The Importance of

Freezing on
Lyophilization Cycle
Development
Thomas W. Patapoff and David E. Overcashier
The freezing method
used during
lyophilization can
substantially affect
the structure of the
ice formed, the watervapor flow during
primary drying, and
the quality of the final
dried product.
Controlling how a
solution freezes can
shorten lyophilization
cycles and produce
more stable
formulations.

L
16

BioPharm

MARCH 2002

yophilization (freeze-drying) is often

used to prepare dry pharmaceutical
formulations to achieve commercially
viable shelf lives. The process
comprises three steps: freezing, primary
drying, and secondary drying. As
water freezes in the first step, the
dissolved
components in the formulation remain in the residual liquid, a phase termed the freeze-concentrate.
At the point of maximal ice formation, the freezeconcentrate solidifies between the ice crystals
that make up the lattice. Under appropriate
lyophiliza- tion conditions, the ice is removed by
sublimation during primary drying, leaving
the remaining freeze-concentrate in the same
physical and chemi- cal structure as when the ice
was present. Residual water in the freezeconcentrate is removed in the secondary drying
step.
Lyophilization cycle development typically
focuses on optimizing the primary drying
step. That is the most time consuming of the
three steps, and the primary drying
parameters are easily adjustable. They can
affect both the time involved and the quality
of the resulting cake. Extensive investigation
of primary drying has demonstrated that two
important parameters are chamber pressure
and shelf temperature (19). They are usually
adjusted to maximize the rate of heat transfer to
each vial (speeding ice sublima- tion) without
causing cake collapse.
Less attention has been paid to the freezing
conditions and their potential effect on the
pri- mary and secondary drying processes and
on the characteristics of the final product.
Kochs et al. reported the effects of freezing

conditions on pri- mary drying

in
a
specially
designed
aluminum and plastic sample
cell (10). They observed variations
in
vapor
diffusion
coefficients (a measure of

the ease of water-vapor flow) as a function

of
position and cooling rate. The
variations appeared to be largely due to
variations in sample morphology. Searles et
al. reported some effects of freezing on the
rate of primary drying in vials (11). They
found that the primary drying rate was
dependent on the ice nucleation
temperature. Faster drying was also
attributed to an increase in lamellar ice
crystal content, formed when ice
nucleants from Pseudomonas syringae
were added in the vial contents. Searles and
colleagues also showed that adding a properly
specified an- nealing step in the freezing
procedure increased the drying rate (12).
Our article describes the effects of
several freezing methods on water-vapor flow
during pri- mary drying and on the final dried
cake structure.

NORMAL
FREEZING
SUBLIMATION

AND

When
freeze-drying
pharmaceutical
products, manufacturers place vials
containing aqueous formulations on shelves
within a lyophilizer. Typi- cally, the shelf
temperature is then decreased (from
5 C) over three hours and held at a low
tempera- ture (typically 40 C to 50 C)
to ensure that all the vials freeze completely.
As the temperature decreases, the liquid cools
below the freezing point of the solution:
Supercooled temperatures range from 10
C to 20 C with ice nucleation typi- cally
occurring at 15 C. Freezing point depression accounts for a lowering of the freezing
tem- perature by only 1.8 kelvin per
molal. Most pharmaceutical manufacturing
relies on supercool- ing because of the lack of
nucleation centers in the formulations (such
as particulates in solution or imperfections
on the inside surface of the vials).
In a supercooled solution at 15 C, ice
can nucleate and propagate through the
solution.

MASS TRANSFER RESISTANCE

An important parameter governing the
relation- ship between independent variables
(the shelf temperature and chamber pressure)
and depen- dent variables (the ice
sublimation rate and the product temperature)
has been defined by Pikal as the resistance to
water-vapor flow or the mass transfer resistance
(MTR) (2).
Determining
MTR. MTR describes the
propor- tionality between the specific
sublimation rate
m and the pressure driving force P
o
Pv:
Ap
Po Pv
MTR =
m/Ap
where m is the sublimation rate, Ap is the crosssectional area of the product in the vial, MTR
is
the normalized dried product resistance, Po is
the equilibrium vapor pressure of ice at the
tempera- ture of the subliming ice, and Pv is
the pressure in the chamber. The relationship is
analogous to that of electrical resistance: For
a given pressure driving force (voltage), a
decrease in MTR (elec- trical resistance)

Freezing
Method

Shelf
Temp (C)

Sublimation
Rate (g/hr)

Standard
Vertical
Vertical
Vertical

10
10
20
30

0.36
0.46
0.48
0.55

(a)
Dried Layer
[Resistance (cm2 mTorr hr g
1
)]

Product
Temp (C)
24.5
27.0
26.0
23.5

Table 1. Effect of freezing

method and primary drying shelf
temperature on sublimation rate
and primary drying product
temperature

Fast freezing
Standard freezing
Vertical freezing
Annealed

10,000
8,000
6,000
4,000
2,000
0
(b)
50,000

Derivative
[Resistance (cm2 mTorr hr g
1
)]

However, the entire solution cannot freeze immediately because the supercooled water can
absorb only 15 cal/g of the 79 cal/g of heat
given off by the ice formation. Therefore, the
ice propagates from the nucleation site, and
about 19% crystal- lizes in multibranching,
tortuous paths while the vial contents warm
to approximately 1 C. After the initial
ice network has formed, addi- tional heat is
removed from the solution by the shelf, and
the remaining water freezes when the
previously formed ice crystals grow.
Primary drying. At the completion of
freezing, primary drying is initiated with the
lowering of the chamber pressure and an
increase in the shelf temperature. The ice
sublimation front moves downward through
the frozen plug, a process that makes the water
vapor transit through the chan- nels left by the
sublimed ice above. Those narrow, often tortuous
channels resist vapor flow, so subli- mation is
slowed. Consideration of the vapor channels
illustrates how the method of freezing, by
affecting ice crystal structure, can affect
primary drying. Decreasing the resistance to
mass transfer of water vapor increases the
sublimation rate, which leads to shorter primary
drying times while lowering the product
temperature.

Fast freezing
Standard freezing
Vertical freezing
Annealed

40,000
30,000
20,000
10,000
0

0.2 0.4 0.6 0.8 1.0

Dried Layer Thickness (cm)

then weighing the amount of the remaining

solu- tion. We previously published a complete
descrip- tion of this method for determining
MTR (9).
Effect of the freezing method on MTR. Figure
1 shows MTRs during primary drying (10 C
shelf temperature, 100 mTorr chamber
pressure) for vials frozen using the standard
freezing process that we described earlier and
for those frozen by alternative methods
described below (plots used
25 mg/mL rhuMAb HER2, 20 mg/mL
trehalose,
0.1 mg/mL polysorbate 20, and 5 mM
histidine, with a 6.0 pH, filled at 5.0 mL in
10cc tubing vials). Following standard freezing,
the resistance initially rose quickly, then more
slowly as the sublimation front moved down
the vial (increas- ing the thickness of the
dried layer). That indi- cates that the upper
layer of the dried cake contributed more to
the resistance than did the lower portion,
suggesting that the structure of the dried layer

Figure 1. Mass transfer

resistance as a function of
dried layer thickness for
material frozen by various
methods: (a) cumulative MTR,
(b) derivative of MTR

BioPharm

MARCH 2002

17

increases the rate of sublimation (electrical

current). To determine MTR, measure the
product temperature (from which we obtain Po
using the equation published by Jancso et al.,
13)
and the sublimation rate m . These
measurements can be obtained using a
thermocouple and sequen- tially stoppering the
vials during primary drying,

18

BioPharm

MARCH 2002

filled vial

add fluorescent dye

lyophilize warm, add black paraffin

under vacuum

cool,
release

image under a

fluorescence
microscope

cut plug

plug

Figure 2. Paraffin dye method

local MTR for various cake depths can

be
determined from the derivative of those
curves (Figure 1b). The derivative curve (for
standard freezing) also shows that the upper
layer of the dried cake contributes more to the
resistance than does the lower portion. That
result is consistent with the results published
by Kochs et al. (10). The relationship
between resistance and dried cake structure
can be investigated by studying cake structure
after lyophilization.

CAKE STRUCTURE
The structure of a lyophilized cake has
been reported several times (1420). In
most cases, cakes came from a supercooled
solution with little or no attempt at
controlling ice nucleation or propagation
direction. Samples were generally cut or pulled
apart and viewed by scanning electron
microscopy (SEM). Because the potentially
fragile cake structure was unsupported, the area
exposed may have been altered by the cutting. If
the cake is pulled apart, the tear will likely
follow a structural weakness in the cake, so the
exposed area may not be representative of the
rest of the cake.
In the paraffin investment technique we developed (Figure 2), paraffin acts as a support for
the physical fine structure of the cake and
also prevents water absorption during

vacuum, the paraffin enters all the pores of

the cake with no observable disturbance to the
cake structure. The vial is then removed
from the vacuum oven, cooled, and
sectioned. The sectioned cakes are then
observed
under
a
fluorescence
microscope.
Interference
from
the
fluorescence deeper in the cake is minimized
by using a dark dye in the paraffin.
Correlating MTR to cake structure. The
tortuous path that supercooled ice takes when
propagating (Figure 3a) shows little
directionality to the chan- nels after
sublimation. Those channels, roughly
25100 m in diameter, serve as conduits
for water-vapor transport from the sublimation
front to the vial headspace. Resistance to
vapor flow
out of the cake is a result of both the small,
tortuous channels and thick skin-like material on
the top of the cake (reaching a thickness greater
than
12 mm). In the standard freezing scenario of
Figures 1a and 1b, high MTR can be seen as
the
sectioning,
observation
, and storage
(9).
The
method uses
a
fluorescent
molecule
(such
as
rhodamine
B) that is
added to the
solution
before
lyophilization
and
distributes
equally
throughout
the
cake
(with
no
visible
phase
separation
or
spatial
partitioning).
After
lyophilization
, the cake in
the vial is
placed
under

vacuum and warmed to 55 C. Then paraf- fin

is poured into the vial. While warm and under

sublimation front moves down through the

first
0.2 cm of the cake, corresponding to the
thick- ness of the skin. Once the
sublimation front passed the skin, the
cumulative MTR increased more slowly
(suggesting lower local MTR).

CONTROLLING ICE NUCLEATION

One way to produce directional freezing is
to freeze the vial contents rapidly by
immersing the vial into a dry-ice/ethanol bath
until it is completely frozen. The resulting
pores left from the rapid freezing and ice
propagation are very small (Figure 3b).
They start at the walls of the vial, move
toward the middle, then move up. Although
directional, the pores are small and
nonvertical. The effect of such fast freezing can
be seen in the MTR: The cumulative MTR
increases rapidly and to a higher level (Figure
1a) and shows very high local MTR at small
cake depths (Figure 1b). For fast freezing, that
high MTR is attributable to the small size of
the pores and to the horizontal direc- tion of the
channels near the surface of the cake. The
restricted vapor flow results in slower ice sublimation.
Vertical freezing. To obtain straight,
vertical channels, solutions were cooled
on wet ice, nucleated at the bottom with dryice, and placed on a 50 C shelf. The ice
crystals propagate vertically, and no thick
skin forms on the cake surface (Figure 3c).
As expected, a vertical chan- nel orientation
and the lack of thick skin on top lowers the
MTR from that of samples from stan- dard
and fast-freezing protocols (Figure 1a).
Figure 1b shows that the local MTR is also
low and constant for all cake depths.
Interestingly, Figure 1b also suggests that
changing the freezing

method did not alter the cake structure at the

vial bottom, because the local MTR values are
equiva- lent for dried layer thickness greater than
0.5 cm.
Vertical freezing produces a higher sublimation rate and lower product temperature
during primary drying (Table 1). The lower
MTR allows faster sublimation, which results in
lower product temperatures
through
increased heat removal from the latent heat
of sublimation. The vertical freezing results
are consistent with those found by Searles et
al. that the presence of lamellar (instead of
spheroidal) ice crystals increases dry- ing rates
(11).

Figure 4a shows an SEM result in which

holes have formed in the walls of the
pores. These porous walls were not
evident in formulations containing protein
(Figure 4b) but were observed

SUPERCOOLING AND ANNEALING

Ice structure and MTR during primary drying can
be altered by freezing the vial contents using
the standard method (supercooling followed by
spon- taneous nucleation), then warming the
frozen solution in the vial to a temperature
just below the melting point (for example
2 C) for
10 hours. This allows the ice crystals to rearrange
and grow to a more stable state. The vials
are then cooled again to 50 C. Figure 3d
shows results from that annealing process.
The cakes produced after annealing tend to
have larger pores than those from the
standard freezing method, which would be
expected
to
facilitate
water-vapor
transmission and lower the MTR during
primary drying. Figure 1a shows that the
annealed material has a much lower MTR
than solutions frozen by the standard or fast
processes and is comparable to solutions that
were nucle- ated on the bottom with vertical
ice propagation. The lower resistance
observed for annealed material is consistent
with a faster sublimation reported by
Searles et al. (12). Figure 1b also shows that
the annealed material has a local MTR that is
low and constant throughout the cake. The
effects of any skin appear to be minimal.

DECREASING MTR BY OTHER METHODS

Depending on solution composition, it may be
possible to adjust the temperature (or the
formu- lation) such that primary drying
takes place at product temperatures near the
glass transition temperature (Tg ) of the
freeze-concentrate. That allows the freezeconcentrate to flow enough to create holes in
the channel walls so water vapor can be
transmitted in a less tortuous path, more
directly up and out of the cake.

Table 2.inEffect
of freezingusing a
only
formulations
method on theplacebo,
dried state
protein-free
which has
a
a therapeutic
protein
astability
lower ofTg
. The holes
were
less evident in the protein-free
material when it was dried at
Freezing
Aggregation
lower
temperatures.Rate
That
b implies
Method
that not all formulations are
Standard
2.3c
amenable
to pore formation.
Annealed
1.6
Figure 5 shows that the MTR
a2.5 mg/mL protein, 123 mg/mL
fortotal
theexcipient
placebo was significantly
bat 50 than
lower
that of the solution
C
cpercentcontains
per month protein
that
(9).
Obviously, the selection of
processing conditions needs to
consider final product quality.
The possibility that such a smallscale collapse might alter the
molecular structure or stability of
the product would need to be
eval- uated by an appropriate
analytical program.

OPTIMIZIN
G
THE
CYCLE

Dried Layer
[Resistance (cm2 mTorr hr g
1
)]

The manner of freezing can have a significant

impact on primary drying characteristics of a
formulation. For a given primary drying
shelf temperature and chamber pressure, a
decrease in MTR will increase the rate of
sublimation. In addition, an increase in the
rate of sublimation will lead to a lower product
temperature because of the enthalpy of ice
sublimation (Table 1). Increasing the primary
drying shelf temperature of vertically frozen
vials increases the product tem-

Figure 3. Freeze-dried cake appearance

corresponding to standard and alternative
freezing methods:
(a) supercooling using standard freezing; (b)
fast freezing;
(c) vertical freezing; and
(d) standard freezing followed by annealing
(photo courtesy of Genentech, Inc.)

6,000
Acti
ve

5,000
4,000
3,000

P
l
a
c
e
b
o

2,000
1,000
0

0
0.2
0.4
0.6
0.8
1
D
r
i
e
d
l
a
y
e
r
t
h
i
c
k
n
e
s
s
(
c
m
)

tion,
recons
titutio
n time
could
be
affecte
d
becaus
e
increas
ing the
surfac
e area
may
allow
faster
recons
titutio
n.

P
R
A
C
T
I
C
A
L
A
S
P
E
C
T
S
Freezi
ng
metho
ds are
seldo
m
investi
gated
becaus
e
freezin
g
is
difficul
t
to

control.
Normally,
pharmaceutic
al
products
supercool
to
roughly
15 C before
nucleating,
which
is

diffic
ult to
overc
ome
by
standa
rd
proces
ses.

0.1 mg/mL
polysorbate
20, and
5 mM
histidine
at 6.0
pH) and
(b) active
protein
(25
mg/mL
rhuMAb of
Figure 4. SEM micrographs
freeze
HER2, 20
mg/mL
dri
trehalose
ed
, 0.1
ma
mg/mL
teri
polysor
al
bate 20,
an
and 5
d
mM
the
histidine
effe
at 6.0
ct
pH)
of
(photo
for
courtes
mu
y of
lati
Genentec
on
h, Inc.)
on
mic
ros
tru
ctu
re:
(a)
pla
ceb
o
(4
5
mg
Correspond
/m
ing author
L
Thomas W.
tre
Patapoff is
hal
a senior
ose
scientist and
director, and
,

Ice might be
induced to
nucleate at
higher
temperatures
(closer
to 0 C) by
conditioning
the bottom
surface of the
David E.
Overcashier
is a senior
process
engineer in
Pharmaceutic
al R&D at
Genentech
, Inc., One
DNA Way,
South San
Francisco,
CA 94080,
650.225.80
06, fax
650.225.72
34,
tomp@gen
e.com,
www.gene.c
om.

Figure 5.
Cumulati
ve mass
transfer
resistance
as
a
function
of dried
layer
thickness
for active
and
placebo
material
perature
for
primary
drying,
which
then
matches
that of a
standardfrozen
material
dried at a
colder
shelf
temperatur
e.
That
higher
shelf
temperatur
e
adds
driving
force to
the heat
transfer,
speeding
primary
drying.
For
equivalent
product
temperatures, the
rate
of
sublimatio
n
was
40%
faster for
vertically
oriented
ice than
for

standardfrozen
solutions
(Table
1).
That leads to
a decrease in
the
time
required for
primary
drying

which constitutes half of

the
time
spent in the
lyophilization
process.
Such
a
decrease
should also
be expected
from
annealed
vials,
so
the
duration of
the
lyophilization
cycle should
be reduced as
well.

OTHER
CONSE
QUENC
ES OF
FREEZI
NG
The freezing
process can
have
unexpected
consequences on
product
quality. For
example,
some forms
of sodium
phosphate
can
crystallize
upon slow
freezing or
annealing,
resulting in
a pH

decrease in
the freezeconcentrate
(2123). A decrease in pH
has been
shown to affect
the stability of
a drug when
frozen and
after
lyophilization
(2426).
Other
excipients that
crystallize
during
freezing (for
example,
mannitol) can
lose their ability to stabilize
proteins in the
dried state (27
30). Some
proteins
undergo cold
denaturation
during slow
freezing or
annealing,
which can have
delete- rious
effects on
product quality
upon
reconstitution.
Freezing
methods can
alter the
voidsolid
interfacial
area of a
lyophilized
cake
and,
inversely, the
thickness of
its
channel
walls.
An
increased
surface area
correlates with
decreased
dried product
stability for
some proteins
(31). Freezing

by
immer
sing a
vial
into
liquid
nitroge
n can
result
in
increas
ed
protein
aggreg
ation
(32) or
decrea
sed
enzym
e
activit
y (33)
when
compa
red
with
freeze
rcooled
sample
s.
Prelimi
nary
studies
indica
te that
the
freezin
g
metho
d has a
signifi
cant
effect
on
dried
protein
stabilit
y
(Table
2). In
addi-

glass
vial.
Nucle
ation
would
then
occur
on the
botto
m
surfac
e, and
the
ice
would
propag
ate in
a
vertic
al
directi
on
resulti
ng in
lower
MTRs.
Vials
with
that
chara
cteris
tic
are
not
yet
availa
ble.
Anne
aling
can
be
more
practi
cal
but
also
has
limita
tions.
Wheth
er
anneal
ing
influe
nces
the
time

for
lyophili
zation
or the
tempera
ture
variations of
the
product
during
freezing
needs
to be
demons
trated.
Short
ening
the
cycle.

The
freezing
method
used
during
lyophili
zation
can
have a
significa
nt effect
on the
structur
e of the
ice
formed
,
affectin
g both
the
watervapor
flow
during
primary
drying
and the
final
product
.
Freezin
g
protein
solution
s
in
vials
with

verticall
y
propagat
ed
crystals
may
prevent
a thick
skin
from
forming
on the
top
of
the
lyophili
zed cake.
The
structure
of
vertically
frozen
material
facilitate
s
fast
watervapor
flow
during
primary
drying,
resulting
in lower
product
temperat
ures. By
increasin
g
the
primary
drying
shelf
temperat
ure, the
rate of
sublimati
on was
40%
faster in
our
studies
for
the
vertically
frozen
material
than for
the
standard
-frozen

material
with
an
equivalent
product
temperature.
We
also
observed
increased
sublimatio
n
in
material
annealed
during the
freezing
segment.
Decreased
surface area
correlates
with
increased
product stability
for
some
proteins.
Understandin
g and controlling how
a
solution
freezes can
lead
to
shorter
lyophilizati
on cycles
and,
in
some cases,
more stable
products.

A
C
K
N
O
W
L
E
D
G
M
E
N
T
S

The
author
s are
indebt
ed to
Jami
e
Moor
e,
Mary
Cromwell,
Anne
Nguyen, Al
Stern, and
Chung Hsu
for technical
assistance
and
consultation.

R
E
F
E
R
E
N
C
E
S

o
n
s
b
y
a
M
i
c
r
o
b
a
l
a
n
c
e
T
e
c
h
n
i
q
u
e
,

J
.

(1) S.L. Nail,

The Effect of
Chamber
Pressure on
Heat
Transfer in
the Freeze
Drying of
Parenteral
Solutions,
PDA J.
Pharm. Sci.
Technol. 34,
358368
(1980).
(2) M.J.
Pikal
et al.,
Phys
ical
Chemi
stry of
Freeze
Dryin
g:
Measu
rement
of
Subli
mation
Rates
for
Froze
n
Aque
ous
Soluti

P
h
a
r
m
.
S
c
i
.
7
2
,
6
3
5

6
5
0
(
1
9
8
3
)
.

(3) M.J. Pikal, M.L. Roy, and S. Shah, Mass

and Heat Transfer in Vial Freeze-Drying of
Pharmaceuticals: Role of the Vial, J.
Pharm. Sci. 73, 12241237 (1984).
(4) M.J. Pikal, Use of Laboratory Data in
Freeze Drying Process Design: Heat and
Mass Transfer Coefficients and the
Computer Simulation of Freeze Drying,
PDA J. Parenteral Sci. Technol. 39, 115
139 (1985).
(5) R.G. Livesey and T.W.G. Rowe, A
Discussion of the Effect of Chamber
Pressure on Heat and Mass Transfer in
Freeze-Drying, J. Parenteral Sci. Technol.
41, 169171 (1987).
(6) M.J. Pikal and S. Shah, The Collapse
Temperature in Freeze Drying: Dependence
on Measurement Methodology and Rate of
Water Removal from the Glassy Phase,
Int. J. Pharm. 62, 165186 (1990).
(7) M.J. Pikal, Freeze-Drying of Proteins, Part
1: Process Design, BioPharm 3(8), 1827
(1990).
(8) B.S. Chang and N.L. Fischer,
Development of an Efficient Single-Step
Freeze-Drying Cycle for Protein
Formulations, Pharm. Res.
12, 831837 (1995).
(9) D.E. Overcashier, T.W. Patapoff, and C.C. Hsu,
Lyophilization of Protein Formulations in
Vials: Investigation of the Relationship
Between Resistance to Vapor Flow During
Primary Drying and Small-Scale Product
Collapse, J. Pharm. Sci., 88, 688695
(1999).
(10) M. Kochs et al., The Influence of the
Freezing Process on Vapour Transport
During Sublimation in Vacuum-FreezeDrying of Macroscopic Samples, Int. J.
Heat Mass Transfer, 36, 17271738
(1993).
(11) J.A. Searles, J.F. Carpenter, and T.W.
Randolph, The Ice Nucleation
Temperature

Determines the Primary Drying Rate

of Lyophilization for Samples Frozen
on
a
Temperature-Controlled
Shelf,
J. Pharm. Sci., 90(7), 860871
(2001). (12) J.A. Searles, J.F. Carpenter,
and T.W.
Randolph, Annealing to Optimize the
Primary Drying Rate, Reduce FreezingInduced Drying Rate Heterogeneity, and
Determine Tg in Pharmaceutical
Lyophilization, J. Pharm. Sci.
90, 872887 (2001).
(13) G. Jancso, J. Pupezin, W.A. Van Hook, The
Vapor Pressure of Ice Between +10-2 and
10+2, J. Phys. Chem. 74, 2984
2989 (1970).
(14) L. Gatlin and P.P. DeLuca, A Study of the
Phase Transitions in Frozen Antibiotic
Solutions by Differential Scanning
Calorimetry, PDA J. Pharm. Sci.
Technol.,
34, 398408 (1980).
(15) E. Tarelli et al., Additives to Biological
Substances III: The Moisture Content and
Moisture Uptake of Commonly Used
Carrier Agents Undergoing Processing
Conditions Similar to Those Used in the
Preparation of International Biological
Standards,
J. Biol. Stand., 15, 331340 (1987).
(16) P.J. Dawson and D.J. Hockley, Scanning
Electron Microscopy of Freeze-Dried
Preparations: Relationship of Morphology to
Freeze-Drying Parameters, International
Symposium on Biological Product FreezeDrying Parameters, Bethesda, MD.
Developments in Biological Standards, Vol.
74 (S. Karger AG, Basel, Switzerland, 1990),
pp.185192.
(17) N. Murase, P. Echlin, and F. Franks, The
Structural States of Freeze-Concentrated and

Info #16

Freeze-Dried Phosphates Studied by

Scanning Electron Microscopy and
Differential Scanning Calorimetry,
Cryobiology, 28,
364375 (1991).
(18) B.S. Chang and C.S. Randall, Use of
Subambient Thermal Analysis to
Optimize Protein Lyophilization,
Cryobiology, 29,
632656 (1992).
(19) R. Haikala et al., Polymorphic Changes
of Mannitol During Freeze-Drying: Effect
of Surface-Active Agents, PDA J.
Pharm. Sci. Technol., 51, 96101 (1997).
(20) N. Milton et al., Evaluation of Manometric
Temperature Measurement as a Method of
Monitoring Product Temperature During
Lyophilization, PDA J. Pharm. Sci.
Technol.,
51, 716 (1997).
(21) L. van den Berg and D. Rose, Effect of
Freezing on the pH and Composition of
Sodium and Potassium Phosphate
Solutions: The Reciprocal System KH2PO4Na2HPO4- H2O, Arch. Biochem. Biophys.,
81, 319329 (1959).
(22) N. Murase and F. Franks, Salt Precipitation
During the Freeze-Concentration of
Phosphate Buffer Solutions, Biophys.
Chem., 34,
293300 (1989).
(23) G. Gomez, M.J. Pikal, and N. RodriguezHornedo, Effect of Initial Buffer
Composition on pH Changes During Farfrom-Equilibrium Freezing of Sodium
Phosphate Buffer Solutions, Pharm. Res.,
18, 9097 (2001).
(24) T.J. Anchordoquy and J.F. Carpenter,
Polymers Protect Lactate
Dehydrogenase During Freeze-Drying by
Inhibiting

Continued on page 72

BioPharm

MARCH 2002

21

Validation
Importance of Freezing continued from page 21
Dissociation in the Frozen State, Arch.
Biochem. Biophys., 332, 231238
(1996).
(25) X.M. Lam et al., Replacing Succinate with
Glycolate Buffer Improves the Stability of
Lyophilized Interferon- , Int. J. Pharm.,
142,
8595 (1996).
(26) K.A. Pikal-Cleland et al., Protein
Denaturation During Freezing and Thawing
in Phosphate Buffer Systems: Monomeric
and Tetrameic -Galactosidase, Arch.
Biochem. Biophys., 384, 398406 (2000).
(27) J.F. Carpenter, S.J. Prestrelski, and T.
Arakawa, Separation of Freezing- and
Drying-Induced Denaturation of
Lyophilized Proteins Using Stress-Specific
Stabilization,
I: Enzyme Activity and Calorimetric
Studies, Arch. Biochem. Biophys., 303,
456464 (1993).
(28) K.-i. Izutsu, S. Yoshioka, and T. Terao,
Decreased Protein-Stabilizing Effects
of Cryoprotectants Due to
Crystallization, Pharm. Res., 10,
12321237 (1993).

72

BioPharm

MARCH 2002

(29) H.R. Costantino et al., Effect of Excipients

on the Stability and Structure of Lyophilized
Recombinant Human Growth Hormone,
J. Pharm. Sci., 87, 14121420 (1998).
(30) L. Kreilgaard et al., Effects of Additives on
the Stability of Humicola Lanuginosa Lipase
During Freeze-Drying and Storage in the
Dried Solid, J. Pharm. Sci., 88, 281290
(1999).
(31) C.C. Hsu et al., Surface Denaturation at
SolidVoid Interface: A Possible Pathway
by Which Opalescent Particulates Form
During the Storage of Lyophilized TissueType
Plasminogen Activator at High Temperatures,
Pharm. Res., 12, 6977 (1994).
(32) B.S. Chang, B.S. Kendrick, and J.F.
Carpenter, Surface-Induced Denaturation of
Proteins During Freezing and Its Inhibition
by Surfactants, J. Pharm. Sci., 85, 1325
1330 (1996).
(33) S. Jiang and S.L. Nail, Effect of Process
Conditions on Recovery of Protein Activity
After Freezing and Freeze-Drying, Eur. J.
Pharma. Biopharma., 45, 249257 (1998).
BP