e degrees of freedom o f the b l o c k are
consumed.
Y i e l d and P u r i t y .
I d e n t i f i c a t i o n of the v a r i a b l e s important i n g e l
chromatography u n i t d e s i g n b e g i n s by d e f i n i n g the c o n c e p t s o f y i e l d
and p u r i t y . These a r e more u s e f u l i n d e s c r i b i n g the performance of
p r o d u c t i o n - t y p e systems than r e s o l u t i o n ,
which i s u s e f u l i n
a n a l y t i c a l chromatography work.
The y i e l d o f p r o d u c t i s d e f i n e d as
the i n t e g r a l o f the e l u t i o n c o n c e n t r a t i o n d i s t r i b u t i o n e q u a t i o n of
t h e p r o d u c t , assumed t o be a G a u s s i a n p r o f i l e , b e t w e e n t h e c u t
v o l u m e s . The y i e l d f o r t h e k e y c o n t a m i n a n t i s d e f i n e d s i m i l a r l y .
The p u r i t y o f t h e p r o d u c t w i t h r e s p e c t t o t h e k e y c o n t a m i n a n t i s
d e f i n e d as t h e r a t i o b e t w e e n t h e p r o d u c t y i e l d and t h e sum o f t h e
y i e l d s of the two key s o l u t e s .
The f i r s t b l o c k ( F i g u r e 4) r e l a t e s
t h e p r o c e s s v a r i a b l e s , ( C > , t h e c o n c e n t r a t i o n s o f t h e two key
s o l u t e s i n the f e e d , the s t a t e v a r i a b l e s ,
( î>
*
^ i * *
production Gaussian
distribution
parameters,
and
the
design
v a r i a b l e s , Y i e l d , P u r i t y , and the c u t volumes, ( V ) and ( V )
i
a n c
i e
Loading.
The p r o d u c t i o n G a u s s i a n d i s t r i b u t i o n p a r a m e t e r s c a n be
r e l a t e d to those found by a n a l y t i c a l l o a d i n g o f the bed by e q u a t i o n s
shown i n F i g u r e 5.
I n a n a l y t i c a l l o a d i n g , the f e e d f r a c t i o n , f , i s
vanishingly small.
The e f f e c t of l a r g e f e e d l o a d s on the e l u t i o n
volume i s g i v e n by the f i r s t e q u a t i o n (18).
F o r a square wave f e e d
(18),
the second e q u a t i o n i s d e r i v e d assuming the p r i n c i p l e of
v a r i a n c e a d d i t i v i t y (19).
G a u s s i a n Parameter C o r r e l a t i o n s .
I n the next b l o c k ( F i g u r e 6) a r e
e q u a t i o n s r e l a t i n g the a n a l y t i c a l G a u s s i a n parameters to a number o f
d e s i g n and s t a t e v a r i a b l e s .
The f i r s t e q u a t i o n i s a rearrangement
of the d e f i n i n g e q u a t i o n f o r the a c c e s s i b i l i t y c o e f f i c i e n t , (
) j
(20).
The s e c o n d e q u a t i o n i s t h e d e f i n i t i o n o f t h e v o l u m e o f a
c y l i n d e r i n t e r m s o f i t s l e n g t h and d i a m e t e r . The t h i r d e q u a t i o n
s t a t e s t h a t the t o t a l a n a l y t i c a l v a r i a n c e i s the sum o f t h a t due to
the bed, end e f f e c t , and t u b i n g . The bed v a r i a n c e i n t e r n has been
r e l a t e d to d e s i g n v a r i a b l e s such as f l o w r a t e , p a r t i c l e d i a m e t e r ,
e l u t i o n volume, and d i f f u s i v i t y , by c o r r e l a t i o n s , such as the van
D e e m t e r (2_1 ) , G i d d i n g s
( 1 5 , 1 6 ) , o r K n o x (2_2) p l a t e
height
e q u a t i o n s . I n p r a c t i c e , the a c t u a l form of the e q u a t i o n r e l a t i n g bed
v a r i a n c e would have to be determined e x p e r i m e n t a l l y . The dependence
o f t h e v a r i a n c e due t o t h e end e f f e c t on f l o w r a t e , t u b i n g and
column diameter can be determined e x p e r i m e n t a l l y or e s t i m a t e d (23).
F i n a l l y , t h e v a r i a n c e c a u s e d by d i s p e r s i o n d u r i n g f l o w t h r o u g h
c o n n e c t i n g t u b i n g and v a l v e s can be e s t i m a t e d by the method o f Go l a y
and A t w o o d (24) and d e p e n d s p r i m a r i l y on t h e t u b i n g d i a m e t e r and
flow rate.
K
S e l e c t i v i t y Equation.
The s e l e c t i v i t y e q u a t i o n ( F i g u r e 7) r e l a t e s
the s t a t e v a r i a b l e s , ( K ) ^ w i t h the p r o c e s s v a r i a b l e s , mw^,
and i s
commonly
f o u n d t o be a p p l i c a b l e o v e r a d e f i n e d m o l e c u l a r w e i g h t
r a n g e w h i c h d e p e n d s on t h e p o r e s i z e d i s t r i b u t i o n o f t h e g e l ( 2 5 ) .
The c o n s t a n t s i n the e q u a t i o n a r e dependent on the d e s i g n v a r i a b l e s ,
G, the g e l type and P, the p a c k i n g method.
a v

K E L L E Y ET AL.
YIELD,,
PURITY =

^(6 )
YIELD
\ 2(6r);
Vc
YIELD, + YIELD,
Ve-
F i g u r e 4.
Y i e l d and P u r i t y E q u a t i o n s f o r G a u s s i a n
Ve-
=
*
(Ve )
:
,
+
* analytical
toit a l y t i c a l
ls2L
2
12
(Ve;)
analytical
F i g u r e 5.
Peaks.
Feed
().a n a l y t i c a l
Loading
Equations.

S E P A R A T I O N , RECOVERY, A N D P U R I F I C A T I O N I N B I O T E C H N O L O G Y
<Ve;>
lytical
analytical
< 4n. c.. = ( ^ d-)+e)

Ve

ly
col
0 1
(6>:
analytical **'bed
Kav
ende
WA,
Packing]
F i g u r e 6.
tubing
End &
ITubing
Design
G a u s s i a n Parameter C o r r e l a t i o n s .
Kav =
+, 0^)|
!A,B = n(G.P)
F i g u r e 7.
Selectivity
Equations.

14.
K E L L E Y ET AL.
Chromatography
201
Productivity.
The p r o d u c t i v i t y o f the p r o c e s s ( F i g u r e 8), e x p r e s s e d
as grams o f p r o t e i n p r o d u c e d p e r u n i t t i m e , i s e q u a l t o t h e y i e l d
m u l t i p l i e d by the t o t a l amount of p r o d u c t mass e n t e r i n g the p r o c e s s ,
(C )
x V ,
d i v i d e d by t h e b a t c h t i m e t .
T h i s form of the
e q u a t i o n i s used when p r o d u c t i o n proceeds by the sequence o u t l i n e d
e a r l i e r i n F i g u r e 2 and when the i n t e r u p t i o n s f o r bed c l e a n i n g and
r e p a c k i n g a r e b r i e f o r n o n - e x i s t a n t due t o t h e u s e o f r e p l a c e m e n t
beds.
The bed d i a m e t e r e q u a t i o n r e l a t e s the p r o c e s s v a r i a b l e , b a t c h
volume, V^, w i t h the d e s i g n v a r i a b l e s , f , the f e e d f r a c t i o n , L, the
bed l e n g t h , i , t h e number o f i n j e c t i o n s p e r b a t c h , and D, t h e bed
d i a m e t e r as shown i n F i g u r e 9.
These b l o c k s can be l i n k e d t o g e t h e r t o form an i n f o r m a t i o n f l o w
system f o r a p r o d u c t i o n - t y p e g e l chromatography system ( F i g u r e 10).
Two a d d i t i o n a l b l o c k s i n f e r r i n g r e l a t i o n s h i p s which have not been
d i s c u s s e d appear i n the model.
One r e l a t e s the p a c k i n g method and
g e l used to the s t a t e v a r i a b l e s , v o i d f r a c t i o n ,
, and e f f e c t i v e
p a r t i c l e diameter, d . The second b l o c k i m p l i e s t h a t the c h o i c e of
g e l c o n s t r a i n s t h e f l o w r a t e and t h e m a g n i t u d e o f t h e e f f i c i e n c y
l o s s due t o e x t r a - c o l u m n causes which can be t o l e r a t e d .
T h i s system i n t u r n can form the b a s i s of a computer program
w h i c h c a n be u s e d f o r i n i t i a l s y s t e m d e s i g n , s u c h a s f o r c o l u m n
s i z i n g , o r f o r o p t i m i z a t i o n o f d e s i g n v a r i a b l e s , s u c h as f e e d
f r a c t i o n and f l o w r a t e .
F o l l o w i n g i s a d e m o n s t r a t i o n o f how t h e
system may be used f o r column s i z i n g .
f

Utility of Large-Scale Gel
Strategy for U t i l i z i n g
f e
the M o d e l
T h i s c o m p l e x s y s t e m c o n t a i n s 42 v a r i a b l e s and 25 e q u a t i o n s .
By
e l i m i n a t i n g the s t a t e v a r i a b l e s , i t can be reduced t o a system of 2
independent e q u a t i o n s w i t h 19 p r o c e s s and d e s i g n v a r i a b l e s , l e a v i n g
17 degrees o f freedom.
I n o r d e r to e s t i m a t e the l e n g t h and diameter
of a c o l u m n r e q u i r e d f o r a p a r t i c u l a r s e p a r a t i o n , t h e d e g r e e s o f
f r e e d o m must be u s e d .
To do t h i s , v a l u e s f o r t h e s i x p r o c e s s
v a r i a b l e s must be a s s i g n e d f i r s t . They are the c o n c e n t r a t i o n , ( C f ) ^ ,
and m o l e c u l a r w e i g h t s , i w ^ , of the two key s p e c i e s i n the f e e d , the
b a t c h volume, V^, and the b a t c h time, t ^ .
Second, v a l u e s f o r s i x d e s i g n v a r i a b l e s , G , P , T , i , f , and u, p l u s
y i e l d and p u r i t y
r e q u i r e m e n t s must be s p e c i f i e d .
Good c h o i c e s f o r
some o f t h e d e s i g n v a r i a b l e s must be made u s i n g h e u r i s t i c s .
For
example, the g e l s h o u l d be d u r a b l e , be a b l e t o be c l e a n e d in
situ,
be n o n - c o m p r e s s i b l e ,
and h a v e a h i g h s e l e c t i v i t y .
The
packing
m e t h o d s h o u l d g i v e r e p r o d u c i b l e , e f f i c i e n t , and s t a b l e p a c k i n g
s t r u c t u r e f o r the g e l used.
The temperature s h o u l d be the h i g h e s t
c o m p a t i b l e w i t h p r o d u c t and s o l i d p h a s e s t a b i l i t y .
The number o f
i n j e c t i o n s per b a t c h s h o u l d minimize
the dead time w i t h
due
c o n s i d e r a t i o n f o r product s t a b i l i t y .
Two more degrees of freedom a r e consumed by making assumptions
c o n c e r n i n g the c o n t r i b u t i o n s of the column ends (23) and t u b i n g (24)
to the system v a r i a n c e .
The f i n a l degree of freedom i s used i n c h o o s i n g an o p t i m i z a t i o n
s t a t e g y . P o s s i b l e s t r a t e g i e s i n c l u d e m i n i m i z i n g bed v o l u m e o r
cost/productivity.
Once the a v a i l a b l e degrees of freedom of the system have been

202
<c),
YIELD (C ^V
2
YIELD
F i g u r e 8.
Productivity
= (
F i g u r e 9.
Equation.
Bed Diameter
Equation.


14.
K E L L E Y ET AL.
203
YIELD AND
PURITY
EQUATIONS
BED
DIAMETER
EQUATION
FEED LOADING
EQUATIONS
GAUSSIAN OR MODIFIED
GAUSSIAN PARAMETER
CORRELATIONS
EFFECTIVE VOIO
FRACTION AND
PARTICLE DIAMETER
LINEAR VELOCITY
ANO EXTRA-COLUMN
CONSTRAINTS
F i g u r e 10. I n f o r m a t i o n F l o w Diagram f o r a P r o d u c t i o n - T y p e G e l
Chromatography System.

204
consumed, e s t i m a t e s f o r ( K
) i
and t h e d e p e n d e n c e o f t h e bed
v a r i a n c e on f l o w r a t e may be o b t a i n e d from t h e o r e t i c a l e x p r e s s i o n s
(15,16,21,22) o r from e x p e r i m e n t s a t the l a b o r a t o r y - s c a l e . Then, the
computer program can be used t o s e a r c h f o r o p t i m a l v a l u e s o f column
l e n g t h (L) and d i a m e t e r (D) as f u n c t i o n s of each o f the o t h e r d e s i g n
variables.
Once a column of the p r e s c r i b e d dimensions i s o b t a i n e d and s e t
up,
t h e a c t u a l c o n t r i b u t i o n o f i t s end e f f e c t s and t u b i n g t o
v a r i a n c e must be d e t e r m i n e d as a f u n c t i o n o f f l o w r a t e . Once t h e
p r o d u c t i o n bed i s packed, the dependence of the a n a l y t i c a l g a u s s i a n
p a r a m e t e r s on f l o w r a t e must be d e t e r m i n e d .
F i n a l l y the computer
program c o u l d once a g a i n be used to f i n d f e e d l o a d i n g and f l o w r a t e s
which o p t i m i z e c o s t / p r o d u c t i v i t y .
The s t e p s i n our proposed s y s t e m a t i c approach are summarized i n
F i g u r e 11. The b e n e f i t s o f s u c h an a p p r o a c h a r e t h a t p r o d u c t i o n s c a l e systems can be more e a s i l y o p t i m i z e d and t h a t s c a l e - u p can be
a c h i e v e d w i t h any s y s t e m .
F u r t h e r m o r e , i n f o r m a t i o n w h i c h may
be
r e q u i r e d by the b i o p r o c e s s e n g i n e e r , such as t h a t which w i l l s i g n a l
bed d e t e r i o r a t i o n o r t r a n s i e n t b e h a v i o r , a r e o b t a i n e d .

Cost A n a l y s i s
As mentioned above, t h e r e i s a l a c k of i n f o r m a t i o n i n the l i t e r a t u r e
c o n c e r n i n g the c o s t s of p r o d u c t i o n - t y p e g e l
chromatography.
C o n s e q u e n t l y , we d e v e l o p e d an economic model f o r t h i s u n i t o p e r a t i o n
which i d e n t i f i e d s o l i d phase l i f e t i m e and c o s t and bed s i z e as the
most important v a r i a b l e s i n the model.
Cost can be o b t a i n e d from
t h e m a n u f a c t u r e r and bed s i z e c a n be e s t i m a t e d f r o m t h e p r o g r a m
g i v e n above f o r any p a r t i c u l a r g e l . However, l i t e r a t u r e e s t i m a t e s
f o r s o l i d phase l i f e t i m e s v a r y from 70 to 2000 i n j e c t i o n s (10).
In
o r d e r to o b t a i n an upper e s t i m a t e we made the f o l l o w i n g assumptions:
1. ) Bed l i f e of 70 i n j e c t i o n s .
2. ) G e l was Sephadex G-50,
s u p e r f i n e , o p e r a t e d a t 10 cm/hr.
3. ) D i f f e r e n c e i n m o l e c u l a r w e i g h t s o f the key s p e c i e s was
2-fold.
4. ) B a t c h t i m e was 10 h r .
5. ) Feed p r o t e i n c o n c e n t r a t i o n was
2%.
6. ) Feed l o a d i n g was 2%.
7. ) C a p i t a l was d e p r e c i a t e d on a
s t r a i g h t - l i n e basis over
10
y e a r s and i n c l u d e d
six
s e c t i o n s of
Pharmacia
s t a c k column (KS370), pumps, p r o c e s s c o n t r o l equipment,
v a l v e s and t u b i n g .
8. ) L a b o r , f i l t e r , and
chemical
c o s t s were e s t i m a t e d by
assuming
t h a t the
r a t i o s between
these c o s t s and the
c o s t o f the
s o l i d phase were the
same as
those g i v e n
by C u r l i n g and Cooney (26).
As a r e s u l t o f t h i s e x e r c i s e we e s t i m a t e t h e t o t a l c o s t o f an
i n d u s t r i a l - t y p e g e l chromatography o p e r a t i o n to be on the o r d e r of
$5/gram o r l e s s .
T h i s f i g u r e c a n be u s e d t o q u i c k l y e s t i m a t e t h e
u t i l i t y o f g e l c h r o m a t o g r a p h y f o r a p a r t i c u l a r p r o c e s s . The m a j o r
c o s t s a r e l a b o r (40%), g e l (25%), f i l t e r s (20%),
chemicals
(10%),
and c a p i t a l ( 5 % ) .
T h i s e s t i m a t e d c o s t o f $5/gram i s q u i t e h i g h
and, a l t h o u g h r e p r e s e n t i n g an upper e s t i m a t e , s t i l l i n d i c a t e s t h a t
g e l chromatography i s an e x p e n s i v e p u r i f i c a t i o n step. T h i s e s t i m a t e


14.
K E L L E Y ET AL.
205
1.
A t lab s c a l e , o b t a i n d e p e n d e n c e of b e d v a r i a n c e
on flow r a t e and Ve of p r o d u c t a n d k e y c o n t a m i n a n t
2.
U s e c o m p u t e r to e s t i m a t e p r o d u c t i o n - s c a l e o p e r a t i n g
conditions
3.
O n c e p r o d u c t i o n - s c a l e s y s t e m is in p l a c e ,
v a r i a n c e due to e n d s , t u b i n g , v a l v e s
4.
Determine dependence
at p r o d u c t i o n s c a l e
5.
U s e c o m p u t e r to s e a r c h for optimum f e e d l o a d and

flow r a t e
F i g u r e 11.
estimate
of b e d v a r i a n c e on flow
Systematic
Scale-up.

rate
206
i s i n agreement w i t h the o b s e r v a t i o n t h a t p r o t e i n f r a c t i o n a t i o n by
g e l chromatography i s o n l y a p p l i e d i n d u s t r i a l l y to p r o t e i n s h a v i n g a
h i g h market v a l u e , such as human p h a r m a c e u t i c a l s .
I t i s not a p p l i e d
t o w a r d f r a c t i o n a t i o n o f b u l k p r o t e i n s s u c h as whey o r o i l s e e d
proteins.

Conclusion
A s y s t e m a t i c approach f o r d e s i g n and o p t i m i z a t i o n of p r o d u c t i o n g e l
c h r o m a t o g r a p h y o p e r a t i o n s w h i c h i s b u i l t up f r o m t h e o r e t i c a l and
e m p i r i c a l e q u a t i o n s i n the l i t e r a t u r e , and which e s t a b l i s h e s u s e f u l
r e l a t i o n s h i p s b e t w e e n p r o c e s s and
design
v a r i a b l e s has
been
p r e s e n t e d . Some of the p r o b l e m s p r e v e n t i n g a more w i d e s p r e a d use of
g e l c h r o m a t o g r a p h y a t t h e i n d u s t r i a l s c a l e , s u c h as r e s t r i c t e d
p r o d u c t i v i t i e s and poor e f f i c i e n c i e s may be improved by the use of
o p t i m i z a t i o n a p p r o a c h e s s u c h as t h e one p r o p o s e d . The p r i n c i p l e s
a r e a p p l i c a b l e t o o t h e r f o r m s of p r o d u c t i o n - s c a l e
linear-isotherm
chromatography c a r r i e d out under c o n s t a n t c o n d i t i o n s .
F i n a l l y , gel
c h r o m a t o g r a p h y r e p r e s e n t s an e s t i m a t e d p r o c e s s c o s t o f $ 5 / g r a m
product p r o t e i n .
Legend o f
Symbols
YIELD
P r o p o r t i o n of component i r e c o v e r e d
volumes r e l a t i v e to the amount of i
P r o p o r t i o n of component i r e c o v e r e d
volumes r e l a t i v e to the sum of the
C o n c e n t r a t i o n of component i i n the
Column Diameter
PURITY
(C )
D
< av>i
Availability Coefficient - (V )
p
"col
between the c u t
a p p l i e d to the column.
between the c u t
key components.
feed.
V
v
Column L e n g t h
Number of c l e a n i n g c y c l e s between r e p a c k i n g s .
P a c k i n g Method
Productivity
(g/h)
Temperature
B a t c h Volume
Cut Volume j
(VColumn Volume
Volume of l i q u i d e f f l u e n t which l e a v e s the column
te
between i n i t i a t i o n of f e e d and e l u t i o n o f the maximum
c o n c e n t r a t i o n of i .
Same as ( V ) ^ , but w i t h v a n i s h i n g l y s m a l l f e e d
ê^ analytical
v o l uume.
V o i d Volume of the packed bed.
e
dp
f
i
mw.^
r
t*
Some measurement of the d i a m e t e r of p a c k i n g p a r t i c l e s ,

P r o p o r t i o n of f e e d volume r e l a t i v e to column volume,
Number of i n j e c t i o n s o f f e e d per b a t c h ,
M o l e c u l a r weight of i .
Number of f e e d c y c l e s between c l e a n i n g s ,
P r o p o r t i o n of volume e l u t e d d u r i n g run regime to column
volume.
Time r e q u i r e d f o r p r o c e s s i n g of one b a t c h .

14.
u
w
KELLEY ET AL.
Utility of Large-Scale Gel
Chromatography
207
Average l i n e a r v e l o c i t y o f l i q u i d phase i n column.

P r o p o r t i o n o f volume e l u t e d d u r i n g wash regime t o column
volume.
V o i d f r a c t i o n o f the p a c k i n g .
V a r i a n c e o f the e l u t i o n c o n c e n t r a t i o n p r o f i l e o f i .

1,2 - r e f e r t o e i t h e r p r o d u c t o r key c o m t a n i n a n t .
1,2 - r e f e r t o f i r s t o r second c u t volume.
Literature Cited
1. Porath, J.; Flodin, P. Nature 1959, 183, 1657.
2. Dunnill, P.; Lilly, M. D. Biotechnol. & Bioeng. Symp. 1972, 3,
97.
3. Durekovic, .; Flossdorf, J.; Kula, M. R. Eur. J. Biochem.
1973, 36, 528.
4. Bruton, C.; Jakes, R.; Atkinson, T. Eur. J. Biochem. 1975, 59,
327.
5. Bascomb, S.; Banks, G. T.; Skarstedt, M. T.; Fleming, .;
Bettelheim, . .; Connors, T.A. J. Gen. Microbiol. 1975, 91,1.
6. Janson, J. C. J. Agr. Food Chem. 1971, 19, 581.
7. Curling, J. M. Amer. Lab. 1976, 8, 26.
8. Hanford, R.; Maycock, W.; Vallet, L. in "Chromatography of
Synthetic and Biological Polymers", vol. 2, R. Epton, Ed.,
Ellis Harwood, Chichester, U.K. 1978, pp. 111-119.
9. Horton, T. Amer. Lab. 1972, 4, 83-91.
10. "The large-Scale purification of Insulin by gel filtration
chromatography", Pharmacia, Uppsala, 1983.
11. Donnelly, E. B.; Delaney, R. A. M. J. Food Technol. 1977, 12,
493.
12. Nichols, R. .; Fox, J. B. J. Chromatogr. 1969, 43, 61.
13. IO-RAD Technical Information. 1985. IO-RAD Laboratories,
pp.111-114.
14. Roumeliotis, P.; Unger, K. J. Chromatogr. 1979, 185, 445.
15. Giddings, J. C. "Dynamics of Chromatography, Part 1, Principles
and Theory", Dekker, New York, 1965.
16. Giddings, J. C.; Mallik, K. L. Anal. Chem. 1966, 38, 997.
17. Charm, S. E.; Matteo, C. C.; Carlson, R. Anal. Biochem.
1969, 30, 1.
18. Personnaz, L.; Gareil, P. Sep. Sci. Technol. 1981, 16, 135.
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20. Kremmer, T.; Boross, L. "Gel Chromatography, Theory, Methodo
logy, Applications", John Wiley & Sons. Chichester. 1979.
21. van Deemter, J. J.; Zuiderweg, F. J.; Klinkenberg, A.
Chem.
Eng. Sci. 1956, 5, 271.
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J.
Chromatogr. Sci. 1981, 19, 1.
24. Golay, M. J. E.; Atwood, J. G. J. Chromatogr. 1979, 186,
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1982, 36, 59.
15

Electron Paramagnetic Resonance Spectroscopy Studies

of Immobilized Monoclonal Antibody Structure and
Function
1
Erik J. Fernandez, Forrest B. Fernandez, Roger B. Jagoda, and Douglas S. Clark

School of Chemical Engineering, Cornell University, Ithaca, NY 14853
Electron paramagnetic resonance (EPR) spectra were

taken of two spin labeled haptens combined with
soluble and immobilized anti-2,4-dinitrophenyl
(anti-DNP) IgG2b and IgE antibodies. Different
association constants between soluble antibodies and
spin labeled haptens were accompanied by differences
in maximum peak-to-peak splittings in EPR spectra.
When the IgG2b antibodies were immobilized using
CNBr-activated Sepharose and immobilized Protein-A,
lowered specific activities were observed (1.2 for
CNBr-Sepharose at .58 mg antibody/ml gel, 0.8 for
Protein- Sepharose at 1.7 mg antibody/ml gel), and
the binding constant of CNBr Sepharose-immobilized
antibody was 44% that of the soluble antibody.
Furthermore, the EPR spectrum of spin label combined
with IgG2b changed measurably upon immobilization of
the antibody to CNBr-Sepharose.
A f f i n i t y chromatography u t i l i z i n g monoclonal a n t i b o d i e s immobilized
t o a s o l i d m a t r i x h a s been used a s a p u r i f i c a t i o n method f o r
s e v e r a l years w i t h c h a r a c t e r i s t i c advantages and disadvantages.(1-3)
A n t i b o d i e s are p r o t e i n s produced by v e r t e b r a t e s "designed" t o b i n d
s p e c i f i c molecules, r e f e r r e d t o as antigens.
When u t i l i z e d i n t h e
laboratory o r production f a c i l i t y , antibodies immobilized t o a s u i t
a b l e s u p p o r t a l l o w a n a n t i g e n t o b e s e p a r a t e d f r o m many o t h e r
components o f a b i o c h e m i c a l p r o c e s s stream.
The s t r o n g i n t e r a c t i o n
between antibody and a n t i g e n ( w i t h e q u i l i b r i u m c o n s t a n t s as h i g h a s
10 M
) (4)c a nbe a great advantage i n product recovery from
f e r m e n t a t i o n b r o t h s , where product c o n c e n t r a t i o n s c a nbe as l o w a s
10
g/i i n e x t r e m e c a s e s . ( 5 )
I t c a n a l s o b e a d i s a d v a n t a g e , how
e v e r , s i n c e p o t e n t i a l l y d e n a t u r i n g c o n d i t i o n s may b e r e q u i r e d t o
1 4
To whom correspondence should be addressed

0097-6156/ 86/ 0314-0208506.00/ 0

15.
FERNANDEZ ETAL.
PR
Spectroscopy of Monoclonal Antibodies
209

c a u s e d i s s o c i a t i o n o f a t i g h t l y bound a n t i g e n .
Another disadvan
tage o f immunoaffinity chromatography i s the c o s t .
Monoclonal
a n t i b o d i e s purchased i n l a r g e q u a n t i t i e s from s p e c i a l i z e d companies
now s e l l f o r a b o u t $ 2 0 0 0 / g , a l t h o u g h t h i s n u m b e r i s e x p e c t e d t o
d r o p s i g n i f i c a n t l y a s more c o s t e f f e c t i v e p r o d u c t i o n methods a r e
developed.
Nonetheless, a wide v a r i e t y o f b i o l o g i c a l products have
been p u r i f i e d u s i n g monoclonal a n t i b o d i e s , a t l e a s t i n the l a b o r a
tory.
(For a n o v e r v i e w , see r e f e r e n c e 6 ) .
In order to maximize the u t i l i t y o f immobilized monoclonal
a n t i b o d i e s and m i n i m i z e c o s t s when t h e y a r e u s e d , i t i s n e c e s s a r y t o
have a m o l e c u l a r - l e v e l understanding o f the e f f e c t s o f i m m o b i l i z a
t i o n on a n t i b o d i e s used i n the p r e p a r a t i o n o f immunosorbents.
S e v e r a l s t u d i e s h a v e a l r e a d y shown t h a t i m m o b i l i z a t i o n c a n h a v e a
s i g n i f i c a n t e f f e c t on antibody a c t i v i t y .
F o r e x a m p l e , B o l t o n and
H u n t e r (7) f o u n d t h a t t h e t o t a l a c t i v i t y o f a n t i - h u m a n g r o w t h
hormone a n t i b o d i e s f e l l when c o u p l e d t o S e p h a r o s e and c e l l u l o s e
supports.
I n a d d i t i o n , they o b s e r v e d a drop i n what they c a l l e d
" s e n s i t i v i t y " , a q u a n t i t y i n v e r s e l y r e l a t e d t o the b i n d i n g s t r e n g t h
of the antibody.
I n a r e l a t e d s t u d y , E v e l e i g h a n d L e v y (8) s t u d i e d
the e f f e c t o f immobilized antibody l o a d i n g on s p e c i f i c a c t i v i t y ,
t h a t i s , t h e number o f a n t i g e n s t h a t c o u l d b i n d t o a n a n t i b o d y
molecule.
They found t h a t a s the l o a d i n g o f a n t i b o d y o n the
s u p p o r t w a s i n c r e a s e d , t h e o v e r a l l b i n d i n g c a p a c i t y o f t h e immuno
sorbent i n c r e a s e d , but the s p e c i f i c a c t i v i t y o f i m m o b i l i z e d a n t i
body dropped.
A s a f i n a l example c o n s i d e r the r e s u l t s o f Weston
a n d S c o r e r , who f o u n d t h a t a s t h e l o a d i n g o f I g G a n t i b o d y o n
S e p h a r o s e was i n c r e a s e d , t h e s p e c i f i c a c t i v i t y o f t h e a n t i b o d i e s
d e c r e a s e d t o t h e e x t e n t t h a t a maximum i n t o t a l b i n d i n g c a p a c i t y
was o b s e r v e d . ( 9 )
Before p o s t u l a t i n g probable causes o f t h i s
b e h a v i o r , i t i s h e l p f u l t o r e v i e w some o f t h e g e n e r a l c h a r a c t e r i s
tics of antibodies.
A n t i b o d i e s , o r immunoglobulins, are globular p r o t e i n s with
m o l e c u l a r w e i g h t s o f 1 5 0 t o 200 k d a l .
T h e y a r e made u p o f f o u r s u b u n i t s , t w o " h e a v y " and t w o " l i g h t " c h a i n s , w h i c h t o g e t h e r f o r m a
"Y"-shaped molecule.
The stem o f the antibody i s r e f e r r e d t o a s
t h e " c o n s t a n t " o r F ^ r e g i o n , w h i l e t h e two b r a n c h e s c o n t a i n i n g t h e
combining s i t e s
are
called
F , fragments.
N o t a b l y , these two
ab
r e g i o n s a r e j o i n e d b y few c o v a l e n t b o n d s , g i v i n g t h e m o l e c u l e a
great deal o fmotional
flexibility.
With these s t r u c t u r a l features i n mind, there are s e v e r a l
p o s s i b l e c a u s e s one m i g h t p u t f o r w a r d a s r e s p o n s i b l e f o r t h e
reduced s p e c i f i c a c t i v i t y o f antibodies on a support, as depicted
s c h e m a t i c a l l y i n F i g u r e 1. F i r s t , a p o r t i o n o f a n t i b o d i e s
c o v a l e n t l y bound i n random o r i e n t a t i o n s can be a t t a c h e d v i a l i n k
ages c l o s e t o a combining s i t e , e f f e c t i v e l y b l o c k i n g i t .
Second,
antibody-antibody s t e r i c e f f e c t s could hinder the a b i l i t y o f an
a n t i g e n t o b i n d t o a c o m b i n i n g s i t e , t h e r e b y r e d u c i n g t h e number o f
"active" sites.
T h i r d , lowered s p e c i f i c a c t i v i t y i s expected f o r
antibody molecules immobilized i n regions o f the support e i t h e r
p a r t i a l l y o r completely i n a c c e s s i b l e t oantigens.
And f i n a l l y , a
d i f f e r e n t type o f i n a c t i v a t i o n w i l l r e s u l t i f adverse conforma
t i o n a l changes occur as a r e s u l t o f the i m m o b i l i z a t i o n p r o c e s s .
The a i m o f t h i s r e s e a r c h , t h e n , i s t o b e t t e r u n d e r s t a n d w h i c h

210
SEPARATION, RECOVERY, A N D
o f t h e s e m e c h a n i s m s i s o r a r e i m p o r t a n t i n f o r m u l a t i n g immunos o r b e n t s o f o p t i m a l a c t i v i t y and b i n d i n g s t r e n g t h .
To do t h i s i t
i s n e c e s s a r y t o d e t e r m i n e t h e a c t i v i t y and c o n f o r m a t i o n o f a n t i bodies immobilized to a support. Unfortunately, the supports o f t e n
used ( e . g . a g a r o s e s ) i n t e r f e r e w i t h most d i r e c t p h y s i c a l measurements o f p r o t e i n s t r u c t u r e and f u n c t i o n .
One m e t h o d l a r g e l y
i n s e n s i t i v e t o t h e n a t u r e o f t h e s u p p o r t , h o w e v e r , i s EPR s p e c troscopy.

M a t e r i a l s a n d M e t h o d s - EPR
Spectroscopy
EPR, o r e l e c t r o n p a r a m a g n e t i c r e s o n a n c e s p e c t r o s c o p y , i s a m a g n e t i c
resonance technique designed to detect species c o n t a i n i n g unpaired
electrons.(10-11)
I t i s s i m i l a r i n p r i n c i p l e t o NMR,
the primary
d i f f e r e n c e b e i n g t h a t EPR i s b a s e d o n t h e m a g n e t i c moments o f
u n p a i r e d e l e c t r o n s r a t h e r t h a n t h e m a g n e t i c moments o f s p e c i f i c
nuclei.
S i n c e a n t i b o d i e s d o n t n o r m a l l y p o s s e s s any u n p a i r e d
electrons, small antigens, i . e . haptens, containing stable free
r a d i c a l s ( " s p i n l a b e l s " ) c a n b e u s e d a l o n g w i t h EPR t o p r o b e t h e
antibody combining s i t e .
B y u t i l i z i n g t h e two d i f f e r e n t 2,4d i n i t r o p h e n y l (DNP) s p i n l a b e l s s h o w n i n F i g u r e 2, we a r e i n v e s t i g a t i n g t h e c o n f o r m a t i o n and a c t i v i t y o f a n t i - D N P m o n o c l o n a l a n t i b o d i e s i n s o l u t i o n and i m m o b i l i z e d by d i f f e r e n t methods.
EPR
s p e c t r o s c o p y i s a p o w e r f u l method f o r t h i s k i n d o f s t u d y because t h e
EPR s p e c t r u m c a n p r o v i d e d e t a i l a b o u t t h e m o t i o n o f t h e s p i n l a b e l
and t h u s t h e c o n f o r m a t i o n o f t h e a n t i b o d y c o m b i n i n g s i t e .
In this
w a y , EPR c a n h e l p t o e l u c i d a t e w h i c h o f t h e p o s s i b l e i n a c t i v a t i o n
mechanisms d i s c u s s e d above i s o r are i m p o r t a n t .
T
Shown i n F i g u r e 3 a r e EPR s p e c t r a o f s p i n l a b e l FDNP-SL i n

s o l u t i o n a n d b o u n d t o a n t i - D N P a n t i b o d y . The m o r e r e s t r i c t e d
m o t i o n o f a n t i b o d y - b o u n d a n t i g e n r e s u l t s i n b r o a d e r l i n e s h a p e s and
a l a r g e r maximum p e a k - t o - p e a k s p l i t t i n g , t e r m e d &
The t h r e e peaked spectrum of s p i n l a b e l i n s o l u t i o n , f o r i n s t a n c e , i s c h a r a c t e r i z e d by an A

o f a b o u t 34G, w h e r e a s t h e s p e c t r u m o f a n t i b o d y max
b o u n d FDNP-SL h a s a n A
o f 61G.
I t s h o u l d a l s o be n o t e d t h a t t h e
max
a n t i b o d y bound s p e c t r u m i s a c o m p o s i t e spectrum c o n t a i n i n g b o t h t h e
c h a r a c t e r i s t i c t h r e e - p e a k e d f r e e s p e c t r u m and a bound s p i n l a b e l
spectrum of broader lineshape.
S i n c e the s i g n a l i n t e n s i t y of each
spectrum i s p r o p o r t i o n a l to the concentration of s p i n l a b e l , the
h e i g h t s o f i s o l a t e d p e a k s a n d / o r t h e d o u b l e i n t e g r a l o f an e n t i r e
a b s o r p t i o n s p e c t r u m c a n be u s e d t o d e t e r m i n e c o n c e n t r a t i o n s o f f r e e
and bound s p i n l a b e l . ( 1 2 )
For i n s t a n c e , i n the case of the f r e e
l a b e l , e i t h e r t h e p e a k h e i g h t o r d o u b l e i n t e g r a l c a n be u s e d .
In
the case of the composite spectrum, however, the i n t e g r a l p r o v i d e s
t h e c o n c e n t r a t i o n o f b o t h f r e e and bound l a b e l , and i t i s n e c e s s a r y
to s u b t r a c t the c o n c e n t r a t i o n of f r e e l a b e l from the t o t a l to
d e t e r m i n e t h e a m o u n t o f s p i n l a b e l b o u n d t o a n t i b o d y . By d o i n g s o
f o r samples c o n t a i n i n g d i f f e r e n t r a t i o s o f f r e e t o bound s p i n l a b e l ,
t h e e q u i l i b r i u m c o n s t a n t b e t w e e n a n t i b o d y and h a p t e n c a n be
o b t a i n e d d i r e c t l y f r o m EPR s p e c t r a .
A more s o p h i s t i c a t e d a n a l y s i s o f s p i n l a b e l m o t i o n i n t h e
c o m b i n i n g s i t e i n v o l v e s t h e a n a l y s i s o f e x p e r i m e n t a l EPR s p e c t r a
w i t h t h e a i d o f c o m p u t e r s i m u l a t i o n s , s u c h a s t h o s e o f F r e e d et at.
m a K

15.
EPR Spectroscopy of Monoclonal Antibodies
F E R N A N D E Z ET AL.
-<

Antibody
Antigen
ORIENTATION
211
Support
STERIC HINDRANCE
Soluble
(^T^ Immobilized)
ACCESSIBILITY
Figure
(A)
1.
CONFORMATION
Causes o f i n a c t i v a t i o n
2 AOV
N
o f immobilized
_^0
antibodies.
DNP-SL
FDNP-SL
(B)
Figure
2.
Spin
labels
used:
( A ) DNP-SL,
( B ) FDNP-SL.


212
(13)
By c o m p a r i n g s i m u l a t e d s p e c t r a w i t h t h o s e o b t a i n e d e x p e r i
m e n t a l l y , i t i s p o s s i b l e t o d i s t i n g u i s h between d i f f e r e n t models o f
t h e m o t i o n o f a s p i n l a b e l . ( 1 4 ) The p r i m a r y m o t i o n a l p a r a m e t e r s
i n v o l v e d a r e " r o t a t i o n a l c o r r e l a t i o n times", c h a r a c t e r i s t i c times
of r o t a t i o n a l d i f f u s i o n around each o f t h e three axes o f a c o o r d i
nate system defined w i t h respect t o the s p i n l a b e l .
A s shown i n
F i g u r e 4 a , t h e x - a x i s i s d e f i n e d a s c o l i n e a r w i t h t h e N-0 b o n d o f
the n i t r o x i d e p o r t i o n o f t h e molecule, t h e z - a x i s as p e r p e n d i c u l a r
to t h e " p l a n e " o f t h e r i n g , and t h e y - a x i s a s p e r p e n d i c u l a r t o t h e
x-z p l a n e .
E f f o r t s t o match c o m p u t e r - s i m u l a t e d and e x p e r i m e n t a l
EPR s p e c t r a r e v e a l e d t h a t t h e b e s t m o d e l i n c o r p o r a t e s a " d i f f u
s i o n a l t i l t " of angle 3 w i t h respect t o the molecular
coordinate
system, a l o n g w i t h t h r e e r o t a t i o n a l c o r r e l a t i o n t i m e s , , , and
a s shown i n F i g u r e 4b.
Y
x
>
R e s u l t s and D i s c u s s i o n
The b e s t f i t t o d a t e b e t w e e n s i m u l a t e d a n d e x p e r i m e n t a l s p e c t r a was
o b t a i n e d w i t h s o l u b l e IgG^,
w i t h e q u a l t o 5 0 , = 5.5 n s , a n d
and
= 33 n s .
This
represents
a fast
r o t a t i o n of the spin
l a b e l around t h e x a x i s and slower m o t i o n around t h e o t h e r axes as

d e p i c t e d i n F i g u r e 4 b . I t i s i n t e r e s t i n g t o n o t e t h a t 33 n s a g r e e s
w e l l w i t h t h e r o t a t i o n a l c o r r e l a t i o n t i m e o f 47 n s f o r t h e F , f r a g ab
m e n t o f a n I g E m o l e c u l e d e t e r m i n e d b y S l a t t e r y , et al.(15)
using
steady s t a t e a n i s o t r o p y measurements.
T h i s agreement suggests t h a t
and r e p r e s e n t time c o n s t a n t s f o r r o t a t i o n a l m o t i o n o f t h e F ,
y
ab
fragment as a whole.
U n f o r t u n a t e l y , because o f t h e complex and
t i m e - c o n s u m i n g n a t u r e o f t h e s i m u l a t i o n s , n o t h i n g f u r t h e r c a n be
r e p o r t e d a t t h i s time about t h e motion o f t h e s p i n l a b e l i n t h e
combining s i t e t h r o u g h c o r r e l a t i o n times and t i l t a n g l e s .
However,
t h e r e i s a l s o u s e f u l i n f o r m a t i o n c o n t a i n e d i n maximum p e a k - t o - p e a k
splittings.
In s t u d i e s t o date, d i f f e r e n t s p i n l a b e l s have produced d i f
f e r e n t s p e c t r a when c o m b i n e d w i t h d i f f e r e n t a n t i b o d i e s .
F o r exam
ple,
t h e s p l i t t i n g s and b i n d i n g c o n s t a n t s f o r t h e two d i f f e r e n t
s p i n l a b e l s b i n d i n g t o s o l u b l e a n t i - D N P IgG, a n t i b o d i e s a r e
zb
s u m m a r i z e d i n T a b l e I . T h e s e d a t a s h o w t h a t EPR i s s e n s i t i v e t o
d i f f e r e n c e s i n t h e m o t i o n o f ( 1 ) t h e same s p i n l a b e l i n d i f f e r e n t
motional environments ( i . e . , d i f f e r e n t antibody combining s i t e s ) ,
Table I .
Maximum p e a k - t o - p e a k s p l i t t i n g s a n d e q u i l i b r i u m b i n d i n g
c o n s t a n t s f o r s o l u b l e I g G , a n d I g E c o m b i n e d w i t h DNP-SL
and FDNP-SL.
A
(gauss)
max
(M )
Spin Labeled Hapten
?
Antibody
DNP-SL
2.2
62
3.9
FDNP-SL
56
1.3
4.8 1 0
^ 2b
^ 2b
IgE
IgE
DNP-SL
FDNP-SL
61

5 6

15.
Figure
3.
E P R s p e c t r a o f (A)
f r e e and
(B)
bound s p i n
213
label.
F i g u r e 4. S c h e m a t i c o f s p i n l a b e l i n a n t i b o d y c o m b i n i n g s i t e :
(A) w i t h o u t " d i f f u s i o n a l t i l t " , ( B ) w i t h " d i f f u s i o n a l
tilt".


214
a n d o f ( 2 ) d i f f e r e n t s p i n l a b e l s i n t h e same e n v i r o n m e n t ( i . e . ,
t h e same a n t i b o d y c o m b i n i n g s i t e ) .
T h i s s u g g e s t s t h a t EPR m i g h t b e
a b l e t o d i s c r i m i n a t e m o t i o n a l d i f f e r e n c e s caused by b i n d i n g s i t e
c o n f o r m a t i o n a l changes.
I f t h i s i s t h e c a s e , EPR s h o u l d be a u s e
f u l tool i n studying structure-function relationships i n immobilized
a n t i b o d i e s as w e l l .
F o r i m m o b i l i z a t i o n s t u d i e s t o d a t e , two d i s t i n c t modes o f
i m m o b i l i z a t i o n have been used.
The f i r s t u t i l i z e s n o n s p e c i f i c
c o v a l e n t b o n d i n g t o C N B r - a c t i v a t e d Sepharose v i a p r i m a r y amino
g r o u p s on t h e a n t i b o d y m o l e c u l e .
S i n c e t h e r e a r e many o f t h e s e
a v a i l a b l e on t h e a n t i b o d y , t h i s i s e x p e c t e d t o r e s u l t i n random
o r i e n t a t i o n o f a n t i b o d y m o l e c u l e s on t h e s u p p o r t .
The o t h e r m e t h o d
i n v o l v e s l i n k a g e t h r o u g h i m m o b i l i z e d p r o t e i n A, a p r o t e i n w h i c h
binds immunoglobulins
i n t h e s t r u c t u r a l F^ p o r t i o n o f t h e m o l e c u l e .
(15)
Because t h i s method s h o u l d r e s u l t i n i m m o b i l i z e d a n t i b o d y
m o l e c u l e s w i t h more o p t i m a l o r i e n t a t i o n s , i t i s e x p e c t e d t o p r o d u c e
s a m p l e s w i t h h i g h e r a c t i v i t y , a l t h o u g h i t w o u l d c e r t a i n l y be a m o r e
e x p e n s i v e and c o m p l e x t e c h n i q u e t o c a r r y o u t on a l a r g e s c a l e .
Shown i n T a b l e I I a r e a c t i v i t y a n d b i n d i n g c o n s t a n t d a t a f o r
s a m p l e s o f i m m o b i l i z e d a n t i b o d i e s p r e p a r e d b y t h e two d i f f e r e n t
methods.
The l o a d i n g o f I g G o n t h e s u p p o r t was d e t e r m i n e d b y a m i n o
a c i d a n a l y s i s , a n d t h e a m o u n t o f a c t i v e a n d a c c e s s i b l e a n t i b o d y was
d e t e r m i n e d b y EPR s p e c t r o s c o p y .
The b i n d i n g c o n s t a n t f o r t h e
C N B r - S e p h a r o s e s a m p l e was d e t e r m i n e d b y f l u o r e s c e n c e t i t r a t i o n . ( 1 6 )
Both samples have lowered s p e c i f i c a c t i v i t y w i t h r e s p e c t to the
i d e a l v a l u e o f 2.0 ( r e m e m b e r t h a t t h e r e a r e two c o m b i n i n g s i t e s p e r
a n t i b o d y m o l e c u l e ) , and t h e b i n d i n g c o n s t a n t f o r t h e C N B r - S e p h a r o s e
sample i s o n l y 44% t h a t o f i t s v a l u e i n s o l u t i o n .
I t i s also very
i n t e r e s t i n g to note that c o n t r a r y to expectations the P r o t e i n - A
s a m p l e h a s a l o w e r s p e c i f i c a c t i v i t y , w h i c h may be d u e t o t h e f a c t
t h a t i t h a s a much h i g h e r l o a d i n g t h a n t h e C N B r - S e p h a r o s e s a m p l e .
C u r r e n t r e s e a r c h i n t h i s l a b o r a t o r y s h o u l d soon p r o v i d e a more
d e f i n i t i v e explanation f o r these r e s u l t s .
Table
II.
L o a d i n g s , s p e c i f i c a c t i v i t i e s , and b i n d i n g c o n s t a n t s f o r
s a m p l e s o f i m m o b i l i z e d IgG_, .
Zb
C o u p l e d IgG
Active/Accessible
by Amino A c i d
I g G b y EPR
Specific
Support
A n a l y s i s (mg/ml)
(mg/ml)
Activity
(1/M)
CNBr-Sepharose
0.58
0.35
1.2
1.7xl0
(44%)
ProteinASepharose
1.7
0.70
0.8

15.
IgG^^
Shown i n F i g u r e 5 a r e E P R s p e c t r a o f FDNP-SL c o m b i n e d w i t h
i n s o l u t i o n and i m m o b i l i z e d t o C N B r - S e p h a r o s e .
Although an
accurate A
cannot be determined from these p a r t i c u l a r s p e c t r a ,
max
the l e f t - m o s t peak i n the spectrum of i m m o b i l i z e d a n t i b o d y i s
s h i f t e d s i g n i f i c a n t l y w i t h respect t o i t s p o s i t i o n i n the spectrum
of antibody i n s o l u t i o n .
This s h i f t i n d i c a t e s that the o v e r a l l
m o b i l i t y o f t h e s p i n l a b e l i s more r e s t r i c t e d when t h e l a b e l
o c c u p i e s the combining s i t e o f i m m o b i l i z e d a n t i b o d y . The
accompanying decrease i n the b i n d i n g c o n s t a n t observed upon
i m m o b i l i z a t i o n i n d i c a t e s t h a t such changes i n s p i n l a b e l m o b i l i t y
are due a t l e a s t i n p a r t t o changes i n combining s i t e c o n f o r m a t i o n ;
h o w e v e r , a l t e r e d m o t i o n o f t h e e n t i r e F , f r a g m e n t may a l s o h a v e
ab
i n f l u e n c e d the spectrum.
C l a r i f i c a t i o n o f t h i s p o i n t , provided by
the use o f computer s i m u l a t i o n s t odetermine r o t a t i o n a l c o r r e l a t i o n
times f o r the immobilized antibody system i s expected i n the near
future.
r

215
Concluding
Remarks
The d a t a p r e s e n t e d a b o v e e s t a b l i s h t h e a p p l i c a b i l i t y o f E P R t o
s t r u c t u r e - f u n c t i o n c h a r a c t e r i z a t i o n o f i m m o b i l i z e d a n t i b o d i e s . The
soluble A
d e t e r m i n a t i o n s i n d i c a t e t h a t EPR i s s e n s i t i v e t o
max
d i f f e r e n t e n v i r o n m e n t s and t o t h e s t r u c t u r e o f t h e p r o b e u s e d .
They a l s o s u g g e s t t h a t d i f f e r e n t p r o b e s m i g h t b e used t o o b t a i n a
more c o m p l e t e p i c t u r e o f t h e c o n f o r m a t i o n o f t h e b i n d i n g s i t e . I n
a d d i t i o n , t h e m e a s u r a b l e d i f f e r e n c e s between t h e EPR s p e c t r a o f
s o l u b l e and i m m o b i l i z e d a n t i b o d i e s i n d i c a t e t h a t i m m o b i l i z a t i o n has
s i g n i f i c a n t l y a f f e c t e d the motion o f the s p i n l a b e l i n the
combining s i t e .
A l t h o u g h the d a t a p r e s e n t e d h e r e do not a l l o w
s p e c i f i c c o n c l u s i o n s t obe drawn about the e f f e c t o f i m m o b i l i z a t i o n
on a n t i b o d y c o n f o r m a t i o n and a c t i v i t y , t h e y do i l l u s t r a t e t h a t EPR
can p r o v i d e m o l e c u l a r l e v e l i n s i g h t s not a v a i l a b l e by t r a d i t i o n a l
methods.
Thus, EPR s p e c t r o s c o p y s h o u l d s e r v e a s a p o w e r f u l t o o l
F i g u r e 5.
IgG .
EPR s p e c t r a o f s o l u b l e and
CNBr
Sepharose-immobilized
2 b

216
for d i r e c t experimental analyses o f immobilized antibody

structure
and f u n c t i o n , a n d t h e r e f o r e h e l p t o e l u c i d a t e t h e b a s i s f o r t h e
s u b o p t i m a l b i n d i n g c a p a c i t i e s f r e q u e n t l y e x h i b i t e d b y immuno
sorbents .

Acknowledgments
The a u t h o r s a r e i n d e b t e d t o D r . B a r b a r a B a i r d , D. D a v i d H o l o w k a ,
and D r . N o r m a l K l i n m a n f o r t h e i r a s s i s t a n c e i n o b t a i n i n g a n t i b o d i e s
and t o Dave S c h n e i d e r a n d G l e n n M i l l h a u s e r f o r t h e i r a d v i c e o n t h e
spectral simulations.
T h i s m a t e r i a l i s based upon work supported
under a N a t i o n a l Science Foundation Graduate F e l l o w s h i p .
Literature Cited
1. Turkova, J. In "Affinity Chromatography"; Elsevier: New York,
1975; p. 95.
2. Chase, H. A. Chem. Eng. Sci. 1984, 39, 1099.
3. Goding, J. W. In "Monoclonal Antibodies: Principles and
Practice"; Academic: New York, 1983; pp. 188-207.
4. Mukkur, T. K. S. Biochem. J. 1978, 172, 39.
5. Dwyer, J. W. Bio/Technology 1984, 2, 957.
6. Low, D. "Proceedings of Biotech 83, International Conference
on the Commercial Applications and Implications of Biotech
nology"; Online: Northwood, U.K. 1983, p. 830.
7. Bolton, A. E.; Hunter, W. M. Biochim. Biophys. Acta 1973,
329, 318.
8. Eveleigh, J. W.; Levy, D. E. J. Solid-Phase Biochem. 1977, 2,
45.
9. Weston, P. D.; Scorer, R. In "Affinity Chromatography";
Hoffman-Ostenhof, et al., Ed.; Pergamon: New York, 1978,
p. 207.
10. Schumacher, R. T. "Magnetic Resonance"; W. A. Benjamin:
New York, 1970.
11. Cantor, C. R.; Schimmel, P. R. In "Biophysical Chemistry";
W. H. Freeman: New York, 1980; pp. 525-536.
12. Bailey, J. E.; Clark, D. S. In "Methods in Enzymology";
Mosbach, K., Ed.; Academic: New York, in press.
13. Meirovitch, E.; Igner, D.; Igner, E.; Moro, G.; Freed, J. H.
J. Chem. Phys. 1982, 77, 3915.
14. Goldman, S. .; Bruno, G. V.; Polnaszek, C. F.; Freed, J. H.;
J. Chem. Phys. 1972, 56, 716; Hwang, J. S.; Mason, R. P.;
Hwang, L. P.; Freed, J. H. J. Phys. Chem. 1975, 79, 289;
J. Phys. Chem. 1975, 79, 2283; Campbell, R. F.; Freed, J. H.
J. Phys. Chem. 1980, 84, 2668; Meirovitch, E.; Freed, J. H.
J. Phys. Chem. 1980, 84, 2459; Shiotani, M.; Moro, G.; Freed,
J. H. J. Chem. Phys. 1981, 74, 15.
15. Slattery, J.; Holowka, D. A.; Baird, B. A. Biochemistry, in
press.
16. Goding, J. W. In "Monoclonal Antibodies: Principles and
Practice"; Academic: New York, 1983; p. 195.
17. Mukkur, T. K. S.; Szewczuk, M. R.; Schmidt, . E., Jr.
Immunochem. 1974, 11, 9.


Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.ix002
Author Index
Asenjo, J . ., 9
B i e r , M i l a n , 185
Chang, Ho Nam, 32
Chung, Bond Hyun, 32
C l a r k , Douglas S., 208
Dove, G. B., 93
D r i o l i , E n r i c o , 52
E r c o l i , ., 43
Fernandez, E r i k J . , 208
Fernandez, F o r r e s t B., 208
F i n n , R. ., 43
F i s h e r , R. R., 109
G l a t z , C. E., 109
Gobie, W i l l i a m ., 169
H a t t o n , . ., 67
Hettwer, David J . , 2
Hunter, J . ., 9
I v o r y , C o r n e l i u s F., 169
Jagoda, Roger ., 208
K a u l , R a j n i , 78
K e l l e y , James J . , 193
Kim, I n Ho, 32
L a d i s c h , M. R., 122
M a t t i a s s o n , Bo, 78
M i t r a , G., 93
Nigam, Somesh C , 153
Rudge, S. R., 122
T h i e n , M. P., 67
Wang, D. I . C , 67
Wang, George Y., 193
Wang, Henry Y., 2,153,193
Subject Index
A
Acetic acid production, extractive

f e r m e n t a t i o n i n aqueous two-phase
systems, 80
Acetone-butanol p r o d u c t i o n , e x t r a c t i v e
systems, 80
Adsorbent, i m m o b i l i z e d a f f i n i t y ,
mathematical modeling o f
b i o p r o d u c t a d s o r p t i o n , 153
Adsorbent bead, i m m o b i l i z e d a f f i n i t y ,
schematic diagram, 156f
Adsorbent m a t r i x , e f f e c t o f b i o p r o d u c t
d i f f u s i v i t y on l i g a n d
consumption, 164f
Aerobic whole-cell immobilization,
d u a l h o l l o w - f i b e r b i o r e a c t o r , 32
A f f i n i t y adsorbent, immobilized
bead, schematic diagram, 156f
h y d r o g e l beads, 158-164
mathematical modeling o f
b i o p r o d u c t a d s o r p t i o n , 153
A f f i n i t y a d s o r p t i o n , t h e o r y , 154-158
A f f i n i t y chromatography, u t i l i z i n g
i m m o b i l i z e d monoclonal
a n t i b o d i e s , 208
Affinity partitioning
aqueous two-phase systems, 8 6 t
p r o t e i n s , aqueous two-phase
systems, 85-87
A f f i n i t y sorbent, p a r t i t i o n i n g i n
two-phase system, 8 8 f
A f f i n i t y support, scale-up o f
chromatographic p r o c e s s e s , 124
Aggregate growth and breakup, p r o t e i n
p r e c i p i t a t i o n , 112
A g i t a t i o n speed, e f f e c t on LEM
separation, 73f
Albumin
c o n c e n t r a t i o n and p a r t i t i o n
c o e f f i c i e n t as f u n c t i o n
of s a l t concentration,
PEG-water s o l u t i o n , 9 9 f
p r o t e i n r e c o v e r y by s a l t
p a r t i t i o n , 107
A l c o h o l i c fermentations, immobilized
systems and aqueous two-phase
systems, 80
A l c o h o l , i o n exchange s e p a r a t i o n , 125f
A l l e r g e n s , RIEF s e p a r a t i o n , I 8 7 , l 8 8 f
A l l o s t e r i c enzymes, g e l l e d membrane
f o r m a t i o n , i m m o b i l i z e d , 61
Amino a c i d s
membrane r e a c t o r , 58
p r o c e s s diagram f o r LEM-based
recovery, 76f
r e c o v e r y from l y s a t e , membrane
p r o c e s s , 56
r e c o v e r y u s i n g l i q u i d emulsion
membranes, 71-75
Amphoteric compounds, IEF, 186
Amylase and c e l l u l a s e , aqueous
two-phase systems, semicontinuous
p r o d u c t i o n , 84
Anaerobic s i n g l e - c e l l p r o t e i n (SCP),
membrane r e a c t o r , 43,44
219

220
SEPARATION, RECOVERY, A N D
A n a l y s i s o f c e l l components, p r o t e i n
r e l e a s e from E. c o l i , 3
A n a l y s i s o f s p i n l a b e l motion,
monoclonal a n t i b o d i e s , 210
A n t i b i o t i c s , c o n c e n t r a t i o n and
p u r i f i c a t i o n by s e q u e n t i a l
UF and R0, 56
Antibodies
d e s c r i p t i o n , 209
EPR s p e c t r o s c o p y s t u d i e s o f
s t r u c t u r e and f u n c t i o n ,
i m m o b i l i z e d monoclonal, 208
m a t e r i a l s and methods, EPR
s p e c t r o s c o p y , 210
s i m u l a t e d and e x p e r i m e n t a l s p e c t r a ,
EPR, i m m o b i l i z e d monoclonal, 212
A n t i g e n s , aqueous two-phase system
Aqueous m u l t i p h a s e systems, phase
p a r t i t i o n i n g , 95
Aqueous two-phase systems
a f f i n i t y p a r t i t i o n i n g , 86t
a l c o h o l i c f e r m e n t a t i o n s compared t o
i m m o b i l i z e d systems, 80
b i o c a t a l y t i c r e a c t i o n , 79
b u l k c h e m i c a l p r o d u c t i o n , 82t
economy, 89
equipment f o r c o n t i n u o u s
e x t r a c t i o n , 90
e x t r a c t i v e b i o c o n v e r s i o n , 81f
f i n e chemicals production
compared w i t h i m m o b i l i z e d
systems, 83
d i s c u s s i o n , 82
i s o l a t i o n o f enzymes, 85t
macromolecules p r o d u c t i o n , 84
product i n h i b i t i o n , f e r m e n t a t i o n
p r o c e s s e s , 79-85
p u r i f i c a t i o n o f p r o t e i n s by
p a r t i t i o n i n g , 85-87
r e c o v e r y and p u r i f i c a t i o n i n
b i o t e c h n o l o g y , o v e r v i e w , 78-90
r e c o v e r y o f p r o t e i n s by s a l t
Aspergillus niger
c u l t i v a t i o n i n dual h o l l o w - f i b e r
b i o r e a c t o r , 37
immobilization i n dual h o l l o w - f i b e r
Asymmetric enzyme c a p i l l a r y membranes,
phase i n v e r s i o n method, 63f
Binding
single-component d i f f u s i o n ,
immobilization i n
h y d r o g e l , 158-163
two-component d i f f u s i o n ,
i m m o b i l i z a t i o n i n h y d r o g e l , 163
B i o - a f f i n i t y , scale-up of
B i o c a t a l y t i c r e a c t i o n , aqueous
two-phase system, 79
B i o c h e m i c a l s e p a r a t i o n s , LEMs,
commodity-type, 70
B i o c o n v e r s i o n , aqueous two-phase
systems, e x t r a c t i v e f e r m e n t a t i o n
p r o c e s s e s , 79-85
Biological materials, colloidal,
e l e c t r o k i n e t i c parameters, l 8 0 t
Bioreactor
advantages and d i s a d v a n t a g e s ,
h o l l o w - f i b e r membrane, 32
aerobic whole-cell immobilization,
d u a l h o l l o w - f i b e r , 32,33
c o n t i n u o u s p r o d u c t i o n o f r i f a m y c i n B,
d u a l h o l l o w - f i b e r , 40f
c r o s s s e c t i o n a l view o f d u a l
h o l l o w - f i b e r , 34f
c u l t i v a t i o n o f A. n i g e r ,
deformed, 39f
c u l t i v a t i o n o f E. c o l i , d u a l
h o l l o w - f i b e r , 35
e l e c t r o n micrographs o f d e n s e l y
packed E. c o l i , d u a l
h o l l o w - f i b e r , 38f
schematic diagram o f e x p e r i m e n t a l
s e t u p , d u a l h o l l o w - f i b e r , 36f
B i o s u r f a c t a n t s , aqueous two-phase
systems, e x t r a c t i v e c o n v e r s i o n , 83
Biotechnology
d e f i n i t i o n , downstream
p r o c e s s i n g , 52
o v e r v i e w , aqueous two-phase systems
f o r r e c o v e r y and
p u r i f i c a t i o n , 78-90
summary o f membrane p r o c e s s e s , 55t
Bonding, i m m o b i l i z a t i o n methods,
monoclonal a n t i b o d i e s , n o n s p e c i f i c
c o v a l e n t , 214
Breakage models, p r o t e i n
Bulk c h e m i c a l p r o d u c t i o n , aqueous
two-phase systems, 80,82t
Buoyancy, e l e c t r o p h o r e s i s s c a l e - u p
c o n s i d e r a t i o n s , 138
Bed diameter e q u a t i o n , g e l
chromatography d e s i g n model, 202f
Calculated elution p r o f i l e
chromatographic p r o t e i n
s e p a r a t i o n , 137f

221

INDEX
Calculated elution p r o f i l e C o n t i n u e d
electrochromatographic protein
s e p a r a t i o n , 145
C a r b o h y d r a t e s , s o l u b l e p r o t e i n and
p e p t i d e s , enzymatic l y s i s and
d i s r u p t i o n o f y e a s t c e l l s , model
simulations,
21-24
Cell, microbial, subcellular location
o f enzyme a c t i v i t i e s , 10
C e l l breakage, mechanical d i s r u p t i o n
o f E. c o l i , 5f
C e l l components, p r o t e i n r e l e a s e from
E. c o l i , 3
C e l l c o n c e n t r a t i o n , e f f e c t on p r o t e i n
release p r o f i l e , 7f
C e l l f r a c t i o n a t i o n , model, enzymatic
l y s i s and d i s r u p t i o n o f y e a s t
c e l l s , 24
C e l l f r a g m e n t a t i o n , m e c h a n i c a l l y based
p r o t e i n r e l e a s e methods, 2
C e l l immobilization, dual hollow-fiber
b i o r e a c t o r , a e r o b i c , 32,33
C e l l l y s i s , sequence, enzymatic l y s i s
and d i s r u p t i o n o f y e a s t c e l l , 11
C e l l permeabilization, protein release
from E. c o l i , 3
C e l l p r e p a r a t i o n , p r o t e i n r e l e a s e from
E. c o l i , 3
C e l l u l a r protein release
c h e m i c a l treatment o f E. c o l i , 4,5f
m e c h a n i c a l d i s r u p t i o n o f E. c o l i , 5f
C e l l u l a s e , amylase and, s e m i c o n t i n u o u s
p r o d u c t i o n i n aqueous two-phase
systems, 84
C e l l u l o s e , enzymatic d e g r a d a t i o n t o
a l c o h o l , membrane r e a c t o r , 58
Chemical p e r m e a b i l i z a t i o n , p r o t e i n ,
DNA, and RNA r e l e a s e from
E. c o l i , 4
Chemicals
bulk, production i n
two-phase systems, 80,82t
e x t r a c t i v e f e r m e n t a t i o n i n two-phase
systems, 80
f i n e , p r o d u c t i o n i n aqueous
two-phase systems, 82
C h l o r i d e c o u n t e r t r a n s p o r t system,
t y p i c a l LEM s e p a r a t i o n , 73f
Chromatographic apparatus f o r p r o c e s s
system, 127f
Chromatographic p r o c e s s e s , s c a l e - u p
c o n s i d e r a t i o n s , 126-129
Chromatographic p r o t e i n f r a c t i o n a t i o n s
c a l c u l a t e d e l u t i o n p r o f i l e , 137f
g e l , i n d u s t r i a l and l a r g e
s c a l e , 195t
Chromatographic s u p p o r t s , s c a l e - u p o f
chromatographic p r o c e s s e s , i o n
exchange r e s i n s , 124
Chromatography
a f f i n i t y , immobilized
monoclonal
ChromatographyContinued
gel
model development, 196-201
p u r i f i c a t i o n of p r o t e i n products,
l a r g e - s c a l e , 193
scale-up processes, t h e o r e t i c a l
considerations i n s i z e
e x c l u s i o n s , 129-135
size exclusion, equilibrium
e x p r e s s i o n s and mass t r a n s f e r
expressions,
131t
C o l l o i d a l and b i o l o g i c a l m a t e r i a l s ,
e l e c t r o k i n e t i c parameters, l 8 0 t
Column a r e a , chromatographic s c a l e - u p
Column l e n g t h , chromatographic
s c a l e - u p c o n s i d e r a t i o n s , 128
Commodity-type b i o c h e m i c a l s ,
s e p a r a t i o n , LEMs, 70
Concentration p r o f i l e of
cycloheximide,
immobilized
adsorbent beads, I60f
Continuous-flow e l e c t r o p h o r e s i s
(CFE)
c l a s s i c , s c h e m a t i c , 172f
c l a s s i c t h i n - f i l m , general
comparison o f s i n g l e - p a s s
models, 175f
d i s c u s s i o n , r e c y c l e , 169,171-173
g e n e r a l d i s c u s s i o n , 170
s c h e m a t i c , c l a s s i c , 172f
Continuous p r o d u c t i o n o f r i f a m y c i n B,
d u a l h o l l o w - f i b e r b i o r e a c t o r , 40f
C o n v e c t i v e d i s p e r s i o n , RCFE,
model, 174
C o n v e r s i o n o f b i o s u r f a c t a n t s , aqueous
two-phase systems, e x t r a c t i v e , 83
Cost a n a l y s i s , g e l chromatography
s c a l e - u p , 204
C o u n t e r t r a n s p o r t system
LEM s e p a r a t i o n , c h l o r i d e ,
73f
LEM-mediated amino a c i d r e c o v e r y , 71
C o v a l e n t bonding, n o n s p e c i f i c ,
monoclonal a n t i b o d y i m m o b i l i z a t i o n
methods, 214
Cross-flow m i c r o f i l t r a t i o n , b i o a c t i v e
compounds from f e r m e n t a t i o n
b r o t h s , 52
C y c l o h e x i m i d e , i m m o b i l i z e d adsorbent
beads, c o n c e n t r a t i o n p r o f i l e , l 6 0 f
Deformed b i o r e a c t o r , c u l t i v a t i o n o f
A. n i g e r , 39f
D i f f u s i o n and b i n d i n g
s i n g l e component, i m m o b i l i z a t i o n i n
h y d r o g e l , 158-163
two component, i m m o b i l i z a t i o n i n
hydrogel,
163


222
D i m e n s i o n l e s s parameters, RCFE
model, 176t
Dispersion c o e f f i c i e n t , e f f e c t i v e ,
electrophoretic mobility,
RCFE, 175f
DNA r e l e a s e
E. c o l i t r e a t e d w i t h g u a n i d i n e HCl
and T r i t o n , 5 f
m e c h a n i c a l l y d i s r u p t e d E. c o l i , 4
D o u b l e - l a y e r e d s t r u c t u r e of the y e a s t
w a l l , 12f
Downstream p r o c e s s i n g
b i o t e c h n o l o g y , d e f i n i t i o n , 52
membrane systems, 53-57
p h e n y l a l a n i n e from f e r m e n t a t i o n
b r o t h , LEM-mediated
s e p a r a t i o n s , 71
Drug d e l i v e r y , LEMs, 70
Dual h o l l o w - f i b e r b i o r e a c t o r
aerobic whole-cell
i m m o b i l i z a t i o n , 32,33
e l e c t r o n micrographs o f densely
packed E. c o l i , 38f
s e t u p , 36f
Effective dispersion c o e f f i c i e n t ,
electrophoretic mobility,
RCFE, 175f
E f f e c t i v e osmolality of c e l l l y s a t e ,
enzymatic l y s i s and d i s r u p t i o n o f
y e a s t c e l l s , 16
Electrochromatography
process considerations for
scale-up,
122
protein separation, calculated
e l u t i o n p r o f i l e , 145
s c h e m a t i c diagram, l 4 0 f
E l e c t r o d i a l y s i s (ED)
b i o a c t i v e compounds from
f e r m e n t a t i o n b r o t h s , 52
summary, b i o t e c h n o l o g y , 55t
E l e c t r o k i n e t i c parameters of c o l l o i d a l
and b i o l o g i c a l m a t e r i a l s , l 8 0 t
E l e c t r o k i n e t i c separations,
e l e c t r o p h o r e s i s , scale-up
E l e c t r o n micrograph
densely packed E, c o l i , d u a l
h o l l o w - f i b e r b i o r e a c t o r , 38f
densely packed N. m e d i t e r r a n e i , d u a l
h o l l o w - f i b e r b i o r e a c t o r , 41 f
E l e c t r o n paramagnetic resonance (EPR),
a n t i b o d i e s , 208,210,212
Electroosmosis, scale-up
considerations,
e l e c t r o p h o r e s i s , 136
Electrophoresis
c o n t i n u o u s flow
g e n e r a l d i s c u s s i o n , 170
r e c y c l i n g e f f l u e n t , 169
factors affecting electrophoretic
m o b i l i t i e s , 135
f i r s t f r e e - f l o w d e v i c e , 169
production-scale, center f o r
s e p a r a t i o n s c i e n c e , 191
scale-up considerations
controlled convection,
138
d i s c u s s i o n , 122,135
electroosmosis,
136
i s o e l e c t r i c f o c u s i n g , 139
j o u l e h e a t i n g , v i s c o s i t y , and
buoyancy, 138
p l a t e h e i g h t and r e s o l u t i o n
c o n c e p t s , 141-143
Electrophoretic mobility
f a c t o r s a f f e c t i n g , 135
RCFE, e f f e c t i v e d i s p e r s i o n
c o e f f i c i e n t , 175f
Elution profile
chromatographic, p r o t e i n
electrochromatographic, protein
s e p a r a t i o n , 145
s i z e e x c l u s i o n chromatography, 132
Enzymatic d e g r a d a t i o n o f c e l l u l o s e t o
a l c o h o l , membrane r e a c t o r , 58
Enzymatic h y d r o l y s i s o f t r i g l y c e r i d e ,
membrane r e a c t o r , 58
Enzymatic l y s i s and d i s r u p t i o n o f
y e a s t c e l l s , model
a p p l i c a t i o n s , 9,24
E n z y m a t i c - m i c r o b i a l methods, f i n e
c h e m i c a l p r o d u c t i o n , 83
Enzyme
a c c u m u l a t i o n , enzymatic l y s i s and
d i s r u p t i o n o f y e a s t c e l l s , 24
i s o l a t i o n i n aqueous two-phase
systems, 85t
l y t i c system
y e a s t c e l l , 11
p r o t e i n r e l e a s e from y e a s t
c e l l s , 10
p a r t i t i o n i n g , m u l t i p h a s e aqueous
systems, 96
r e l e a s e o f s i t e s p e c i f i c by y e a s t
l y s i s , 25f
RIEF, 191
Enzyme membrane r e a c t o r s
asymmetric c a p i l l a r y , phase
i n v e r s i o n method, 63f
f o r m a t i o n , S. s o l f a t a r i c u s ,
g e l l e d , 61-65
r e s e a r c h and development, 59
Enzyme r e c o v e r y from s u b c e l l u l a r
s t r u c t u r e s , y e a s t l y s i s , 28f
Enzyme r e l e a s e s i m u l a t i o n , y e a s t l y s i s
model, p r o c e s s c o n d i t i o n s , 26

223

INDEX
Equilibrium expressions, size

e x c l u s i o n chromatography, 131t
Escherichia c o l i
c u l t i v a t i o n , dual hollow-fiber
e l e c t r o n micrographs, dual
hollow-fiber bioreactor, 38f
immobilization i n dual h o l l o w - f i b e r
Ethanol, e x t r a c t i v e fermentation i n
aqueous two-phase systems, 80
E x t r a c t i o n , c o n t i n u o u s , aqueous
Extractive bioconversion
b i o s u r f a c t a n t s , aqueous two-phase
systems, 83
p r i n c i p l e , aqueous two-phase
systems, 8 1 f
product i n h i b i t i o n , aqueous
two-phase systems, 79-85
E x t r a c t i v e fermentation, chemicals,
F a c i l i t a t e d transport
LEMs, 68
p h e n y l a l a n i n e - c h l o r i d e system,
mechanism, 6 9 f
Feed l o a d i n g e q u a t i o n s , g e l
chromatography d e s i g n model, 199f
F e r m e n t a t i o n b r o t h , LEM-mediated
s e p a r a t i o n s , downstream p r o c e s s i n g
o f p h e n y l a l a n i n e , 71
F e r m e n t a t i o n chambers, simultaneous
p r o d u c t i o n o f SCP and methane, 46
Fermentation processes, e x t r a c t i v e
b i o c o n v e r s i o n s , product
i n h i b i t i o n , 79-85
Fermentation products
RIEF, 191
t o t a l market v a l u e s , 54t
F i n e c h e m i c a l s , p r o d u c t i o n i n aqueous
Fractionation
i n d u s t r i a l and l a r g e s c a l e , g e l
chromatographic p r o t e i n , 195t
RIEF
i l l u s t r a t i v e , 187-189
l a r g e - s c a l e p r o t e i n , 185
Gaussian parameter c o r r e l a t i o n s , g e l
chromatography d e s i g n
model, 198,200f
G e l chromatography
c u r r e n t d e s i g n methods, 194
d e s i g n model, u s e , 201-204
model development, 196-201
protein fractionation
i n d u s t r i a l and l a r g e s c a l e , 195t
s c a l e - u p methods, 195f
protein purification
i n h e r e n t problems, 194
l a r g e - s c a l e , 193
s c a l e - u p c o s t a n a l y s i s , 204
sequence o f o p e r a t i o n s ,
p r o d u c t i o n - t y p e , 197f
G e l l e d enzyme membrane f o r m a t i o n ,
S. s o l f a t a r i c u s , 61-65
Glucan h y d r o l y s i s r a t e , enzymatic
c e l l s , 16
Glucose
i o n e x c l u s i o n s e p a r a t i o n , 127f
and pH h i s t o r i e s , e f f l u e n t , d u a l
G l y c e r i n e and a c i d s , membrane
r e a c t o r , 58
G r a d i e n t s , pH, RIEF, 189-191
Guanidine HC1 and T r i t o n , s y n e r g i s t i c
e f f e c t on p r o t e i n r e l e a s e p r o f i l e ,
E. c o l i , 4,6f
Hollow-fiber bioreactor
dual
aerobic whole-cell
i m m o b i l i z a t i o n , 33
continuous production o f
r i f a m y c i n B, 4 0 f
c r o s s s e c t i o n a l v i e w , 34f
c u l t i v a t i o n o f E. c o l i , 35
e l e c t r o n micrograph o f d e n s e l y
packed N. m e d i t e r r a n e i , 4 1 f
setup, 36f
i m m o b i l i z a t i o n o f enzymes, 59
membrane, advantages and
d i s a d v a n t a g e s , 32
Hydrocortisone, transformation to
p r e d n i s o l o n e , aqueous two-phase
system, 83
Hydrogel, e f f e c t of bioproduct
d i f f u s i v i t y on l i g a n d
consumption, l 6 4 f
Hydrogel beads
bioproduct separation, immobilized
a f f i n i t y a d s o r b e n t s , 153
simulation studies, small a f f i n i t y
adsorbent
i m m o b i l i z a t i o n , 158-164

224

Hydrolysis
c e l l w a l l , enzymatic l y s i s and
g l u c a n and s o l u b l e p r o t e i n ,
enzymatic l y s i s and d i s r u p t i o n
o f y e a s t c e l l s , 16
t r i g l y c e r i d e , membrane r e a c t o r ,
enzymatic, 58
Hydrophobic chromatography, polymer
removal, p r o t e i n p u r i f i c a t i o n , 89
Immobilization
aerobic w h o l e - c e l l , dual
h o l l o w - f i b e r b i o r e a c t o r , 33
e f f e c t on a n t i b o d y a c t i v i t y , 209
enzymes i n membranes, 59
s p e c t r o s c o p y , 210
monoclonal a n t i b o d i e s , l i n k a g e
through p r o t e i n A, 214
simulation studies, small a f f i n i t y
adsorbents i n hydrogel
beads, 158-164
Immobilized a f f i n i t y adsorbents
mathematical modeling o f b i o p r o d u c t
a d s o r p t i o n , 153
schematic diagram, 156f
Immobilized a l l o s t e r i c enzymes, g e l l e d
membrane f o r m a t i o n , 61
Immobilized monoclonal a n t i b o d i e s
EPR s t u d i e s o f s t r u c t u r e and
f u n c t i o n , 208
s i m u l a t e d and e x p e r i m e n t a l s p e c t r a ,
EPR, 212
Immobilized systems
a l c o h o l i c f e r m e n t a t i o n s compared t o
f i n e c h e m i c a l s p r o d u c t i o n compared
w i t h aqueous two-phase
systems, 82
Immunoglobulin
d e s c r i p t i o n , 209
p a r t i t i o n , 107
Immunoglobulin G
c o n c e n t r a t i o n i n s a l t phase as
function of s a l t concentration,
PEG-water s o l u t i o n , 100f
p a r t i t i o n c o e f f i c i e n t as a f u n c t i o n
o f pH, PEG-water s o l u t i o n , 101f
p a r t i t i o n c o e f f i c i e n t as f u n c t i o n o f
s a l t c o n c e n t r a t i o n , PEG-water
s o l u t i o n , 100f
I n h i b i t i o n , fermentation processes,
extractive bioconversions i n
aqueous two-phase systems,
p r o d u c t , 79-85
I n t e r f e r o n , RIEF, 191
Ion exchange chromatography, polymer
removal, p r o t e i n p u r i f i c a t i o n , 89
Ion exchange r e s i n s as chromatographic
supports, scale-up o f
I s o e l e c t r i c focusing (IEF)
a p p a r a t u s , r e c y c l i n g , 187
e l e c t r o p h o r e s i s scale-up
s c a l e - u p , d i s c u s s i o n , 185
I s o l a t i o n o f enzymes i n aqueous
two-phase systems, 85t
Joule heating
c l a s s i c t h i n - f i l m CFE, 170
v i s c o s i t y , and buoyancy,
e l e c t r o p h o r e s i s scale-up
L a r g e - s c a l e g e l chromatography,
p u r i f i c a t i o n of protein
p r o d u c t s , 193
Large-scale protein f r a c t i o n a t i o n ,
RIEF, 185
Linear equilibrium, size exclusion
chromatography, 132
Linkage through i m m o b i l i z e d p r o t e i n ,
L i q u i d chromatography, p r o c e s s
c o n s i d e r a t i o n s f o r s c a l e - u p , 122
L i q u i d emulsion membrane (LEM)
amino a c i d r e c o v e r y
d i s c u s s i o n , 71-75
p r o c e s s diagram, 7 6 f
a p p l i c a t i o n s , 70
b i o c h e m i c a l s e p a r a t i o n s , 67
chloride countertransport
system, 7 3 f
concept, 67
e f f e c t o f a g i t a t i o n speed, 7 3 f
system, 6 9 f
v e r s a t i l i t y , 75
L o a d i n g , g e l chromatography d e s i g n
model, 198
L y s a t e , amino a c i d , membrane r e c o v e r y
p r o c e s s , 56
L y s i n g y e a s t c e l l , s c h e m a t i c , 12f
Lysis
sequence o f c e l l , enzymatic l y s i s
and d i s r u p t i o n o f y e a s t c e l l , 11
y e a s t , p r o c e s s c o n d i t i o n s f o r enzyme
r e l e a s e s i m u l a t i o n , 26

225
INDEX
L y t i c enzyme system
y e a s t c e l l , 11
p r o t e i n r e l e a s e from y e a s t c e l l s , 10
y e a s t l y s i s , 16

Macroraolecules, p r o d u c t i o n from
Mass t r a n s f e r e x p r e s s i o n s , s i z e
e x c l u s i o n chromatography, 131t
M e c h a n i c a l s t a b i l i t y , LEMs, 68
M e c h a n i c a l l y based p r o t e i n r e l e a s e
methods, u n d e s i r a b l e p r o p e r t i e s , 2
Membrane
asymmetric enzyme c a p i l l a r y , phase
i n v e r s i o n method, 63f
enzyme, r e s e a r c h and development, 59
l i q u i d e m u l s i o n , amino a c i d
r e c o v e r y , 71-75
Membrane b i o r e a c t o r s
amino a c i d from l y s a t e , 56
enzyme, 58
growth o f rumen b a c t e r i a
g l u c o s e , 45t
sugar beet p u l p , 46t
h o l l o w f i b e r , advantages and
d i s a d v a n t a g e s , 32
SCP and methane, rumen
fermentation, 47f
simultaneous p r o d u c t i o n o f SCP and
methane, 43
Membrane d i s t i l l a t i o n , summary,
b i o t e c h n o l o g y , 55t
Membrane f o r m a t i o n , S. s o l f a t a r i c u s ,
g e l l e d enzyme, 61-65
Membrane f o r m u l a t i o n , LEM-mediated
amino a c i d r e c o v e r y , t y p i c a l , 72
Membrane p r o c e s s e s
downstream, 57f
summary, b i o t e c h n o l o g y , 5 5 t
Membrane s w e l l , LEMs, 70
Membrane t e c h n o l o g y , i n t e g r a t i o n w i t h
Methane
membrane b i o r e a c t o r p r o d u c t i o n , 43
SCP and, rumen f e r m e n t a t i o n
p r o d u c t i o n i n membrane
b i o r e a c t o r , 47f
Microbial c e l l s , subcellular location
o f enzyme a c t i v i t i e s , 10
M i c r o f i l t r a t i o n , summary,
b i o t e c h n o l o g y , 55t
M i t o c h o n d r i a , r e l e a s e o f c y t o s o l and,
Model
c o n v e c t i v e d i s p e r s i o n , RCFE, 174
yeast c e l l s
a p p l i c a t i o n s , 24
background, 11
discussion, 9
s o l u b l e p r o t e i n , p e p t i d e s , and
c a r b o h y d r a t e s , 21-24
y e a s t mass, 21-24
particle size distribution, protein
p r e c i p i t a t i o n , 112-116
p r e c i p i t a t i o n phenomena, p r o t e i n
r e c o v e r y , 109
yeast l y s i s
r e a c t i o n pathways f o r
s t r u c t u r e d , 17f
s i m p l e and s t r u c t u r e d , 13
s t r u c t u r e d , 19
v a r i a b l e s and parameters, 15t
Monoclonal a n t i b o d i e s
i m m o b i l i z e d , EPR s t u d i e s o f
s t r u c t u r e and f u n c t i o n , 208
s p e c t r o s c o p y , 210,214
M u l t i p h a s e aqueous systems, phase
Nocardia mediterranei
e l e c t r o n micrograph, d u a l
immobilization, dual hollow-fiber
p r o d u c t i o n o f r i f a m y c i n B, d u a l
Nucleic acid
p a r t i t i o n i n g , m u l t i p h a s e aqueous
systems, 96
O l i g o s a c c h a r i d e s , i o n exchange
Organic s o l v e n t , e f f e c t , enzymatic
s y n t h e s i s o f f i n e c h e m i c a l s , 83
Osmolality of c e l l lysate, e f f e c t i v e ,
Osmotic s w e l l , LEMs, 70

226

P a r t i c l e formation, primary, p r o t e i n
Particle size distribution, protein
p r e c i p i t a t i o n , model, 112-116
P a r t i t i o n c o e f f i c i e n t , PEG-water
s o l u t i o n , immunoglobulin as a f u n c t i o n
of pH, 101f
P a r t i t i o n e d phases, a p p l i c a t i o n s , 96
Partitioning
a f f i n i t y , aqueous two-phase
systems, 86t
a f f i n i t y s o r b e n t , two-phase
system, 8 8 f
plasma p r o t e i n s , m u l t i p h a s e aqueous
systems, 96
p r o t e i n s , aqueous two-phase systems,
a f f i n i t y , 85-87
P e c l e t numbers, RCFE, performance
e v a l u a t i o n , 178-181
P e p t i d e s and c a r b o h y d r a t e s , e n z y m a t i c
c e l l s , 21-24
Permeabilization, chemical, protein,
DNA, and RNA r e l e a s e from
E. c o l i , 2-4
pH
e f f e c t on s a l t p a r t i t i o n r e c o v e r y o f
p r o t e i n s , 103
e f f l u e n t , dual hollow-fiber
b i o r e a c t o r , g l u c o s e , 36f
g r a d i e n t s , RIEF, 189-191
Phase i n v e r s i o n , asymmetric enzyme
c a p i l l a r y membranes, 63f
Phase p a r t i t i o n i n g , m u l t i p h a s e aqueous
systems, 95
P h e n y l a l a n i n e , downstream p r o c e s s i n g
from f e r m e n t a t i o n b r o t h ,
LEM-mediated s e p a r a t i o n s , 71
P h e n y l a l a n i n e - c h l o r i d e system,
mechanism f o r f a c i l i t a t e d
t r a n s p o r t , 69f
Plasma p r o t e i n s , p a r t i t i o n i n g ,
m u l t i p h a s e aqueous systems, 96
P l a t e h e i g h t and r e s o l u t i o n concepts
in electrophoresis,
s c a l e - u p , 141-143
P o l y ( e t h y l e n e g l y c o l ) (PEG)-dextran
e x t r a c t i v e f e r m e n t a t i o n , two-phase
system, 80
two-phase systems i n
b i o t e c h n o l o g y , 78
P o l y ( e t h y l e n e g l y c o l ) (PEG)-water,
r e c o v e r y o f p r o t e i n s by s a l t
p a r t i t i o n , 93,104-107
P o l y c l o n a l antibody f r a c t i o n a t i o n ,
RIEF, I89,190f
Polymers, removal from p u r i f i e d
p r o t e i n , aqueous two-phase
systems, 87
Population balances, modeling, p r o t e i n

P r e c i p i t a t e b e h a v i o r , models, p r o t e i n
Precipitation
model
particle size distribution,
p r o t e i n , 112-116
p r o t e i n r e c o v e r y , 109
P r e d n i s o l o n e , aqueous two-phase
system, t r a n s f o r m a t i o n o f
h y d r o c o r t i s o n e , 83
Product i n h i b i t i o n , f e r m e n t a t i o n
processes, e x t r a c t i v e
b i o c o n v e r s i o n s i n aqueous
two-phase systems, 79-85
Production
b u l k c h e m i c a l s , two-phase
systems, 80
f i n e c h e m i c a l s , aqueous two-phase
systems, 82
macromolecules, aqueous two-phase
systems, 84
p r o t e c t i v e a n t i g e n s , aqueous
r i f a m y c i n by N. m e d i t e r r a n e i , d u a l
Production-scale electrophoresis
system, c e n t e r f o r s e p a r a t i o n
s c i e n c e , 191
P r o d u c t i o n - t y p e g e l chromatography
system, sequence o f
o p e r a t i o n s , 197f
P r o d u c t i v i t y , g e l chromatography
d e s i g n model, 210,202f
Protein
a n a e r o b i c SCP, membrane r e a c t o r
aqueous two-phase systems, removal
of polymers, 87
l a r g e - s c a l e g e l chromatography
f r a c t i o n a t i o n , 193
p a r t i t i o n i n g o f plasma, m u l t i p h a s e
aqueous systems, 96
recombinant, problems i n
production, 9
r e l e a s e , E. c o l i , 4
RIEF, 191
Protein fractionation
RIEF, l a r g e - s c a l e , 185
s c a l e - u p methods, g e l
chromatography, 195 f
P r o t e i n h y d r o l y s i s , enzymatic l y s i s
and d i s r u p t i o n o f y e a s t c e l l s , 16
Protein precipitation
d i s c u s s i o n , 110-112
p a r t i c l e s i z e d i s t r i b u t i o n , 112-116
p r e c i p i t a t e b e h a v i o r , 109,116
Protein purification
i n h e r e n t problems, g e l
chromatography, 194
p a r t i t i o n i n g i n aqueous two-phase
systems, 85-87

227

INDEX
P r o t e i n recovery
e f f e c t o f pH and s a l t c o n c e n t r a t i o n
on s a l t p a r t i t i o n i n g , 103
salt partitioning
effect of s a l t
c o n c e n t r a t i o n , 97,98
PEG-water s o l u t i o n , 93,104-107
Protein release
chemically permeabilized
E. c o l i , 2-7
T r i t o n and g u a n i d i n e HC1 treatment
o f E. c o l i , 6f
undesirable properties,
mechanical, 2
Protein separation
chromatographic, c a l c u l a t e d e l u t i o n
p r o f i l e , 137f
electrochromatographic, c a l c u l a t e d
e l u t i o n p r o f i l e , 145
P r o t e o l y s i s , a f t e r mechanical
d i s r u p t i o n , 10
P u r i f i c a t i o n o f p r o t e i n s by
p a r t i t i o n i n g i n aqueous two-phase
systems, 85-87
R e a c t i o n pathways f o r s t r u c t u r e d
model, y e a s t l y s i s , 17f
Recombinant p r o t e i n s
problem i n p r o d u c t i o n , 9
r e c o v e r y from E. c o l i , 2
Recovery and p u r i f i c a t i o n i n
b i o t e c h n o l o g y , aqueous two-phase
systems, 78-90
Recycle continuous-flow
e l e c t r o p h o r e s i s (RCFE)
d i s c u s s i o n , 171-173
model, 173-178
performance e v a l u a t i o n , e l e v a t e d
P e c l e t numbers, 178-181
r e g e n e r a t o r s , s c h e m a t i c , I82f
schematic, 172f
R e c y c l i n g i s o e l e c t r i c f o c u s i n g (RIEF)
illustrative fractionation
r e s u l t s , 187-189
p o l y c l o n a l r a b b i t a n t i b o d i e s , 190f
schematic p r e s e n t a t i o n , I 8 8 f
Regenerators, RCFE, 181
Reverse osmosis
Rifamycin
continuous p r o d u c t i o n , d u a l
hollow-fiber bioreactor, 40f
p r o d u c t i o n by N. m e d i t e r r a n e i , d u a l
RNA r e l e a s e
E. c o l i t r e a t e d w i t h g u a n i d i n e HC1
and T r i t o n , 5 f
m e c h a n i c a l l y d i s r u p t e d E. c o l i , 4
Rumen b a c t e r i a , growth on g l u c o s e ,
membrane r e a c t o r , 45t
Rumen f e r m e n t a t i o n , membrane
b i o r e a c t o r , SCP and methane
p r o d u c t i o n , 47f
Rumen m i c r o o r g a n i s m s , a n a e r o b i c SCP
Salt concentration, effect, s a l t

p a r t i t i o n i n g recovery o f
p r o t e i n s , 98,103
Salt partition
applications, protein
r e c o v e r y , 104-107
e f f e c t o f pH and s a l t c o n c e n t r a t i o n ,
p r o t e i n r e c o v e r y , 103
methodology, p r o t e i n r e c o v e r y , 97
Scale-up c o n s i d e r a t i o n s
chromatographic p r o c e s s e s , 126-129
cost a n a l y s i s , g e l
chromatography, 204
g e l chromatography, p r o t e i n
f r a c t i o n a t i o n , 195f
IEF, 185
S e l e c t i v i t y equation, g e l
chromatography d e s i g n
model, 198,200f
Separations
product from key contaminant, g e l
chromatography, 195 f
u s i n g LEMs, 68
Separations science, center f o r ,
production-scale electrophoresis
system, 191
S i n g l e - c e l l p r o t e i n (SCP)
a n a e r o b i c , membrane r e a c t o r , 43,44
methane, rumen f e r m e n t a t i o n i n
membrane r e a c t o r , 4 7 f
Single-component d i f f u s i o n and
binding, immobilization i n
h y d r o g e l , 158-163
S i n g l e - p a s s c o n t i n u o u s flow
e l e c t r o p h o r e s i s , model
comparison, 175f
S i z e - e x c l u s i o n chromatography
i n i t i a l and boundary
c o n d i t i o n s , 130t
mass t r a n s f e r and e q u i l i b r i u m
e x p r e s s i o n s , 1311
scale-up, t h e o r e t i c a l
c o n s i d e r a t i o n s , 129-135
S o l f o l o b u s s o l f a t a r i c u s , g e l l e d enzyme
membrane f o r m a t i o n , 61-65


228
SEPARATION, RECOVERY, A N D PURIFICATION IN B I O T E C H N O L O G Y
S o l u b l e p r o d u c t s , model, enzymatic
c e l l s , 18
Soluble protein
h y d r o l y s i s , enzymatic l y s i s and
p e p t i d e s , and c a r b o h y d r a t e s ,
enzymatic l y s i s and d i s r u p t i o n
o f y e a s t c e l l s , model
s i m u l a t i o n s , 21-24
Solvents, production, extractive
systems, 80
S p i n l a b e l , EPR s e n s i t i v i t y ,
S p i n l a b e l motion, monoclonal
Sugar, i o n exchange s e p a r a t i o n , 125f
Sugar beet p u l p , membrane r e a c t o r ,
growth o f rumen b a c t e r i a , 46t
Sulfuric acid, ion exclusion
S w e l l i n g , LEMs, 70
T h i n - f i l m continuous-flow
electrophoresis, general
T o x i c i t y , macromolecules on b a c t e r i a l
c e l l s , 84
T r a n s p o r t , LEMs, 68
T r i g l y c e r i d e s , degree o f c o n v e r s i o n
w i t h t i m e , membrane p r o c e s s e s , 6 0 f
T r i t o n , c e l l u l a r p r o t e i n r e l e a s e from
E. c o l i , 4
Two-component d i f f u s i o n and b i n d i n g ,
i m m o b i l i z a t i o n i n h y d r o g e l , 163
Two-phase system
a f f i n i t y sorbent, p a r t i t i o n i n g , 88f
aqueous
b i o c a t a l y t i c r e a c t i o n , 79
b u l k c h e m i c a l p r o d u c t i o n , 82t
examples o f a f f i n i t y
p a r t i t i o n i n g , 86t
e x t r a c t i v e b i o c o n v e r s i o n s , product
i n h i b i t i o n i n fermentation
p r o c e s s e s , 79-85
f i n e c h e m i c a l s p r o d u c t i o n , 82
i s o l a t i o n o f enzymes, 85t
macromolecules p r o d u c t i o n , 84
principle for extractive
b i o c o n v e r s i o n , 81 f
p u r i f i c a t i o n o f p r o t e i n s by
p a r t i t i o n i n g , 85-87
r e c o v e r y and p u r i f i c a t i o n i n
b i o t e c h n o l o g y , o v e r v i e w , 78-90
Two-phase systems, p u r i f i c a t i o n and
r e c o v e r y i n b i o t e c h n o l o g y , 78
U l t r a f i l t r a t i o n (UF)
V i s c o s i t y , j o u l e h e a t i n g , and
buoyancy, e l e c t r o p h o r e s i s s c a l e - u p
Volumetric p r o d u c t i v i t i e s , i n d u s t r i a l
and l a r g e - s c a l e g e l chromatography
p r o t e i n f r a c t i o n a t i o n s , 195t
W a l l h y d r o l y s i s e q u a t i o n s , enzymatic
c e l l s , 18
W a t e r - s o l u b l e polymers, two-phase
systems i n b i o t e c h n o l o g y , 78
Yeast c e l l s
a p p l i c a t i o n s , model o f enzymatic
l y s i s and d i s r u p t i o n , 24
models, enzymatic l y s i s and
disruption, 9
r e l e a s e o f c y t o s o l and
m i t o c h r o n d r i a , 18
schematic o f l y s i n g , 12f
s o l u b l e p r o d u c t s , model, enzymatic
l y s i s and d i s r u p t i o n , 18
s t r u c t u r e model, enzymatic l y s i s and
d i s r u p t i o n , 11
Yeast l y s i s
enzyme r e c o v e r y from s u b c e l l u l a r
s t r u c t u r e s , 27f
l y t i c system, enzymes, 16
model, p r o c e s s c o n d i t i o n s f o r enzyme
r e l e a s e s i m u l a t i o n , 26
r e l e a s e o f s i t e - s p e c i f i c enzymes,
simulation, 25f
s i m p l e model, 13,17f
s t r u c t u r e d model, 13
v a r i a b l e s and parameters, model, 15t
Yeast mass, enzymatic l y s i s and
d i s r u p t i o n o f y e a s t c e l l s , model
s i m u l a t i o n s , 21-24
Yeast w a l l , d o u b l e - l a y e r e d
s t r u c t u r e , 12f
Y i e l d and p u r i t y , g e l chromatography
d e s i g n model, 198


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Separation, Recovery, and

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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Separation, Recovery, and

Juan Hong, EDITOR

Developed from a symposium sponsored by

American Chemical Society, Washington, D C 1986

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

Library of Congress Cataloging-in-Publication Data

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Includes bibliographies and index.

American Chemical Society

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

ACS Symposium Series

M. Joan Comstock, Series Editor

Marye Anne Fox

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

The ACS SYMPOSIUM SERIES was founded in 1974 to provide a

medium for publishing symposia quickly in book form. The

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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Illinois Institute of Technology

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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David J. Hettwer and Henry Y. Wang

0097-6156/ 86/ 0314-0002$06.00/ 0

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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HETTWER AND WANG

Protein Release from Chemically Permeabilized E. coli

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SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

F i g u r e 1 shows the p r o t e i n , DNA,

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

HETTWER AND WANG

Protein Release from Chemically Permeabilized E. coli

EXTENT OF RELEASE OF CELL COMPONENTS (*),

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DISRUPTION TIME (min.)

EXTENT OF RELEASE OF CELL COMPONENTS (*),

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SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

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TRITON X100 (*v/v), GUANIDINE HC1 (M)

and g u a n i d i n e HC1 on the p r o t e i n

PROTEIN RELEASE (*)

2t1 GUANIDINE HC1

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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HETTWER AND WANG

Protein Release from Chemically Permeabilized E. coli

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

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Edebo, L. In 'Fermentation Advances'; Perlman, D., Ed.;

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.;

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J. B. Hunter and J. A. Asenjo