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NOVEL MODEL FORMULATION FOR
VIRUS REMOVAL BY TANGENTIAL FLOW

ULTRAFILTRATION
By
Angayar Kumari Pavanasam
A thesis submitted in fulfilment of the requirement for the
degree of
Doctor of Philosophy
School of Chemical Engineering
The University of New South Wales
Sydney 2052 AUSTRALIA
March 2011
STATEMENT OF ORIGINALITY
‘I hereby declare that this submission is my own work and to the best of my knowledge
it contains no materials previously published or written by another person, or substantial
proportions of material which have been accepted for the award of any other degree or
diploma at UNSW or any other educational institution, except where due acknowledge-
ment is made in the thesis. Any contribution made to the research by others, with whom
I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also
declare that the intellectual content of this thesis is the product of my own work, except
to the extent that assistance from others in the project’s design and conception or in style,
presentation and linguistic expression is acknowledged.’
COPYRIGHT STATEMENT
‘I hereby grant the University of New South Wales or its agents the right to archive and to
make available my thesis or dissertation in whole or part in the University libraries in all
forms of media, now or here after known, subject to the provisions of the Copyright Act
1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in
future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in
Dissertation Abstract International. I have either used no substantial portions of copyright
material in my thesis or I have obtained permission to use copyright material; where per-
mission has not been granted I have applied/will apply for a partial restriction of the digital
copy of my thesis or dissertation’.

AUTHENTICITY STATEMENT
‘I certify that the Library deposit digital copy is a direct equivalent of the final officially
approved version of my thesis. No emendation of content has occurred and if there are any
minor variations in formatting, they are the result of the conversion to digital format.’
Signed: .......................... Date: .........

Dedicated to
My teachers &
Mom - Gnanam, Dad - Pavanasam, Sister - Selvi, & Brother - Prabhu

ACKNOWLEDGEMENTS
The journey of Ph.D. is a constant discovery and transformation of one’s untapped potential
and ability in the chosen field. There are many instrumental persons who made my journey
a memorable one. I am extremely glad to thank my supervisor Professor Vicki Chen for
the total freedom she bestowed on me and for her timely guidance. Her support at the
critical time helped me a lot to push myself to complete on time.
The continued discussions with Dr. Ali Abbas, my supervisor at the University of Sydney,
about population balance theory was thought provoking which got deciphered into this
thesis. His constant motivation, ever-encouraging words, and access to gPROMS made it
possible for me to take every step ahead. He helped me immensely in analyzing, inter-
preting and presenting the results precisely. My visits to University of Sydney was always
like visiting my home. This is not just for the sake of acknowledging but it is a heartiest
thankyou to Dr. Ali. He remained a constant source of inspiration since the day one of this
journey.
I would like to thank A/Professor Greg Leslie, Dr. Pierre LeClech, and Professor Rose
Amal for being a part of my review committee and for their valuable comments which
helped in shaping up the thesis. I would like to thank the UNESCO Center for Membrane
Science and Technology and the School of Chemical Engineering for giving me the great
opportunity to learn and prosper.
Thanks Eisham for the cross flow rig without which I would have struggled to carry out
the experiments. Maylim, thankyou so much for the training on particle size measurement.
I would like to thank Dorothy and Rebaya for helping me in ICP analysis and Katie for
ii
the imaging analysis. Thanks to all my UNESCO membrane mates. Very special thanks to
Dr. Alice Antony for the lunch time discussions, lunches and constant calls - I owe a lot to
her especially for the initial settling-in recipes. I would like to thank all the technical and
administrative staff of the Chemical Engineering School in general and Deyan in particular
for facilitating my access to laboratory equipments and the chemicals, and Ling for her
timely reminders regarding the reviews and courses.
The time and support offered by Sashi during the illness time, I cannot thank her enough
for that. The regular reminders about my deadline always came from my friends. Thanks
to Chandra and Simit for their kind and moral support and Kiet for the computer support.
Ofcourse, my special thanks to Raghav and Jose from Univ. of Sydney for their thoughtful
help throughout - it meant a lot. I am running short of words to express my sincere gratitude
to my friends - distance never mattered - Prof. Raman (India), Prof. Raghavan (India), Anu
(US) and Suganthi (US), Dr. Sundar (US), Dr. Bala (Germany), Dr. Kumaran (India), and
Balaji (Dubai) for their words of confidence and motivation. Balaji (Singapore), eternal
thanks for those (silent) words of wisdom and memories.
It is a heavenly bliss to have two wonderful and dedicated souls as my parents. My mother
known for her intelligence and my father a superb human being and for instilling the sense
of discipline and conviction, I am blessed to be their daughter. My sister Selvi and brother
Prabhu are my constant source of inspiration and lot more. Thanks for being very patient
with all my tantrums.
My namaskarams with thanks to the almighty for showering only good things on me!.
ANGAYAR KUMARI PAVANASAM
August 2010
ABSTRACT
Products of human and animal origin (biologicals and pharmaceuticals) and drinking water
pose unique health and safety problems, especially due to viral contaminations, and have
traditionally been a key concern both for regulators and industrial operations. They have
also proven to be a stumbling block for early product research and development and inex-
perienced small start-up firms. Membrane filtration, being non-invasive, non-destructive
and a robust technique, is one of the most preferred choices for virus filtration with ul-
trafiltration being an efficient process for removing macromolecules, colloids, endotoxins,
viruses, and bacteria.
The challenge, in virus filtration, is to remove virus particles from products to close to
zero levels especially in emerging water and heavily regulated multi-billion biopharmaceu-
tical industries. In virus capture or clearance operations, the virus particles are distributed
in the range of 15nm - 300nm depending upon their protein coat and family, hence it is crit-
ical to study impact of particle size in ultrafiltration. A number of previous studies have
been concerned with particle characteristics and their relation to flux decline behavior.
However the relationship between, on one hand, particle properties of the feed and operat-
ing parameters like transmembrane pressure and cross flowrate and on the other hand, the
filtration efficiency (log reduction value), are still not adequately established.
This thesis investigates the effect of feed particle size, transmembrane pressure and
cross flowrate on the filtration efficiency for tangential flow ultrafiltration. An empirical
model is developed to quantify the dependencies of the above said parameters. For this
study, a cross flow ultrafiltration rig (built in-house) is used with 30 and 100 kDa polyether
iv
sulphone membranes for three different particle sizes of silica (model virus particles) at
three different transmembrane pressures (20-60 kPa) and three cross flowrates (0.3-1.0
L/min). The investigation shows that among feed particle size, transmembrane pressure
and cross flowrate, feed particle size and transmembrane pressure are significant param-
eters in controlling the filtration efficiency. In the studied experimental range, higher log
reduction values are obtained at lower transmembrane pressure (20 kPa) and at higher
cross flowrate (1 L/min). The experimental statistical analysis also gives an insight on the
cross interactions of the feed particle size, transmembrane pressure and cross flowrate. It
shows that the effect of transmembrane pressure and feed particle size seems to be more
significant than that of the interaction of cross flowrate with feed particle size.
The experimental investigations identified that feed particle size is one of the key in-
fluential factors on the filtration efficiency. Far less attention is given to this effect in the
literature both experimentally and theoretically. This thesis attempts to fill this gap by
developing a rigorous model formulation for tangential flow ultrafiltration to predict the
evolution of particle size in the filtered stream. Population balance theory is employed to
describe the population density of each particle size class in the output streams. In this
formulation, the population balance equation is coupled with filtration kinetic constitutive
relations and with mass balance equations and solved using discretisation method. The
model predicts permeate particle size distribution and the log reduction value as primary
model outputs. A novel approach in the form of optimization-based parameter estimation
is employed to estimate filtration kinetic parameters. This approach which directly uses
the population balance model, as well as particle size data, represents a stark deviation
from previous filtration kinetic calculations that only utilize flux data and no models that
v
describe the particle state. As such, this approach offers more comprehensive description
of filtration kinetics incorporating the effects of particle size. The model is validated and is
found to be in good agreement with experimental results. The model serves as a predictive
tool for filtration efficiency and filtrate particle size distribution. Overall, this model-based
prediction and estimation capability serves well and facilitates the design, operation and
scale-up of ultrafiltration processes.
Keywords: Ultrafiltration, population balance, virus filtration

AUTHOR’S PUBLICATIONS
Publications based on this work
Refereed Journal Articles

(i) Angayar K Pavanasam, Ali Abbas, "Ultrafiltration and Virus Removal: A Mini Re-
view of Recent Patents", Recent Patents on Chemical Engineering, 151-156, 1(2),
2008.
(ii) Angayar K Pavanasam, Ali Abbas, Vicki Chen, "Influence of Particle Size and Oper-
ating Parameters on Virus Ultrafiltration Efficiency", Water Science and Technology:
Water Supply, 31-38, 11(1), 2011.
(iii) Angayar K Pavanasam, Ali Abbas, Vicki Chen, "An Empirical Modeling of Particle
Size and Polydispersity of Model Virus Filtration", Biotechnology and Bioprocess
Engineering (Submitted).
(iv) Angayar K Pavanasam, Ali Abbas, "Predictive Model Formulation and Simulation of
Tangential Flow Ultrafiltration: A Novel Approach", Chemical Engineering Science
(Under review for submission).
Conference Articles
(v) Angayar K Pavanasam, Ali Abbas, Santosh Ansumali, Vicki Chen, "A Population
Balance Model for Ultrafiltration Processes", Engineering With Membranes (EWM
2008), Portugal, May 2008.

vii
(vi) Angayar K Pavanasam, Ali Abbas, Santosh Ansumali, Vicki Chen, "Modeling virus
ultrafiltration processes: A population balance approach, International Congress on
Membranes and Membrane Processes (ICOM 2008),Hawaii, July 2008.
(vii) Angayar K Pavanasam, Ali Abbas, Vicki Chen, "Influence of particle size and oper-
ating parameters on virus ultrafiltration efficiency, International Water Association -
Membrane Technology Conference (IWA-MTC 2009), Beijing, September 2009.
(viii) Angayar K Pavanasam, Ali Abbas, Vicki Chen, "Modeling Particle Size in Ultrafil-
tration, EuroMembrane (EM 2009), France, September 2009.
(ix) Angayar K Pavanasam, Ali Abbas, Vicki Chen, "Particle Size in Membrane Filtra-
tion: Novel Strategy for Better Insights", Membrane Society of Australasia - Stu-
dents Conference, Wollongong, Australia, February 2010.
Award Articles
(x) Angayar Kumari Pavanasam, Ali Abbas, Vicki Chen, "Novel Model Formulation for
Virus Removal by Tangential Flow Ultrafiltration", Dean’s Award for Excellence in
Postgraduate Research, University of New South Wales, Sydney, November 2008.
(xi) Angayar Kumari Pavanasam, Ali Abbas, Vicki Chen, "Population Balance Modeling
Approach: Better Insights into Virus Removal Mechanism", Dean’s Award for Ex-
cellence in Postgraduate Research, University of New South Wales, Sydney, October
2009.
GLOSSARY
AeDNV Aedes aegypti DensoNucleosisVirus
AF4 Asymmetric Flow Field Flow Fractionation
ALV Avian Leukemia Virus
ATCC American Type Culture Collection
BHK Born Hamster Kidney cells
BVDV Bovine Viral Diarrhea Virus
B19 Human Parvovirus
CFR Cross Flow Rate
CHO Chinese Hamser Ovary
CJD CreutzfeldJacob disease
CMR Comprehensive Microbial Resource
CPV Canine Parvo Virus
DEF/NFF Dead End Filtration/Normal Flow Filtration
DLS/MALS Dynamic Light Scattering/MultiAngle Light Scattering
EBV Epstein-Barr Virus (also known human herpesvirus 4)
EMC Encephalo MyoCarditis
FCS Fetal Calf Serum
FDA Food and Drug Administration
FFF Field Flow Fractionation
FPS Feed Particle Size

ix
GMP Good Manufacturing Practices
HAV Hepatitis A Virus
HBV Hepatitis B Virus
HCV Hepatitis C Virus
HIV Human Immunideficiency Virus
HPTFF High Performance Tangential Flow Filtration
HSA Human Serum Albumin
ICH International Conference on Harmonization
IgG Immunoglobulin G
LRV/LTR Log Reduction Value/Log Titre Reduction
MAB Monoclonal AntiBody
MLV Murine Lukemia Virus
MVM Minute Virus of Mice
MWCO Molecular Weight Cut Off
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PPV Porcine Parvo Virus
PSD Particle Size Distribution
PSR PSeudo Rabies Virus
PVDF Poly Vinylidene Fluoride
RTP Recombinant Therapeutic Protein
SV40 Simian Virus 40
TFF/CFF Tangential Flow Filtration/Cross Flow Filtration

x
TMP Transmembrane Pressure
UF Ultrafiltration
USP United States Pharmacopeia
VSV Vesicular Stomatis Virus
Symbols
A - Membrane area, m2
B, G - Nucleation and growth rates, #/m3 min
C f - Feed concentration, weight of solute per weight of the suspension, w/w%
C p - Permeate concentration - weight of solute per weight of the suspension, w/w%
Cr - Retentate concentration, weight of solute per weight of the suspension, w/w%
D(t) - Permeate particle size, m
F - Filtration kinetics
F f - Feed flow rate, L/min
F p - Permeate flow rate, L/min
Fr - Retentate flow rate, L/min
k1 , k2 , k3 - Kinetic parameters
L - Membrane thickness, m
La - Average particle size, nm
L pa - Particle size, nm
L po - Average pore size, nm
N - Number of measurements taken during experiment
NE - Number of experiments
NMi j - Number of measurements of the jth variable in the ith experiment

xi
NVi - Number of variables measured in the ith experiment
n p - Particle number density in permeate stream, #/m3
nr - Particle number density in retentate stream, #/m3
n f - Particle number density in feed stream, #/m3
P pa - Probability of particle permeation
t - Time, min
U - Manipulated variables
V - System volume, m3
σ - Standard deviation
σ2i jk - Variance of kth measurement of variable j in experiment i
ω2i jk - Standard deviation of the measurement error
θ - Vector of parameters
φ - Objective function
Contents
ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
AUTHOR’S PUBLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . vi
GLOSSARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
1 Introduction 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Literature Review 7
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Virus Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.1 In the context of biopharmaceutical industry . . . . . . . . . . . . 12
2.2.2 In the context of water industry . . . . . . . . . . . . . . . . . . 14
2.3 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4 Regulatory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.4.1 Regulations governing biopharmaceutical industry . . . . . . . . 16
2.4.2 Regulations governing water industry . . . . . . . . . . . . . . . 20
2.5 Virus Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.5.1 Membrane modes and configurations . . . . . . . . . . . . . . . 23
2.5.1.1 Normal flow filtration . . . . . . . . . . . . . . . . . . 23
2.5.1.2 Tangential flow filtration . . . . . . . . . . . . . . . . . 26

Contents xiii
2.5.1.3 High performance tangential flow filtration . . . . . . . 28
2.5.1.4 Field flow fractionation . . . . . . . . . . . . . . . . . 29
2.5.1.5 Remarks on membrane modes and configurations . . . 31
2.5.2 Membrane modules . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.5.2.1 Flat sheet . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.5.2.2 Spiral wound . . . . . . . . . . . . . . . . . . . . . . . 34
2.5.2.3 Hollow fibre . . . . . . . . . . . . . . . . . . . . . . . 34
2.5.3 Particle characterization . . . . . . . . . . . . . . . . . . . . . . 36
2.5.3.1 Dynamic light scattering . . . . . . . . . . . . . . . . . 36
2.5.3.2 Imaging and image analysis . . . . . . . . . . . . . . . 41
2.5.3.3 Asymmetrical flow field flow fractionation and multi-
angle light scattering . . . . . . . . . . . . . . . . . . . 42
2.5.3.4 Electro-spray differential mobility analysis . . . . . . . 42
2.6 Economics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.7 Patent Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3 Experimental Section 68
3.1 Membrane Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2 Characterization Techniques . . . . . . . . . . . . . . . . . . . . . . . . 72
3.2.1 Particle concentration measurement by inductively coupled
plasma technique . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.2.2 Particle morphology studies by transmission electron microscopy 74

Contents xiv
3.2.3 Particle size and distribution studies by dynamic light scattering
technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.3 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4 Influence of Particle Size and Size Distribution on Filtration Efficiency and
Separation Mechanism 82
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.3 Cross-Flow Filtration Experimental Details . . . . . . . . . . . . . . . . 89
4.4 Results and Discussion: Filtration Efficiency Study . . . . . . . . . . . . 89
4.4.1 Experimental design and surface response modeling . . . . . . . 90
4.4.2 Pearson’s correlation analysis . . . . . . . . . . . . . . . . . . . 92
4.4.3 Model prediction results . . . . . . . . . . . . . . . . . . . . . . 95
4.4.4 Effect of process parameters and input condition on filtration effi-
ciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.4.4.1 Effect of TMP on filtration efficiency . . . . . . . . . . 98
4.4.4.2 Effect of CFR on filtration efficiency . . . . . . . . . . 99
4.4.4.3 Effect of particle and pore size on filtration efficiency . 101
4.4.5 Concluding remarks for filtration efficiency study . . . . . . . . . 105
4.5 Results and Discussion - Influence of Particle Size and Size Distribution
on Filtration Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.5.1 Study of particle size characterization based on particle concentration109
4.5.2 Experimental design and statistical analysis . . . . . . . . . . . . 112

Contents xv
4.5.3 Model prediction results . . . . . . . . . . . . . . . . . . . . . . 116
4.5.3.1 Effect of TMP on permeate particle size and polydisper-
sity index . . . . . . . . . . . . . . . . . . . . . . . . 116
4.5.3.2 Effect of CFR on permeate particle size and polydisper-
sity index . . . . . . . . . . . . . . . . . . . . . . . . 117
4.5.4 Particle size distribution (PSD) analysis . . . . . . . . . . . . . . 119
4.5.4.1 Temporal effect . . . . . . . . . . . . . . . . . . . . . 119
4.5.4.2 Effect of TMP on particle size distribution in permeate
streams . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.5.4.3 Effect of CFR on particle size distribution in permeate
streams . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.5.4.4 Modeling particle size distribution . . . . . . . . . . . 125
4.5.4.5 Size polydispersity index and its effects . . . . . . . . . 126
4.5.5 Concluding remarks for particle size and distribution investigation
section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
5 Modeling and Simulation 137
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
5.2 Earlier Modeling patterns . . . . . . . . . . . . . . . . . . . . . . . . . . 142
5.2.1 Macro-scale modeling: A quick review . . . . . . . . . . . . . . 143
5.2.1.1 Flux decline prediction models . . . . . . . . . . . . . 145
5.2.2 Particle property models . . . . . . . . . . . . . . . . . . . . . . 147
5.3 Novel Model Formulation: Population Balance Approach . . . . . . . . . 150

Contents xvi
5.3.1 Statement of the problem . . . . . . . . . . . . . . . . . . . . . . 151
5.3.2 Modeling of size distribution of suspended particles through a
cross-flow UF process . . . . . . . . . . . . . . . . . . . . . . . 153
5.3.2.1 Input and output streams . . . . . . . . . . . . . . . . 154
5.3.2.2 Constitutive equations . . . . . . . . . . . . . . . . . . 155
5.3.2.3 Permeation probability . . . . . . . . . . . . . . . . . 156
5.3.2.4 Expression of solid permeate flow . . . . . . . . . . . . 157
5.3.2.5 Balance equations . . . . . . . . . . . . . . . . . . . . 157
5.4 Model Solution and Simulation Results . . . . . . . . . . . . . . . . . . 160
5.4.1 Discretisation of the model . . . . . . . . . . . . . . . . . . . . . 161
5.4.1.1 Discretisation issues . . . . . . . . . . . . . . . . . . . 161
5.4.1.2 The internal coordinate and its significance . . . . . . . 163
5.4.1.3 The method of moments . . . . . . . . . . . . . . . . . 166
5.4.1.3.1 Different mean sizes . . . . . . . . . . . . . . 169
5.4.1.4 Number and volume distributions . . . . . . . . . . . . 171
5.4.1.5 Useful quantity predicted from the above model . . . . 171
5.4.2 Simulation using gPROMS . . . . . . . . . . . . . . . . . . . . . 172
5.4.2.1 Model entities of the entire process . . . . . . . . . . . 173
5.4.2.2 Parameters and variables employed in the model devel-
opment . . . . . . . . . . . . . . . . . . . . . . . . . . 174
5.4.3 Simulation results . . . . . . . . . . . . . . . . . . . . . . . . . 174
5.4.3.1 Representative particle size distribution in output streams 174
5.4.3.2 Effect of feed particle size on particle size distribution . 177

Contents xvii
5.4.3.3 Effect of transmembrane pressure drop on the particle
size distribution . . . . . . . . . . . . . . . . . . . . . 178
5.4.3.4 Effect of cross flow rate on particle size distribution . . 178
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
6 Optimization-based Parameter Estimation 191
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
6.2 Overview of Parameter Estimation . . . . . . . . . . . . . . . . . . . . . 196
6.3 Filtration Kinetic Parameter Estimation . . . . . . . . . . . . . . . . . . 197
6.4 Estimation Results and Discussion . . . . . . . . . . . . . . . . . . . . . 200
6.4.1 Parameter estimation summary . . . . . . . . . . . . . . . . . . . 200
6.4.2 Overlay plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.5 Model Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.5.1 Validation against literature data . . . . . . . . . . . . . . . . . . 208
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
7 Conclusions and Future Directions 214
7.2 Recommendations for Future Directions . . . . . . . . . . . . . . . . . . 218
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
List of Figures
1.1 A typical bioprocess flowchart with indicative operations . . . . . . . . . 2
2.1 Different types of virus particles giving a snapshot of size and shape of
viruses (Pic. courtesy: antibac2k.com, iah.bbsrc.ac.uk.com, innovations-
report.de). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2 Virus Clearance is a critical step in biotechnolgical/biopharmaceutical
manufacturing. It ensures removal of viruses found to have contaminated
the bioprocess. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3 Schematic representation of dead end flow (NFF), cross flow (TFF) and
flow field fractionation (FFF) modes. . . . . . . . . . . . . . . . . . . . . 24
2.4 TFF modules for small and large scale applications. (Courtesy: Millipore
Corporation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.5 Particle size distribution of high molecular weight, low molecular weight,
and virus like particle (MIX) solutions by DLS [Lipin et al., 2008]. . . . . 38
2.6 Particle size distribution in the retentate (right) and permeate (left) streams
through 0.1 μ membrane during the study of HAV by TFF system. . . . . 39
2.7 Particle size distribution in the retentate (right) and permeate (left) streams
through 0.2 μ membrane during the study of HAV by TFF system. . . . . 40
2.8 Particle size distribution of diethylaminoethyl (DEAE) and packaged virus
like particles by dynamic light scattering technique. . . . . . . . . . . . . 40

List of Figures xix
2.9 Membrane filtration’s place in the Pharmaceutical/Biotech industry -
World Market Share . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.1 Tangential flow UF experimental setup employed for this study . . . . . . 69
3.2 An outline of various methods employed for the analysis of silica samples. 72
3.3 TEM Micrograph of small silica particles at x200K magnification. . . . . 74
3.4 TEM Micrograph of medium silica particles at x100K magnification. . . . 75
3.5 TEM Micrograph of big silica particles at x200K magnification. . . . . . 75
3.6 Schematic diagram depicting the main components in a typical dynamic
light scattering setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.1 Schematic diagram of the tangential flow filtration experimental setup. . . 88
4.2 Cross flow filtration: Sample flowing parallel to the membrane surface. . . 89
4.3 Log reduction values: predicted vs experimental. . . . . . . . . . . . . . 94
4.4 Rankit Plot showing the residuals are normally distributed. . . . . . . . . 94
4.5 Model prediction showing the effect of cross flowrate at 20 kPa on LRV. . 95
4.8 Model prediction vs experimental trend for varying TMP at 0.6 L/min. . . 97
4.9 Experimental results showing the effect of TMP at 0.3 L/min on LRV (lines
drawn to indicate trends). . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.10 Experimental results showing the effect of TMP at 0.6 L/min on LRV (lines

List of Figures xx
4.11 Experimental results showing the effect of TMP at 1 L/min on LRV (lines
4.12 Model prediction showing the effect of TMP at 0.6 L/min on LRV. . . . . 101
4.13 Experimental results showing the effect of feedflow rate at 40 kPa on LRV
(lines drawn to indicate trends). . . . . . . . . . . . . . . . . . . . . . . . 102
4.14 A comparison of experimental results (points joined by dotted lines to in-
dicate the trend) with our model and Ferry-Faxen model. . . . . . . . . . 103
4.15 Efficiencies (LRV) for the two MWCO membranes as a function of the
PPSR. Data for lower TMP (20 kPa) and higher flowrate (1 L/min). . . . 105
4.16 Two different representation of particle size distribution of the original
feed sample: multimodal size distribution (top) and log normal distribu-
tion (bottom). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.17 Illustration of the correlation function - Exponential decay of auto-
correlation function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
4.18 Counts per second versus concentration plot for three different particles.
This shows the range of suitable concentration below 800 kcps threshold
as specified by the BIC particle sizer. . . . . . . . . . . . . . . . . . . . . 111
4.19 Observed vs Predicted: Permeate Particle Size. . . . . . . . . . . . . . . 115
4.20 Observed vs Predicted: Permeate PDI. . . . . . . . . . . . . . . . . . . . 116
4.21 Experimental (points joined by dashed lines to indicate the trend) Vs
Model Prediction: Effect of TMP at the middle CFR (0.6 L/min) on the
permeate particle size. . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

List of Figures xxi
Model Prediction: Effect of TMP at the middle CFR (0.6 L/min) on the
permeate PDI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Model Prediction: Effect of CFR at the middle TMP (40 kPa) on the per-
meate particle size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Model Prediction: Effect of CFR at the middle TMP (40 kPa) on the per-
meate PDI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.25 Representative distribution of feed stream sample of all three particles. . . 120
4.26 An illustration of the particle size distribution evolution in the permeate
stream at TMP=20 kPa. . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.27 An illustration of the particle size distribution evolution in the permeate
stream at TMP=60 kPa. . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.28 Particle size distribution of the permeate stream at various TMPs (20-60
kPa), time=9 mins, CFR=0.6 L/min. . . . . . . . . . . . . . . . . . . . . 122
4.29 Particle size distribution of the retentate stream at various TMPs (20-60
kPa), time=9 mins, CFR=0.6 L/min. . . . . . . . . . . . . . . . . . . . . 123
4.30 Particle size distribution of the permeate stream at various CFRs (0.3-1
L/min), time=9 mins, TMP=40 kPa. . . . . . . . . . . . . . . . . . . . . 124
4.31 Particle size distribution of the retentate stream at various CFRs (0.3-1
L/min), time=9 mins, TMP=40 kPa. . . . . . . . . . . . . . . . . . . . . 124

List of Figures xxii
4.32 Representative particle size distribution of observed (points joined by
dotted lines to show the trend) and predicted plots for smaller perme-
ate particles at (a)-(c) at TMP=20,40,60 kPa and 0.3 L/min (d)-(f)at
TMP=20,40,60 kPa and 0.6 L/min (g)-(i)at TMP=20,40,60 kPa and 1 L/min.127
dotted lines to show the trend) and predicted plots for medium perme-
dotted lines to show the trend) and predicted plots for bigger perme-
4.35 Effect of feed and permeate PDIs on filtration efficiency at middle TMP
(40 kPa) and CFR (0.6 L/min). . . . . . . . . . . . . . . . . . . . . . . . 130
5.1 Illustrations of (a) dead-end filtration and (b) Tangential flow filtration. . . 142
5.2 A schematic diagram of the tangential flow filtration: Particles sieving
through the membrane (permeate) and those being excluded (retentate). . 152
5.3 A schematic diagram of the membrane filtration and its relationship with
the proposed PBE model. . . . . . . . . . . . . . . . . . . . . . . . . . . 152
5.4 An overview of the model encapsulating the set of balances involved in the
model formulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

List of Figures xxiii
5.5 Plot showing the behavior of the probability of particles permeating
through the membrane: Smaller the particles higher the probability and
vice-versa. Also the plot shows that the particles approaching membrane
pore reflects zero probability of particle permeation. . . . . . . . . . . . . 158
5.6 Solid permeate flow indicating the bounding condition of no particle flow
of bigger particles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
5.7 The discretisation of the density function and the relationship between size
class, and density. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
5.8 Plots indicating the significance of the geometric constant (a-c) and num-
ber of size classes (d-f) for the model discretisation. . . . . . . . . . . . . 167
5.9 Representative plot indicating the relation of particle size and the number
of size classes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
5.10 Representative particle size distribution in the permeate at TMP=90 kPa
and CFR=0.045 L/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5.11 Representative particle size distribution in the retentate at TMP=90 kPa
and CFR=0.045 L/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5.12 Particle size distribution of three range of feed particle sizes. . . . . . . . 177
5.13 Particle size distribution of permeate for three feed particle size ranges. . 177
5.14 Permeate particle size distribution with changing transmembrane pressure
at CFR=0.045 L/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
5.15 Retentate particle size distribution with changing transmembrane pressure
at CFR=0.045 L/min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

List of Figures xxiv
5.16 Permeate particle size distribution with changing crossflow rate at
TMP=90 kPa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
5.17 Retentate particle size distribution with changing crossflow rate at
TMP=90 kPa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1 Flowchart showing the various steps involved in parameter estimation:
pre-estimation and post-estimation checks in arriving at the accurate values. 195
6.2 Comparison of measured values and predicted values during the parameter
estimation step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
6.3 Comparison of measured values and predicted values during the parameter
estimation step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
6.4 Simulation results of permeate particle size distribution for bigger sized
particles (140nm avg.) at TMP=40 kPa and CFR=0.6 L/min. . . . . . . . 205
6.5 Simulation results of retentate particle size distribution for bigger sized
particles (140nm avg.) at TMP=40 kPa and CFR=0.6 L/min. . . . . . . . 205
6.6 Simulation results of permeate particle size distribution for medium sized
particles (80nm avg.) at TMP=40 kPa and CFR=0.6 L/min. . . . . . . . . 206
6.7 Simulation results of retentate particle size distribution medium sized par-
ticles (80nm avg.) at TMP=40 kPa and CFR=0.6 L/min. . . . . . . . . . 206
6.8 Model predictions vs. experimental results of filtration efficiency at
TMP=40 kPa and CFR=0.6 L/min. . . . . . . . . . . . . . . . . . . . . . 207
6.9 Model predictions vs. experimental results of permeate particle size for all
three particle size ranges at TMP=40 kPa and CFR=0.6 L/min. . . . . . . 208
List of Figures xxv
6.10 Model predictions vs. experimental results of permeate particle size distri-
bution at TMP=40 kPa and CFR=0.6 L/min of bigger sized particles. . . . 209
bution at TMP=40 kPa and CFR=0.6 L/min of medium sized particles. . . 209
bution at TMP=40 kPa and CFR=0.6 L/min of smaller sized particles. . . 210
List of Tables
2.1 Virus Characteristics: Type, family and size used in virus clearance study. 11
2.2 Reported incidents of virus contamination. . . . . . . . . . . . . . . . . . 13
2.3 Virus contamination and their cell lines in the production of biological
products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.4 Virus contamination and their sources resulting in a possible outbreak in
drinking water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.5 Timeline of global regulations for viral safety of licensed products. . . . . 19
2.6 Summary of various operating modes of FFF. . . . . . . . . . . . . . . . 31
2.7 Typical cross flowrates for various membrane configurations. . . . . . . . 31
2.8 Summary of commercially available membrane modules for virus removal. 35
2.9 Summary of various analytical techniques available for virus particle char-
acterization studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.10 A mini review of patents on the use of UF for the removal of virus particles. 47
3.1 Characteristics of Silica Colloid. . . . . . . . . . . . . . . . . . . . . . . 71
4.1 Full Factorial, 3 level experimental design for cross flow UF of silica par-
ticles using 100 kDa MWCO membrane (b : coded variables). . . . . . . . 91
4.2 Pearson’s correlation analysis. . . . . . . . . . . . . . . . . . . . . . . . 93
4.3 Analysis of variance summary. . . . . . . . . . . . . . . . . . . . . . . . 93

List of Tables xxvii
4.4 Efficiencies (LRV) for the two MWCO membranes against particles of
different size ranges. Data for lower TMP (20 kPa) and higher flowrate (1
L/min). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.5 Full Factorial, 3 level experimental design for cross flow UF of three par-
ticle size ranges of silica colloid. . . . . . . . . . . . . . . . . . . . . . . 113
4.6 Summary of Coefficients, Analysis of variance (ANOVA). . . . . . . . . 114
5.1 Summary of various models considering feed particle size as a factor to
study the permeate flux. . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
5.2 Summary of the partitioned sections available for the model entity. . . . . 175
5.3 Simplified summary of the partitioned sections available for the process
entity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5.4 Input data for the simulation of the model. . . . . . . . . . . . . . . . . . 175
5.5 Various input conditions utilised for the model simulations. . . . . . . . . 175
5.6 LRV at various TMPs and crossflow rates of smaller particles (80 nm avg.). 180
6.1 The types of parameter estimation problems. . . . . . . . . . . . . . . . . 194
6.2 Specification of the variance model in the gEST file. . . . . . . . . . . . . 200
6.3 Parameter estimation summary showing the number of experiments used
for estimating the parameters, the variables D(t) and C(t) measured, vari-
ance model employed and the objective function contribution. . . . . . . 201
6.4 Model parameter estimates. . . . . . . . . . . . . . . . . . . . . . . . . . 201
6.5 Correlation matrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.6 Lack of fit test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

List of Tables xxviii
6.7 Validation of the model with the Sano et al.’s [Sano et al., 2006] results. . 210
Chapter 1
Introduction
1.1 Introduction
Products of human and animal origin products (biologicals and pharmaceuticals) and
drinking water pose unique health and safety problems, especially due to viral contami-
nations, and have traditionally been a key concern both for regulators and industrial op-
erations. They have also proven to be a stumbling block for early product research and
development and inexperienced small start-up firms. In a biopharmaceutical industry, a
typical bioprocess consists of various unit operations including media preparation, cell
harvesting, followed by three or four stages of purification using a series of chromatogra-
phy columns and ultrafiltration or diafiltration steps. Those operations performed before
chromatographic operation are referred to as upstream while those post-chromatographic
are referred to as downstream processing (Fig. 1.1).
Membrane filtration, being non-invasive, non destructive and a robust technique, is
the most preferred choice for virus removal and which is used extensively throughout the
production, purification, and formulation of biotechnology products, including recombi-
nant proteins and nucleic acids [Brandwein and Aranha-Creado, 2000; Davis, 2008]. Over
the past few years, viral safe products have been manufactured by the development and
availability of biocompatible viral ultrafiltration systems. Ultrafiltration is an efficient pro-

1.1. Introduction 2
Figure 1.1: A typical bioprocess flowchart with indicative operations
cess for removing macromolecules, colloids, endotoxins, pyrogens, viruses, and bacteria
[van Reis and Zydney, 2007]. It uses membranes of pore size ranging up to 1000 kDa
MWCO and these systems are specifically designed to allow proteins and retain viruses,
and typically more than 4 logs of virus removal are achieved while ensuring good pro-
tein permeability and recovery [Arkhangelsky et al., 2008; Hirasaki et al., 2002; Manabe,
1996]. During filtration, viruses smaller than the pore size carried along with the fluid,
pass through the membrane pores and when the pore size is smaller than the virus parti-
cles, then the particles get retained. Size exclusion is considered the main mechanism of
clearance by filtration.
Viral retentive membrane filtration systems have been evolving tremendously owing to
their efficient removal of numerous bacterial and mammalian viruses, including human and
murine retroviruses, simian virus (SV40), various hepatitis viruses and parvoviruses such
as minute virus of mice and B19. New research offers promising data on retention of trans-
missible spongiform encephalopathy (TSE) agents known as prions. The total US market
1.1. Introduction 3
value for ultrafiltration is expected to reach US$908 million by 2011, following its steady
growth from US$579 million in 2005 and US$635 dollars million in 2006, according to
ultrafiltration membrane markets, MST044B from BCC Research [Hanft, 2010]. These
data suggest an even wider application of membrane filtration to enhance viral safety in
manufacturing biological and biopharmaceutical products, including the ability to scale
reliably and efficiently from the lab to production.
Quality by Design highlights the importance of identifying and controlling all the pa-
rameters that can influence final product performance: Specifically, International Confer-
ence on Harmonization (ICH) Topic Q6A identifies particle size as a potentially important
variable [ICH, December 2000]. The specific need for a particle size specification is deter-
mined by assessing if and how this parameter affects the performance in purification and
fractionation. In virus capture or clearance operations, the virus particles are distributed
in the range of 15nm - 300nm depending upon its protein coat and its family, hence the
particle size study in ultrafiltration becomes inevitable because of its distribution nature.
Filtration is most effective when operated as tangential flow (TFF) or otherwise known
as cross-flow mode and is being exploited in several virus removal and purification oper-
ations [Guo et al., 2009; Michalsky et al., 2009] as it is less expensive compared to other
techniques like chromatography and are easier to operate. But extensive experimentation is
required prior to design and implementation especially for biotechnological applications.
Although various efficient experimental techniques for flux prediction have been presented
and used, there still is a lack of theoretical studies particularly those aiming at predicting
filtration efficiency in terms of log reduction value. Thus there is strong incentive for new
approaches to predict final permeate quality of tangentially ultrafiltered products.

1.1. Introduction 4
While others have attempted modelling systems such as deep bed filtration [Santos and
Bedrikovetsky, 2004], this work, we believe, is a maiden attempt to illustrate the appli-
cation of the population balance framework to predict the final product efficiency in the
context of tangential flow ultrafiltration (UF). The main objective of this research is to de-
velop a novel model for the TFF based on the population balance theory. Such predictive
modelling will serve as a promising tool for optimizing and controlling the TFF process.
The innovation of this work lies in the above fact, whereas previous ultrafiltration models
have been dominated by flux modeling techniques, i.e. at the macroscale.
The thesis is organized as follows: first, a literature survey covering various sources of
virus contamination and different regulatory requirements is presented. This is followed by
an overview of the UF process and its modes of operation. Particle characterization, a quick
economic sketch and a mini patent review on virus removal are then presented. Chapter
3 outlines the experimental setup and the methods employed for particle size and particle
concentration ultrafiltration studies. Chapter 4 gives a detailed account on the undertaken
filtration efficiency study and the influence of feed particle size and size distribution on
separation mechanism. Chapter 5 discusses already available models before introducing
our modeling approach based on population balances. The model simulation results are
presented to illustrate the applicability of our model to simulate the virus filtration mech-
anism. Chapter 6 presents optimization-based parameter estimation and discusses its nov-
elty in the field of filtration in general, and specifically shows the usefulness of such an
approach in the area of ultrafiltration by way of validation against our experimental data.
Finally, Chapter 7 summarizes the significant findings and recommends future directions
of this work.
Bibliography 5
Bibliography
Arkhangelsky, E., Steubing, B., Ben-Dov, E., Kushmaro, A., Gitis, V., 2008. Influence of
ph and ionic strength on transmission of plasmid dna through ultrafiltration membranes.
Desalination 227 (1-3), 111–119. 2
Brandwein, H., Aranha-Creado, H., 2000. Membrane filtration for virus removal. Devel-
opments in biological standardization 102, 157–163. 1
Davis, K., 2008. Biopharmaceutical manufacturing: The challenge of global regulatory
compliance. Pharmaceutical Technology 32 (6), 60–68. 1
Guo, Y., Cheng, A., Wang, M., Zhou, Y., 2009. Purification of anatid herpesvirus 1 par-
ticles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation. Journal
of Virological Methods 161 (1), 1 – 6. 3
Hanft, S., 2010. Ultrafiltration membranes: Technologies and the u.s. market. BCC Report
ID: MST044C. 3
Hirasaki, T., Yokogi, M., Kono, A., Yamamoto, N., Manabe, S., 2002. Removal and deter-
mination of dispersion state of bacteriophage [phi]x174 in aqueous solution by cupram-
monium regenerated cellulose microporous hollow fiber membrane (bmm®). Journal of
Membrane Science 201 (1-2), 95 – 102. 2
ICH, December 2000. Ich topic q6a specifications: Test procedures and acceptance criteria
for new drug substances and new drug products. Chemical Substances. 3
Bibliography 6
Manabe, S., 1996. Removal of virus through novel membrane filtration method. Develop-
ments in biological standardization 88, 81–90. 2
Michalsky, R., Passarelli, A., Pfromm, P., Czermak, P., 2009. Purification of the bac-
ulovirus autographa californica m nucleopolyhedrovirus by tangential flow ultrafiltra-
tion. Desalination 245 (1-3), 694 – 700. 3
Santos, A., Bedrikovetsky, P., 2004. Size exclusion during particle suspension transport in
porous media: stochastic and averaged equations. Computational and Applied Mathe-
matics 23 N. 2-3, 259–284. 4
van Reis, R., Zydney, A., 2007. Bioprocess membrane technology. Journal of Membrane
Science 297 (1-2), 16–50. 2

Chapter 2
Literature Review
Contents
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Virus Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.1 In the context of biopharmaceutical industry . . . . . . . . . . . . . 12
2.2.2 In the context of water industry . . . . . . . . . . . . . . . . . . . 14
2.3 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4 Regulatory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.4.1 Regulations governing biopharmaceutical industry . . . . . . . . . 16
2.4.2 Regulations governing water industry . . . . . . . . . . . . . . . . 20
2.5 Virus Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.5.1 Membrane modes and configurations . . . . . . . . . . . . . . . . 23
2.5.1.1 Normal flow filtration . . . . . . . . . . . . . . . . . . . 23
2.5.1.2 Tangential flow filtration . . . . . . . . . . . . . . . . . . 26
2.5.1.3 High performance tangential flow filtration . . . . . . . . 28
2.5.1.4 Field flow fractionation . . . . . . . . . . . . . . . . . . 29
2.5.1.5 Remarks on membrane modes and configurations . . . . 31

8
2.5.2 Membrane modules . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.5.2.1 Flat sheet . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.5.2.2 Spiral wound . . . . . . . . . . . . . . . . . . . . . . . . 34
2.5.2.3 Hollow fibre . . . . . . . . . . . . . . . . . . . . . . . . 34
2.5.3 Particle characterization . . . . . . . . . . . . . . . . . . . . . . . 36
2.5.3.1 Dynamic light scattering . . . . . . . . . . . . . . . . . . 36
2.5.3.2 Imaging and image analysis . . . . . . . . . . . . . . . . 41
2.5.3.3 Asymmetrical flow field flow fractionation and multi-
angle light scattering . . . . . . . . . . . . . . . . . . . . 42
2.5.3.4 Electro-spray differential mobility analysis . . . . . . . . 42
2.6 Economics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.7 Patent Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Preface
Ultrafiltration (UF) is having an increasing role in virus filtration and is becoming a
significant operation for virus removal or virus purification/concentration operations. It is
mainly employed in the biopharmaceutical and water industry to meet strict regulations
(i.e. those of the Food and Drug Administration’s and Safe Drinking Water Act respec-
tively) requiring validation of viral clearance. In order to obtain the final products safe
2.1. Introduction 9
from viral contamination, the proper insight of the source material and the subsequent pro-
duction and purification stages are essential. This review covers various aspects including
possible ways of virus contamination, reviewing various regulatory agencies’ guidelines,
mechanism and modes of virus UF, and particle size characterization techniques. It finally
assesses the economic significance of virus UF before finishing with a review of commer-
cial patent literature in the field. The content of this chapter has been published in Recent
Patents on Chemical Engineering, 2008 [i].
2.1 Introduction
Viruses are simple life forms, potentially pathogenic organisms with a small number
of genes (DNA or RNA) encased in a coat of protein (capsid) or a lipid membrane, i.e.
RNA or DNA enveloped/non-enveloped types, and normally fall in the size range 15nm
- 300nm. Table 2.1 shows types of viruses and the sizes which are normally involved in
virus filtration studies (Fig. 2.1). Viruses are incapable of independent replication and
need a host cell where newly replicated viral particles infect other cells. In some cases, the
viral DNA becomes an integral part of the host cell chromosome.
There are many reported incidents where some products, including drinking water,
vaccines, hormones and blood clotting factors, have resulted in the transmission of infec-
tious disease. Hence, virus filtration is a major concern in the biopharmaceutical, blood
products and water industries [Burnouf and Radosevich, 2003; Charcosset, 2006; Hensgen
et al., 2010; Huang and Peterson, 2001; Soluk et al., 2008] as risk assessment and safety
assurance are critical elements of the starting materials. Globally, inadequate supply of
water and increased consumption of contaminated drinking water create a big dent on the
health safety and developing countries are the worst hit. This necessitates the policy mak-
2.1. Introduction 10
Figure 2.1: Different types of virus particles giving a snapshot of size and shape of viruses
(Pic. courtesy: antibac2k.com, iah.bbsrc.ac.uk.com, innovations-report.de).
Table 2.1: Virus Characteristics: Type, family and size used in virus clearance study.
Type Viruses Family Size (nm)
RNA enveloped HIV Lentivirus 80-110
Xenotropic, ecotropic, Retrovirus 80-110
recombinant murine retro-
viruses
Bovine diarrhea virus Flavivirus 50-70
Vesicular stomatits virus Rhabdovirus 70-150
RNA non enveloped Reovirus Reovirus 60-80
Polio virus Picornavirus 25-30
EMC virus Picornavirus 25-30
Hepatitits A virus Picornavirus 25-30
DNA enveloped Pseudorabies virus Herpesvirus 120-200
Herpes simplex virus Herpesvirus 120-200
DNA non enveloped Adenovirus Adenovirus 70-90
SV-40 virus Papovavirus 45-55
Porcine parvovirus Parvovirus 18-24
Minute virus of mice Parvovirus 18-24
ers and production companies to quantitatively assess the health risk of existing or planned
catchment, treatment, and distribution system of drinking water [Henley, 2009].
Virus filtration is one of the emerging markets for membranes, because it is considered
a robust and effective virus clearance technology and a common unit operation in various
industries. Early work in this field dates back to 1971 when virus retention using mem-
branes was first attempted [Scutt, 1971]. The impetus behind the continuous evolution of
virus filtration technology is due to two important factors; (i) liability of virus contami-
nation is large and there are numerous cases where virus contamination has led to serious
illness, and (ii) viruses must be removed, in any case, say drinking water or during pro-
duction in a biotech process. In virus filtration, it is very important that attention is paid to
meet the regulatory requirements, selection criteria, optimization, and validation of virus
filtration steps and to process scale-up.
Among various filtration processes available, UF is emerging as a powerful tool for
virus filtration. Number of works demonstrate UF being employed for virus purifica-
2.2. Virus Contamination 12
tion/concentration studies where the degree of recovery of virus is less critical in compar-
ison with virus removal operation. Viruses purified by UF include Aedes aegypti, Minute
Virus of Mice, Autographa californica M nucleopolyhedrovirus which are industrially im-
portant vectors [Guo et al., 2009; Hensgen et al., 2010; Michalsky et al., 2009]. In virus
removal or protein separation studies, the main aim is to achieve higher retention of viruses
with complete recovery of protein product. Say, during the manufacturing of diaspirin
crosslinked haemoglobin (DCLHb), UF is identified to be one of the crucial stages where
filtration of red blood cell haemolysate through hollow fibre membrane cartridges of 500
kDa results in stroma free haemoglobin [Azari et al., 2000; Ogle and Azari, 2001]. Thus
virus filtration is efficiently achieved by UF and which is dependent upon the contaminant
source, the regulatory requirements, and the configuration.
In this chapter, we discuss various aspects of virus filtration giving an overview of the
possible ways of virus contamination, reviewing various regulatory agencies’ guidelines,
mechanism and modes of virus UF, and types of virus filters. We also assess the economic
significance of virus UF before finishing with a mini review of commercial patent literature
in the field. This chapter is organized to cover the above outlined aspects of virus filtration
considering biopharmaceutical and water industry as major players.
2.2 Virus Contamination
2.2.1 In the context of biopharmaceutical industry

The occurrence of biopharmaceutical contamination by viruses happens at various lev-
els starting from the source material to the final product preparation. Throughout the his-
tory of biologics administered for human use, one can find numerous examples of products
that have been contaminated with potential human pathogens. Table 2.2 summarizes some
of the instances of viral contamination. The risk of the contamination is considered based
on the source, i.e. if its animal derived, human blood or plasma derived. It is very sur-
prising to note that the diphtheria epidemic paved the way for the inception of regulatory
affairs for health care industries and the maiden one dates back to 1902 Biologics Control
Act [Bren, Jan-Feb 2006]. With the advent of the biotechnology industry and the use of
continuous animal cell culture to produce therapeutic monoclonal antibodies, viral vac-
cines and recombinant proteins for human use, there are many concerns relating to the
potential transmission of animal viruses present in the cell lines, particularly retroviruses,
to humans [Garnick, 1989]. For this reason, regulatory authorities both in the United States
and in Europe formulated guidelines designed to minimize any potential risk of viral trans-
mission. This involved extensive testing of the cell banks, unpurified bulk material, and
final product for the presence of infectious agents. In addition, it was requested that the
production processes used in the purification of these biopharmaceuticals were tested for
their ability to remove or inactivate any potential infectious virus contamination.
Table 2.2: Reported incidents of virus contamination.

Disease (Year) Contaminated bio- Virus Possible Source
logical
Diptheria (1900’s) Antitoxin Unknown Horse
Yellow Fever (World Vaccine HBV as HSA Human blood
War II)
1950’s and 1960’s Vaccine ALV Embryonated hens
– Poliovirus Vaccine SV40 Monkey primary cell
lines
Restricted growth syn- – CJD Human growth hor-
drome (1970’s) mones
1980-2000 MABs, RTP HIV, HAV, HBV, HCV Human blood / plasma
– Veterinary Vaccine Bluetongue Unknown
Despite the stringent controls that are already in place, specific instances of virus con-
tamination of biotechnology production processes still occur. In each case, contamination
is thought to be either adventitious, that is, being introduced from an external source such
as the medium / serum used in the culture process or from an operator through a break-
down in current Good Manufacturing Practice (cGMP) procedures. Viral contamination
of cultured cells is associated with several problems including detection of virus particles,
capacity to replicate, risk for the operators/patients/non-contaminated cell cultures. Thus,
it is becoming evident that all biological products are prone to virus contamination (Table
2.3). These are thus characterized and rigorously tested for the presence of retroviral or
latent viruses and require purification and validation for complete removal. For a complete
review on the various sources of virus contamination, their cell lines and the final products
and viral detection techniques, the interested readers are referred to the article by Merten
[Merten, 2002].
Table 2.3: Virus contamination and their cell lines in the production of biological products.
Virus Cell line Product Contamination
Retrovirus Hybridoma Monoclonal anti- Endogenous
body / recombi-
nant protein
Retrovirus BHK Recombinant Endogenous
protein / vaccines
EBV Namalva Interferons EBV transforma-
tion
2.2.2 In the context of water industry

Traditionally, the human health impact of drinking water is assessed via epidemiology
and it is generally considered that pathogens pose higher health risks than chemicals. Ac-
cording to such studies, the consumption of drinking water is associated with waterborne
outbreaks of diseases such as Hepatitis A & E, gastroenteritis, Cryptosporidium, and Gia-
rdia [Grabow, 1996, 1997; Guthmann et al., 2006; Teunis et al., 1997]. Enteric viruses are
2.3. Definitions 15
increasingly seen as the most important pathogens in drinking water in developed regions
(from a health outcome point of view), versus Cryptosporidium that is recognized to cause
the majority of outbreaks [Ashbolt, 2004]. Table 2.4 gives an overview of various viruses
capable of contaminating drinking water and the possible outbreaks.
Table 2.4: Virus contamination and their sources resulting in a possible outbreak in drink-
ing water.
Disease and Transmission Virus Contaminant Source
Adenovirus infection Adenovirus Self manifestation in improperly
treated water
Gastroenteritis Calicivirus, Enteric Aden- Self manifestation in improperly
ovirus, and Parvovirus treated water
SARS (Severe Acute Respi- Coronavirus Self manifestation in improperly
ratory Syndrome) treated water
Hepatitis A Hepatitis A virus (HAV) Self manifestation in improperly
treated water
Poliomyelitis (Polio) Poliovirus Through the feces of infected indi-
viduals
Polyomavirus infection Two of Polyomavirus: JC Self manifestation in improperly
virus and BK virus treated water
It is reported that there were 642 outbreaks of waterborne disease in the United States
(1971-1996 period) of which 58% of it is associated with ground water sources, especially,
the identified agent is virus which accounted to 9% of the outbreaks [E.P.A., 2000]. As
per 1999-2000 report, 70% infectious disease outbreaks are attributed to groundwater, and
virus is identified as one of those [Lee et al., 2002]. Thus, virus contamination is becoming
increasingly a threat, especially with new virus candidates evolving (like H5N1), to a safe
drinking water.
2.3 Definitions
Several terms relevant to the UF process are defined here to facilitate understanding of
discussions that follow. Primarily are the synonymous terms Log Reduction Value (LRV)
or Log Titer Reduction (LTR) (Equation 2.1) defined as the logarithmic10 of the ratio of
2.4. Regulatory Requirements 16
the virus concentration (titer) in the feed (C f ) and the virus concentration (titer) in the
permeate stream (C p ). In some literature, LRV is also referred to as reduction factor.

Cf
LRV = log10 (2.1)
Cp
The filtration recovery (S) refers to the ratio of the permeate flow (Q p ) to the feed flow
(Q f ):
Qp
S = (2.2)
Qf
Another term is the retention or rejection (R) coefficient of a membrane which is expressed
as:
Cp
R=1− (2.3)
Cf
2.4 Regulatory Requirements

Stringent regulatory requirements are to be met during the course of processing stage,
and the regulations are quickly summarized for biopharmaceutical industry and water in-
dustry.
2.4.1 Regulations governing biopharmaceutical industry

To ensure the safety of biopharmaceutical/biological products for human use, the bio-
pharmaceutical companies need to implement adequate technologies in their manufactur-
ing process and demonstrate the capability of their processes to remove or inactivate con-
taminants based on a process-specific virus clearance strategy [Ray and Tarrach, 2008].
Another major challenge to enter the global market is to follow geographic based regula-
tions.
Various regulations, prior to harmonizing, could be quickly summarized as:
• U.S. Food and Drug Administration (FDA) requires extensive information on facil-
ities and validation processes. FDA’s Center for Biologics Evaluation and Research
is responsible for most of the drug products for virus contamination. They mainly
target to protect and enhance public health through regulation of biological products
including blood, vaccines, therapeutics and related drugs and devices according to
statutory authorities.
• The European Agency for the Evaluation of Medicinal Products looks for less vali-
dation data but rigorous on inspection data. It has various centers but the Center for
Proprietary Medicinal Products is concerned with virus clearance validation.
• Japanese Ministry of Health, Labor and Welfare has a role in the international con-
ference on harmonization towards quality, safety and efficacy.
• The Paul Ehrlich Institute provides advisory functions at the national (Federal Gov-
ernment, German states) and international level (World Health Organization, Euro-
pean Medicines Agency, European Commission, Council of Europe and others) for
the safety of biological medicinal products. It mainly establishes validation practices
for virus clearance.
Tracing the inception of these regulations on viral clearance in the family map shows
that the European Union was the first to provide written guidance on biopharmaceutical
Figure 2.2: Virus Clearance is a critical step in biotechnolgical/biopharmaceutical manu-

facturing. It ensures removal of viruses found to have contaminated the bioprocess.
manufacturers (Table 2.5). Various guidelines now exist that detail the requirements nec-
essary to ensure these products are free from viral contamination ([Hesse and Wagner,
2000]). Manufacturers should select and test source materials for the absence of viruses,
check for the capability of the production process to remove or inactivate viruses and at
each and every stage of the production till the final product stage (Fig. 2.2).
The process of harmonizing global regulatory requirements began more than 20 years
ago (1990), when industry groups and regulatory authorities in the US, Western Europe,
and Japan formed the International Conference on Harmonization (ICH) of Technical Re-
quirements for the Registration of Pharmaceuticals for Human Use to discuss scientific and
Table 2.5: Timeline of global regulations for viral safety of licensed products.
Year Agency
1989 European Union - First guide for viral clearance for bio-pharmaceutical
manufacturers
1991a Committee for Proprietary Medical Products - First published document
1991b Committee for Proprietary Medical Products - Human blood and plasma
derived products
1993 US FDA’s center for Biologics Evaluation and Research - General
framework for process evaluation studies
1994 FDA - Guide for viral clearance and validations
1994 Paul Erlich Institute - Recommendation for viral clearance from plasma
derived products
1996 Committee for Proprietary Medical Products - Viral validation studies
1996 International Conference on Harmonization - Viral safety evaluation of
biotechnology products from human/animal origin cell lines
technical aspects of pharmaceutical product registration. According to these regulations,
the manufacturers need to meet the safety margin, i.e. less than 1 virus particle per 106
doses. It also depends on the type of viruses and the product. For example, endogenous
retroviruses log reduction value should be in the range of 12-18 log clearance whereas for
adventitious viruses it is around 6 log removal. Paul Ehrlich’s recommendations state that
at least 4 log clearance should be achieved in one of two robust steps in a process while a
total of 6 log clearance should be achieved in the overall process. This being with the log
clearance of 10 for enveloped viruses and 4 for nonenveloped viruses. So, depending upon
the respective regulatory requirements, either type of retrovirus clearance or parvovirus
clearance is to be chosen [EMEA, 2006].
To summarize, the major guidelines for viral safety stipulate that a licensed product
must be assured by three complimentary approaches:
• The source/raw materials and the cell lines are to be tested thoroughly for viral con-
tamination.
• The capacity of downstream processing of clear infectious viruses are to be assessed.

• Each and every stage of the product manufacturing is to be tested for virus contami-
nation and virus clearance levels.
These indicators are aimed to serve as good guidelines, however the committee for
proprietary medical product guidelines emphasize more on the robustness of steps rather
than clearance values. Hence, each process is reviewed on a case-by-case basis by the
regulatory authorities [Trijzelaar, 1993].
2.4.2 Regulations governing water industry

In 1974, the US congress passed the Safe Drinking Water Act, applicable for all public
water systems, which published enforceable regulations. These regulations formulated
by Environmental Protection Agency (EPA), applicable for virus contamination, can be
enumerated under two categories : (i) Surface Water Treatment Rule (SWTR) and (ii)
Ground Water Rule (GWR).
• The Surface Water Treatment Rule (54 FR 27486; 29 June 1989) highlights the
importance of safe public water systems that use surface water, or groundwater under
the direct influence of surface water, against water borne pathogens, specifically
including Giardia lamblia, viruses, and Legionella [Berger et al., 2009].
• The Groundwater Rule (71 FR 65574; 8 November 2006) applies to all public water
systems that use groundwater (about 142 000 systems). However, systems that use
groundwater that are under the direct influence of surface water must instead comply
with the SWTR [Berger et al., 2009]. Under the GWR, the State will determine
whether a system is fecally contaminated or is vulnerable to fecal.

2.5. Virus Filtration 21
Both the rules (SWTR and GWR) require systems to use sufficient water treatment (re-
moval, inactivation, or both) to reduce enteric viruses by at least 99.99% of their source
water densities. Apart from the above US regulations, WHO’s Guidelines for drinking wa-
ter quality (2006) gives a comprehensive strategy for ensuring safe drinking water [WHO,
2008]. Other notable countries providing such guidelines include Canada (www.hc-
sc.gc.ca), Australia (www.waterquality.crc.org.au), and New Zealand (www.mfe.govt.nz).
In addition, the US Environmental Protection Agency (USEPA) has a constant vigilance
on the contaminant candidate and potential ones include adenoviruses, caliciviruses, cox-
sackieviruses, echoviruses posing a threat to virologically safe water. It is also identified
that the modeling tools for such water borne pathogens are in its infancy.
2.5 Virus Filtration

Products of human/animal origin products (biologicals and pharmaceuticals) and
drinking water pose unique safety problems and have traditionally been a key concern
both for regulators and industry as already discussed. It has also proven to be a stumbling
block for early product research and development, and inexperienced small start-up firms.
Filtration, being non-invasive, non-destructive and a robust mechanism, is the most
preferred choice for virus removal and purification [Brandwein and Aranha-Creado, 2000;
Hensgen et al., 2010; Kitis et al., 2003; Michalsky et al., 2009]. Over the past few years,
a major leap towards viral safe products has been made, and accomplished by the de-
velopment and availability of biocompatible viral UF systems. Membranes of pore size
ranging upto 1000 kDa are utilized and these UF systems typically provide >4 logs of
virus removal in protein-viral mixtures ensuring good protein permeability and recovery
[Arkhangelsky et al., 2008; Hirasaki et al., 2002; Manabe, 1996] and in the case of water
treatment, log removal values range between 1.5-10 which exhibit tremendous application
of UF membranes for virus removal.
In the case of biopharmaceutical applications, D’Alisa et al. [Chandra et al., 2002] are
the first to show the potential of UF for a range of therapeutics and varied virus removal
efficiencies. Their paper showed results of single filtration of a factor IX concentrate pro-
ducing a 7.3 log reduction in vaccinia virus, 11.2 log reduction in HIV, 7.05 log reduction
in VSV, 4.19 log reduction in Sindbis virus, and 3.90 log reduction in EMC virus. Thus vi-
ral retentive membrane filtration systems demonstrate removal of numerous bacterial and
mammalian viruses, including human and murine retroviruses, SV40 virus and various
hepatitis viruses and parvoviruses such as MMV and B19. New research offers promis-
ing data on retention of transmissible spongiform encephalopathy (TSE) agents known as
prions [Terrab and Pawlak, 2007]. These data suggest an even wider application of mem-
brane filtration to enhance viral safety in manufacturing biological and biopharmaceutical
products, including the ability to scale reliably and efficiently from the lab to production.
In water treatment applications, more than 4.44 log removal of influenza viruses
(H5N1) and more than 3.85 log removal of MS2 phage through a hollow fibre polyether
sulphone membranes of 150kDa are reported [Lenes et al., 2010]. MS2 phage is an ac-
cepted model virus particle for enteric viruses such as poliovirus, hepatitis A or calicivirus
and for highly-pathogenic influenza viruses as well. UF membranes exhibited more than
7.1 log removal of MS2 virus when pre-coagulated / flocculated [Fiksdal and Leiknes,
2006]. Similar results have been reported with MS2 removal achieving more than 6 log
removal with 100 kDa membranes by Otaki et al. [Otaki et al., 1998].
2.5.1 Membrane modes and configurations

A number of examples of filtration work found in the literature aimed at removal and
concentrating viruses in final products using two main modes, namely normal flow filtra-
tion (NFF) (otherwise known as dead end flow filtration (DEF)) and cross flow filtration
(CFF) (otherwise known as tangential flow filtration (TFF)). Recently, two technologies
are evolving namely high performance tangential flow filtration (HPTFF) and field flow
fractionation (FFF) (Fig. 2.3). All the above modes differ in their operation and perfor-
mance characteristics. The challenging aspect of separation of viruses from the product
to be recovered lies in the fact when the size of viruses is similar to the size of proteins,
such as in the case of parvovirus clearance from the protein immunoglobulin G (IgG). The
membrane configuration in this case should be chosen to allow the separation of viruses
while maintaining the passage of proteins. Hence, the selection of the membrane mode of
operation, membrane type and configuration are crucial and specific to each case.
2.5.1.1 Normal flow filtration
Normal flow filtration is mostly employed for virus filtration and especially for labora-
tory scale applications [Bohonak and Zydney, 2005]. In NFF, the feed passes through the
membrane perpendicularly and hence there is more tendency for particle accumulation on
the membrane surface. When particles, in this NFF configuration, are trapped within the
pores or body of filter medium, it is called depth filtration or clarifying filtration, whereas
when the particles are stopped at the surface of the medium and accumulated as a cake of
increasing thickness, the separation is called cake filtration [Russell et al., 2007]. Normal
flow filtration for virus removal involves the following sequence of steps: loading the filter
modules within their housings, flushing, pre-use integrity testing, flushing, sanitization,
Figure 2.3: Schematic representation of dead end flow (NFF), cross flow (TFF) and flow
field fractionation (FFF) modes.
buffer flushing, challenge fluid processing, product recovery flushing, cleaning, post-use
integrity testing and module removal. The product quality in terms of permeability, LRV,
and retention characteristics is influenced by several mechanisms while operated in NFF
mode, including:
• Adsorption of protein products or other components in the fluid constricting the
pores, leading to reduction in the permeability and increase in the retention of larger
components.
• Particle capture associated with cake formation on the surface reduces the perme-
ability and retention is more complex and is normally conceived as initial increase
in passage followed by a drop in passage because of the balancing effect of prefer-

ential plugging and protein and or virus formation on the surface as a cake layer.
• Dynamic changes in the passage during filtration due to the concentration polariza-
tion effect leading to reduction in hydraulic driving force and protein precipitation.
The above effects can change the flow patterns within the membrane structure, in
some cases, reorienting the overall pore size distribution and a reduction in LRV
with throughput. The protein recovery could be well enhanced in NFF by buffer
rinse which is carried out at either constant transmembrane pressure (TMP) or fil-
trate flux.
Number of works illustrates the application of NFF mode for virus filtration. For exam-
ple, PlanovaT M 15N/35N (Asahi Kasei Medical Co. Ltd., Japan) constructed of cuprammo-
nium regenerated cellulose hollow fibers of 15nm and 35nm pore size showed more than
5.7 log removal for both the enveloped (eg. HIV, BVDV, PSR) and non-enveloped viruses
(eg. bovine parvovirus, poliovirus, SV40, reovirus). This is operated in dead end mode
while concentrating Factor IX and Factor XI [Radosevich et al., 1994]. The UF mem-
branes DV20/50 (Pall Corporation, USA) demonstrated virus removal of 50nm and larger
size viruses with higher LRVs (> 6 LRV) whereas smaller viruses like bacteriophage and
poliovirus were retained less in a complex medium [Brandwein and Aranha-Creado, 2000;
Roberts, 1997]. This is achieved using a polyvinylidene fluoride membrane of 20nm and
50nm pore size, operated in dead end mode for processing cell culture medium or phos-
phate buffered saline albumin. Proteins as large as IgGs were able to permeate through
these filters showing good recovery rates. Similarly, the Millipore Viresolve NFP is a nor-
mal flow parvovirus removal filter with a patented membrane structure for strength and
high flow. The filter retains >4 logarithmic reduction value (LRV) of 20 nm diameter
parvovirus.
2.5.1.2 Tangential flow filtration
Tangential flow or cross flow filtration involves passing of the fluid across the surface
of the filter medium, in a direction tangent to the filter. Industrial UF processes are usually
carried out in cross flow mode, the advantage being the lower extent of concentration po-
larization, filter plugging, high permeability and component passage [Belfort et al., 1994;
Guo et al., 2009; Powell and Timperman, 2005]. In addition to that, the cross flow mode
allows recirculation of the retentate stream which offers operational advantages [Ghosh,
2008]. During TFF operation, depending upon the particle membrane interaction, fluc-
tuations in LRV as a function of process variations maybe observed. It is claimed that a
region of high protein concentration known as boundary layer results which aids in retain-
ing viruses leading to improved LRV. UF in this mode finds a variety of applications in
concentrating (removal of solvent from protein solutions), fractionation (protein-protein
separation), clarification/removal (removal of particles from protein solutions), and desalt-
ing (removal of low molecular weight compounds from protein solutions).
The fluid flow dynamics in a typical TFF system used for virus filtration are controlled
by the pressure gradients, flowrate, fluid properties and if applicable, the polarization of
the membrane by solute or solids. A TFF system can be operated in constant TMP mode
or in constant flux mode. The TMP is the average driving force for the feed to pass through
the membrane and is given by:
P f + Pr
T MP = − Pp (2.4)
2
where P f is the feed inlet pressure, Pr is the retentate pressure and P p is the permeate
pressure. In a TFF system, the permeate side pressure is commonly not restricted and
hence can be considered to be at atmospheric pressure. Generally, the fluid flow through
the pores is laminar, and therefore proportional to the TMP. The nature of the flow through
the pores is dependent on the flow regime (i.e. on Reynolds number) of the retentate flow
and on the presence of screens disrupting the steady flow. The performance of the virus
removal filters which is graded by LRV is typically more influenced by the membrane
configuration, operating range and feedstock.
Number of researchers have employed cross flow filtration for virus filtration studies
TM
and have reported good removal efficiency. For example, Planova 20N/75N (Asahi
Kasei Medical Co. Ltd., Japan) membranes are operated in tangential mode for prothrom-
bin complex concentrates and IVIG resulting in more than 6 log removals with both small
and large virus particles. Similarly, ViresolveT M modules (Millipore Corporation, USA)
demonstrated a good range of removal starting 3.5 LRV with 70nm pore size membranes
which is a patented composite PVDF membrane that is specifically geared towards virus
removal applications. The membrane is designed so that not only a high LRV (>4) is
achieved in combination with a high protein recovery (>98 percent), but also that the ap-
plication aspects are incorporated: water wettable, a simple and predictable scale-up and
the process flux is competitive and economical. Aranhe-Creado et al. [Aranha-Creado
and Fennington Jr., 1997] showed the use of dead end in conjunction with tangential flow
to achieve > 10 log removal with the above membranes where some virus aggregation
is found to happen. Zydney [Ho and Zydney, 2003] and coworkers suggested that the
Millipores’ Viresolve membrane is efficient enough for the study of virus filtration but
concluded that membrane morphology and orientation play a critical role in determining
the overall performance of virus filtration membranes. It is quite evident from the above
survey that the filtration modes are operated in both the modes extensively but the choice
is dependent on the feedstock, operating parameters, expected log removal values and the
cost measures.
2.5.1.3 High performance tangential flow filtration
High performance tangential flow filtration, first introduced by Robert van Reis in
1997, is an emerging technology which enables simultaneous protein purification, con-
centration, and buffer exchange in a single unit operation [van Reis et al., 1997]. It can
potentially be used throughout the downstream purification process to remove specific im-
purities, clear viruses, and/or eliminate protein oligomers. HPTFF is a two dimensional
unit operation that exploits both size and charge mechanisms. Several factors make this
process a natural choice: Operating in the pressure-dependent flux regime, optimized oper-
ating parameters and input conditions lead to highest selectivity, resolution and throughput
[van Reis, 1996, 2000]. Charged membranes accentuate the resolution of both charged
and neutral solutes [van Reis et al., 1999]. In this mode, the permeate collected is re-
turned to the permeate side of the module such that the permeate flows co-current to the
feed. This aids in maintaining constant TMP throughout the module leading to finer frac-
tionation of proteins. Many researchers have worked on this, for example, Grzenia et al.
[Grzenia et al., 2008, 2007] demonstrated the use of both the modes i.e. TFF and HPTFF
for virus (AeDNV) purification and reported that with 100 kDa membrane, TFF exhibited
complete rejection whereas in HPTFF, smaller viral fragments permeate through the mem-
brane. But with respect to protein passage, HPTFF possessed better passage for proteins
that are similar in size with the molecular weight cutoff of the membranes and better sep-
aration factors for proteins with size differences less than an order of magnitude. Hensgen
et al. employed HPTFF for purification of Minute Virus of Mice using 50,100 and 300
kDa Sartorius membranes, in which they have virus particles being excluded completely
in 50 & 100 kDa membranes whereas 300 kDa is found to pass virus particles through the
membrane [Hensgen et al., 2010]. They have also reported more flux decay with 300 kDa
than 50 & 100 kDa which they have attributed to the virus particles’ access to the pores and
the cell culture medium. For a comprehensive review on HPTFF, the readers are referred
to the article by Zydney et al. [Zydney and van Reis, 2001].
2.5.1.4 Field flow fractionation
Field flow fractionation is a highly efficient technique for separating and estimating
physical parameters of different materials including biopolymers, biological cells, mi-
croorganisms, and colloidal and solid particles. FFF technique is briefly discussed here
as it is considered to be the fingerprint of UF owing to similar sieving mechanisms, foulant
membrane interactions, and membrane relative sizes [van de Ven et al., 2009]. The FFF
technique is successfully employed to fractionate macromolecules in the wider range of
molecular weights (103 - 1016 ) and particle sizes (10−3 - 102 μm) [Giddings, 1993]. The
separation occurs by differential retention in a stream of liquid flowing through a thin trape-
zoidal channel by the application of an external field. Broad methods of separation are af-
forded via FFF and are classified depending on the nature of the external field, i.e. whether
it is sedimentation, thermal, electrical, cross-flow, magnetic, or other field [Schimpf et al.,
2000]. The field is applied at right angles to the flow forcing particles to move towards the
walls of the channel at different velocities which in turn cause the separation [Giddings,
1993; Ratanathanawongs and Giddings, 1993]. Due to the parabolic velocity profile gen-
erated in the channel by the combined effects of field and flow, larger particles stay close to
the membrane whereas the smaller particles diffuse further away from the membrane and
are carried faster along the channel [Caldwell et al., 1980]. Ideally, the laminae occupied
by each particle type would be held essentially at a fixed position within the parabolic flow
profile. More realistically, Brownian motion and other disturbances cause some broaden-
ing of the regions. Several different types of distributions, arise out of different operating
modes, and are dependent on the balancing of primary and opposing forces (equilibrium
distribution) which establishes the selectivity, resolution and elution time. Various operat-
ing modes and the relevant opposing forces are summarized in Table 2.6. Among all the
techniques listed, the cross flow FFF is considered the most universal technique and is used
to fractionate different synthetic polymers and particles of natural samples. The retention
is dependent on the fields applied and defined as:
Cross-flow:
6D
R= (2.5)
Uw
Electric:
6D
R= (2.6)
μEw
Thermal:
6T
R= (2.7)
αT (dT/dx)w
where R is the retention, D is the diffusion coefficient, U is the cross flow velocity, w is
the channel thickness, μ relates to the electrophoretic mobility, E relates to electric field
strength, αT is the thermal diffusion and T is the temperature.
Table 2.6: Summary of various operating modes of FFF.

Operating mode Opposing force Sample distribution
Normal (up to 1 μ) Diffusion Exponential
Steric (1-100 μ) Repulsion from wall Thin layer at wall
Lift hyperlayer Hydrodynamic lift Thin layer above wall
forces
Gradient hyperlayer Secondary gradients Thin layer above wall
Cyclical Friction coefficient Oscillating
Chromatographic Surface forces Thin layer at wall
hybrid
Effect of cross flow
The effect of cross-flow in FFF which plays a major role in achieving an optimized sep-
aration is well covered by Giddings [Giddings et al., 1987]. However, it is briefly discussed
here. Cross flowrate is a key parameter in the design and operation of a cross flow mode
as it has a major influence on the concentration gradient buildup near the membrane wall
and the proper range depends on the configuration (Table 2.7). Increase in cross-flow im-
proves the permeate flux due to increasing sweeping effect resulting in less concentration
polarization. Whereas, it has a relatively less effect on the retention data.
Table 2.7: Typical cross flowrates for various membrane configurations.

Configuration cross flow velocity (ap-
proximate)
Flat sheet <3 m/s
Hollow fiber <5 m/s
Spiral (1 cm by 16 cm) <0.4 L/s
2.5.1.5 Remarks on membrane modes and configurations
All the above modes find extensive applications in virus filtration and the choice is de-
pendent upon the virus dimensions and characteristics, protein recovery, filter capacity and
process issues such as ease of use, as well as process economics. The NFF modes have
fewer components, are easier to operate, lower in capital costs (however the cost is depen-
dent on larger membrane areas if plugging is rapid). The thickness of the membrane in this
mode may be important when considering rejection. The TFF mode has the capacity to
suppress cake formation and the decline in flux thereby larger feed volumes can be filtered
before cleaning and or disposal. The thickness of the membrane is of secondary impor-
tance as rejection is primarily on the surface. The TFF mode is robust and is the most
preferred mode, for industrial applications, with respect to consistency during the entire
course of filtration and while considering capacity to handle different feedstocks without
plugging. If size exclusion alone is aimed at, more emphasis is on the virus and membrane
characteristics. On the other hand, while trying to achieve higher LRV and or retention,
more consideration is put on operating parameters, feed properties and relevant input con-
ditions. The HPTFF mode finds enormous number of applications in the purification of
small virus particles (like parvovirus) where contaminating host cell proteins and DNA
may be of similar size. The FFF mode has an unique advantage of characterizing the sam-
ples in their native form. The promise from this technology see it continuously attracting
interest and advancing. This method is still considered to be commercially sensitive in the
field of bioseparations. Compared to thermal and centrifugal force, flow field is considered
to be the leading technique and when coupled with other characterization techniques will
serve as a powerful separation method for protein and virus purification and is likely to
replace the other techniques, in future.
2.5.2 Membrane modules

The UF membranes employed for virus filtration are cast into different modules: flat
sheets, spiral wound, and hollow fibre membranes, the latter traditionally being employed
in major industries including bio-pharmaceutical and water industry [Li et al., 2008] (Table
2.8). Many of these modules are also available either as cartridges or capsules which may
use pleated structures to maximize filter area for high particle retention capacity while still
allowing high flowrates. The selection, design, and operation of membrane modules are
highly dictated by the techno-economic factors. These include cost of supporting materials
and enclosures, power consumption, ease of cleaning and replaceability, good mass trans-
fer characteristics, feed stream components, product volume, and scaling needs [Gagne
and Vaccaro, 2003; Schwinge et al., 2004].
2.5.2.1 Flat sheet
Flat sheet membranes are most sought after module because of ease of use and ease of
cleaning. They have relatively high packing density, provide good cross flow performance,
can handle viscous feeds and suspensions, and are amenable to incorporating turbulence-
promoting spacers. Limitations to this design are labour intensive fabrication, low mem-
brane area to module volume ratio, high hold up volume of the feed side, and lower pack-
ing densities compared to hollow fibre membranes. Several basic flat sheet membranes are
connected within a cassette in series or in parallel which have a wider application range
from small to large scale operations in virus filtration (Fig. 2.4). DiLeo et al. demonstrated
filtration of mammalian virus spiked protein solutions in flat sheet module and reported ef-
ficient flowrates with reproducibility and high retention [DiLeo et al., 1993]. Biesert et
al. attempted removing both enveloped and non-enveloped viruses from protein solutions
using flat sheet modules and obtained a high level of log removal with various viruses
[Biesert et al., 1997]. Similar results obtained with flat sheet modules at small and large
scales are reported elsewhere [Brough et al., 2002; Choi and Kim, 2008].
Figure 2.4: TFF modules for small and large scale applications. (Courtesy: Millipore
Corporation)
2.5.2.2 Spiral wound
Spiral wound membranes are difficult to clean and sterilize, are subject to plugging,
have a limited range of scalability, and their long flow distance makes high flow with low
pressure drop difficult. However they offer relatively high packing density, are inexpen-
sive and very successful for particulate free process streams. Generally, nanofiltration and
reverse osmosis popularly employ spiral wound configuration than UF and MF [Pearce,
2007]. But literatures are available with UF spiral wound configuration like Lute et al.
reporting efficient virus removal with ultipors DV20 membranes in spiral wound configu-
ration [Lute et al., 2008].
2.5.2.3 Hollow fibre
Hollow fibre membranes are self supporting, do not need spacers, have low dead vol-
umes on the feed side, have low pumping cost, have greater membrane surface area per
volume. However their disadvantage is their lower mass transfer coefficients and possi-
bility of fiber breakage. The inside-out hollow fibre membranes are the preferred ones as
they afford controlled flow hydraulics and cross flow along the membrane surface limits
membrane fouling . In this mode, the feed enters one end of the fiber, retentate leave the
other, while the permeate pass through the outside of the membrane fiber. Hollow fibre
is reported to be widely employed in biopharmaceutical industries wherein all types of
viruses are efficiently removed. Viruses larger than 35 nm in size (eg. BVDV, HIV) are
removed using hollow fibre Planova 35N membranes with 5.9 - 7.3 log removal whereas
significant removal of bigger viruses is easily accomplished using Planova 15N. Smaller
viruses (eg. PPV) showed 4.4 log removal with Planova 15N owing to very close virus
and pore size. Hirasaki et al. demonstrated that hollow fibre Planova 20N provides high
levels of parvovirus and porcine removal from human IgG solutions with minimal fouling
[Hirasaki et al., 2006].
Table 2.8: Summary of commercially available membrane modules for virus removal.
Name (Manufacturer) Material Module type Pore size, nm LRV
Viresolve 70 (Milli- Hydrophilic PVDF Flat sheet TFF – PV > 3.5
pore)
SV40 > 5.6
SBV > 7.4
RV > 7.2
MLV > 6.6
HIV > 8.5
Viresolve 180 (Milli- Hydrophilic PVDF Flat sheet TFF – PV > 2
pore)
HAV > 4
SV > 4.5
RV > 5.5
MLV > 6
Planova 15 N (Asahi Hydrophilic cupram- Hollow fiber - 15 ± 2 PPV > 4.4
Kasei) monium regenerated NFF
cellulose
PV > 7.8
Planova 20N (Asahi Hydrophilic cupram- Hollow fiber - 19 ± 2 PPV > 4.2
cellulose
ECM > 5.4
Planova 35N (Asahi Hydrophilic cupram- Hollow fiber - 35 ± 2 BVDV > 5.9
cellulose
HIV > 7.3
Virosart Polyether sulphone Spiral wound - 20 RV > 6
CPV(Sartorius) NFF
Bacteriophage >
4
Ultipor DV20 (Pall) Hydrophilic PVDF Spiral wound - > 20 Bacteriophage >
NFF 3
Ultipor DV50 (Pall) Hydrophilic PVDF Spiral wound - > 50 Bacteriophage >
NFF 6
Based on the above survey (Table 2.8), it is evident that all the three modes find numer-
ous applications for varying virus particles. But literature indicates hollow fibre is largely
employed in industrial applications [Li et al., 2008] whereas for small/lab scale, flat sheet
membranes maybe employed.
2.5.3 Particle characterization

Quality by Design highlights the importance of identifying and controlling all the pa-
rameters that can influence final product performance. Specifically, ICH Topic Q6A iden-
tifies particle size as a potentially important variable [ICH, December 2000]. The specific
need for a particle size specification is determined by assessing if and how this parameter
affects purification and separation performances. In virus clearance or purification opera-
tions, the virus particles are distributed in the range of 15nm - 300nm depending upon virus
protein coat and virus family. Various analytical techniques are available for virus particle
characterization which are summarized in Table 2.9. The prominent methods being either
light scattering and/or image analysis. However, field-flow fractionation coupled with mul-
tiple angle light scattering and electrospray differential mobility analysis (ES-DMA) are
slowly emerging as a powerful technique for virus particle characterization [Chuan et al.,
2008; EP, 2006; USP, 2008].
2.5.3.1 Dynamic light scattering
Particle size distribution is typically presented as number weighted (number of particles
in each size class) or volume/mass weighted (volume/mass of particles in each size class)
or intensity weighted distributions. If coarser particles are predominant, then volume/mass
weighted distribution is the appropriate presentation. Number based distributions are sen-
sitive to presence of finer particles and more so when dispersion dominates [USP, 2008].
Table 2.9: Summary of various analytical techniques available for virus particle character-
ization studies.
Method Brief description (manufacturer exam- References
ple)
Electron microscopy [Kuo, 2007; Lengyel et al.,
2008; Pelchen-Matthews and
Marsh, 2007]
- Transmission Electron (TEM) TEM – Particle morphology and size [Gillock et al., 1997]
(JOEL)
- Scanning Electron/Probe (SEM/SPM) SEM/SPM – Particle morphology and [Johnson and Chiu, 2000]
size
- Cryo Electron Tomography (CET) CET – 3D molecular structure [Harris et al., 2006]
Light scattering [Citkowicz et al., 2008]
- Dynamic Light Scattering (DLS) DLS – Measures stoke’s (hydrodynamic) [Tsoka et al., 1999]
radius of particles (Malvern, Brookhaven)
- Multi Angle Light Scattering (MALS) MALS – Measures root mean square ra- [Shortt et al., 1996]
dius, molar/molecular mass (Wyatt Tech
Corp –Heleos)
Asymmetric Flow Field Flow Fractiona- Analytical tool for characterization [Pease III et al., 2009]
tion (AF4) of biological samples (Wyatt Tech
Corp—Eclipse)
Electrospray differential mobility analy- Particle size distribution (TSI Inc) [Pease III et al., 2009]
sis (ES-DMA)
Diode Array Detector (DAD) Quantification and identification of parti- [Citkowicz et al., 2008]
cles (Agilent 1100)
Fluorescent Detector (FD) Quantifies and identifies particles like [Citkowicz et al., 2008]
proteins, DNA impurities (Agilent 1100)
Refractive Index Detector (RID) Measures absolute refractive index (RI) [Citkowicz et al., 2008]
and dn/dc (differential RI) of the solution
(Wyatt Tech Corp - Optilab REX)
In optical laser based particle characterization, repeatability and reproducibility are ex-
acting requirements but which depends on the choice of suitable sample concentration
and dispersion conditions. The US and European pharmacopeias specify a target repro-
ducibility of 10% for volume median diameters of particles >10μm in diameter and 20%
for those smaller than this [EP, 2006; USP, 2008]. Dynamic light scattering (DLS) thus
finds extensive application in particle size studies and especially for size distribution anal-
ysis. For example, Lipin et al. [Lipin et al., 2008] reported the use of DLS for particle
size distribution characterization of viral particles obtained during protein purification by
chromatographic separation column (Fig. 2.5). They showed the versatility of the DLS
technique in quantifying changes in size distribution that result from changes made to the
process.
Figure 2.5: Particle size distribution of high molecular weight, low molecular weight, and
virus like particle (MIX) solutions by DLS [Lipin et al., 2008].
In another study [Wickramasinghe et al., 2005], tangential flow UF is used to capture

human influenza virus particles using 100 and 300 kDa UF membranes. Particle size dis-
tributions were measured successfully in the permeate and retentate streams using laser
diffraction light scattering demonstrating fractionation of the original suspension (Figs.
2.6,2.7). In their study, Citkowicz et al. [Citkowicz et al., 2008] elaborated on the charac-
terization techniques for gene therapy from human polyoma JC virus, demonstrating the
online and offline use of DLS technique for measurement of particle size to the order of
25nm (Fig. 2.8).
Figure 2.6: Particle size distribution in the retentate (right) and permeate (left) streams
through 0.1 μ membrane during the study of HAV by TFF system.
It is very surprising to note that though particle size plays a significant role in the
membrane filtration, very limited literature is available relating the particle size and final
product quality. All the works cited above do not give or relate the particle properties
on the filtration efficiency values. Especially, quantifying the dependency of particle size
parameter on the filtration mechanism and studying its influence is still an unearthed area.
Figure 2.7: Particle size distribution in the retentate (right) and permeate (left) streams
through 0.2 μ membrane during the study of HAV by TFF system.
Figure 2.8: Particle size distribution of diethylaminoethyl (DEAE) and packaged virus like
particles by dynamic light scattering technique.
Hence, to bridge this knowledge gap we investigate and quantify this effect which will
lead to a better understanding and modeling particle size distribution, which is still in its
infancy.
2.5.3.2 Imaging and image analysis
Image analysis is another excellent choice as it provides statistically relevant particle
size and shape data in addition to detailed visual insight about shape and size. Several
imaging techniques are available for the study of viral size and morphology including
transmission electron microscopy, scanning electron/probe microscopy, atomic force mi-
croscopy, cyto electron microscopy and electron tomography. The choice of imaging tech-
nique is dependent on cell lines used, and safety aspects [Brandenburg and Zhuang, 2007;
Gaczynska and Osmulski, 2008; Kolin and Wiseman, 2007; Lengyel et al., 2008; Pelchen-
Matthews and Marsh, 2007]. Transmission electron microscopy (TEM) is the most com-
monly used technique for virus particle studies as it readily allows visualization of particle
morphology and size. However its limited resolution and possible artifacts caused by sam-
ple preparation may limit its accuracy. The use of cryo electron tomographic approach
to analyze and quantitate the structural complexity of viruses give an insight about the
structural information and bridges a critical gap in virus imaging analysis. Alongside the
imaging techniques, image processing which refers to the algorithms and software that
analyse the imaging data has been evolving rapidly in recent years. Several applications
are now commercially available including the popular freeware ImageJ (image process-
ing software inspired by National Institute of Health, USA) [Sun et al., 2007]. These can
perform several techniques for particle analysis including projected particle pixel area and
chord length measurement. Other algorithms such as that developed by Korath et al. [Ko-
rath et al., 2008] can perform more complex analyzes including high-fidelity separation of
touching particles.
Thus, image analysis gives an accurate information about the morphology of the virus
particles. Whereas for size distribution studies, image analysis method becomes quite
tedious and hence light scattering method is found to be more suitable.
2.5.3.3 Asymmetrical flow field flow fractionation and multi-angle light scattering
The availability of commercial field flow fractionation (FFF) equipment coupled with
sophisticated analytical instruments like multi-angle light scattering (MALS) has started
slowly penetrating into various biotechnological applications especially for the analysis of
viruses and virus like particles for vaccines and gene therapy [Wei et al., 2007]. Inspite of
FFF’s presence for more than 30 years, the technique is yet to gain momentum at various
levels viz. academic, commercial and research [Roda et al., 2009] for virus analysis appli-
cations. Specifically, the hybrid technique, i.e. asymmetric flow FFF (AF4) coupled with
MALS, is extremely useful for fractionation of wider range of particles of different species,
and is expected to make a big way in fractionation and removal. Chuan et al. [Chuan et al.,
2008] demonstrated the use of AF4-MALS technique for characterization of virus-like
particles giving cumulative size distribution at optimized condition and could, using such
data, show the influence of cross flowrates on fractionation. Pease et al. [Pease III et al.,
2009] demonstrated AF4-MALS as a valuable method to characterize virus-like particle
distributions rapidly and quantitatively.
2.5.3.4 Electro-spray differential mobility analysis
One of the emerging techniques for virus sample characterization is the electro-
spray differential mobility analysis (ES-DMA) which is capable of analyzing viruses like
2.6. Economics 43
MS2,T2, T4 and adenoviruses [Cole et al., 2009; Hogan Jr. et al., 2005; Thomas et al.,
2004]. In this technique, the concentration of samples are obtained. Samples are elec-
trosprayed into a gas stream where particles are detected using a condensation particle
counter via various stages including evaporation, charge neutralization, size separation.
This method is preferable for spherical viruses because of its ability to retain their viability
after electrospraying thus helping consistent sizing measurements. Cole et al. [Cole et al.,
2009] demonstrated the concentration measurement of the virus particles MS2, PP7 and
φX174 by ES-DMA and recommended PP7 to be used for virus filter standardization. ES-
DMA technique also provides information on the aggregation state of the sample and has
a wide range of concentration measurement from small viruses including MS2 and larger
ones including adenovirus (used for gene therapy) [Hogan Jr. et al., 2006].
Thus, to summarize, virus particle characterization maybe largely achieved by image
and dynamic light scattering analysis. Electron microscopic methods can accurately pro-
vide size and morphology information of the virus particles, however imaging techniques
are prone to image artifacts and are not optimised for high throughput analysis. Whereas,
dynamic light scattering technique provides size and size distribution with a fair level of
accuracy, but when the sample available is limited, this method is restricted as the sample
concentration sufficient to obscure the laser needs to be determined. A more recent hybrid
technique namely AF4 with MALS is expected to replace the current techniques in a big
way, leading to a more qualitative and accurate separation and characterization.
2.6 Economics
UF technology is constantly evolving with newer types of membranes and materials to
meet the regulations, hence variedly employed in the booming pharmaceutical and biotech
2.6. Economics 44
fields (Fig. 2.9 [Vacura, 2008]). In general, the market for membrane technology used in
biotechnological discovery, development and commercial production, is expected to rise at
a compound annual growth rate (CAGR) of 7.4% to $908 million in 2011 [Hanft, 2010].
Specifically, UF a multi-million dollar market, is growing at an annual average growth rate
(AAGR) of 7.6%. The growth of the UF method for bioprocessing expands because of the
constant demand of protein purification, gene therapy, vaccine production, juice and wine
industry, pharmaceutical grade water wherein virus filtration is a vital operation.
Figure 2.9: Membrane filtration’s place in the Pharmaceutical/Biotech industry - World

Market Share 2008 ($ million) (UPW-UltraPure Water, MaF-Macro Filtration, SAC-
Sedimentation and Centrifugation).
According to the updated technical market research report on UF Membrane Markets -
MST044C (May 2010) - from BCC Research, there is a clear indication of an accelerated
growth rate of UF. Specifically, the US market value for UF was $882 million in 2009
and is expected to reach $932 million by the end of 2010. As UF is found to be the most
appropriate technology for producing dairy and juice products, protein fractionation, corn
and other mill product processing, nutraceuticals manufacture, and waste water treatment,
2.6. Economics 45
the challenge is to obtain most efficient membranes but at reasonably reduced cost. The
capital costs involved in membrane filtration industry are higher whereas resources like
company space and energy consumption are less compared to other chemical processes.
This enables companies to reduce costs by upwards of 65%.
The success of biotechnology for bulk product manufacturing, thus, heavily depends
on engineering solutions in downstream processing in which separation and purification
have a crucial role to play. Interestingly, biotechnology sector is expected to grow robustly
over the next 5 years. Growth forecast for the period 2010-2015 is expected to rise at a
compound annual growth rate of 10.2% [Hanft, 2010]. Whereas in drinking water treat-
ment, UF is becoming competitive pretreatment system for RO, where the feed water is
not too high in terms of organic content. And UF has become the preferred alternative to
conventional technology to remove water borne pathogens in the preparation of drinking
water [Clever et al., 2000; Laine et al., 2000; Rautenbach and Vobenkaul, 2001]. Consis-
tently, UF seems to efficiently achieve current water regulation values for turbidity, Giardia
and virus removal. UF applications, in full-scale plants, represent about 74% of the total
installed capacity. This is due to the fact of no loss in the performance in terms of pro-
duction capacity, membrane material over time and water quality. UF proves to be quite
economic including power, membrane replacement, consumable, maintenance, capital and
labor costs, treating surface water costs around $ 0.235 per m3 reports Drouiche et al.
[Drouiche et al., 2001].
These data clearly confirm that UF technology is gaining a stronghold position meet-
ing the international acceptable standards in biotechnology and water sectors. With this
tremendous growth in the upward direction, regulatory authorities depend specifically on

2.7. Patent Review 46
well-designed, optimized process and cost-efficient polymers to remove contaminants at
the end product.
2.7 Patent Review

Accelerated developments are taking place in the manufacturing methods of UF mem-
branes, their configurations and material makeup towards achievement of appropriate re-
tention values, throughput and permeability for virus removal. Viruses, either those oc-
curring in nature, or recombinant versions thereof, are extensively used in the field of
gene therapy and vaccinations. Purification of such viruses, being a vital operation, is
efficiently achieved by UF and widely employed in industries. It is observed that high re-
tention values are obtained with porous polyvinylidene fluoride membranes and cellulosic
membranes [Degen et al., 1998b; DiLeo et al., 1991; Handlin et al., 2009; Masayuki and
Shin-ichi, 2009]. Defect free cellulosic UF membranes made from microporous polymeric
substrates, are capable of maintaining desirable flux values as well as good retention per-
formance [Tucelli and McGrath, 1996; Walter et al., 2010]. Wilson’s patent [Wilson and
Mikhail, 2007] includes an additional step of autoclaving the membrane in boiling water
and/or steam during the fabrication stage. This provides a caustic resistance and which can
be of hollow fiber or sheet type. These hydrophobic polysulfone composite polymer mem-
branes, having its surface rendered hydrophilic with a hydroxyalkyl cellulose, are capable
of achieving higher LRV value and throughput. Miranda’s patent claims that purification
of viruses is best achieved by applying back pressure in the permeate side [Miranda, 2009].
A patent review covering inventions on the use of UF for virus removal is summarized in
Table 2.10.
A TFF system has been considered by van Reis [van Reis, 1996, 2000] to separate
Table 2.10: A mini review of patents on the use of UF for the removal of virus particles.
Year/US Patent Patent Title Patent Inventor(s) Patent Assignee
Number
2009/20090123989 Virus purification using UF Weggeman Miranda Crucell Holland B.V.
(Leiden, NL)
2007/7226429 Method for removal of viruses from Tullis Richard H Aethlon Medical Inc.
blood by lectin affinity hemodialy- (San Diego, CA)
sis
2007/7223585 Viral purification methods Coffey Matthew Oncolytics Biotech Inc.
(Calgary, CA)
2006/7118675 Process for removing protein aggre- Siwak Martin, An Millipore Corporation
gates and virus from a protein solu- Hong, Cormier Jason (Billerica, MA)
tion R, Kinzlmaier Dana
2006/7108791 High resolution virus removal Tkacik Gabriel, Kazan Millipore Corporation
methodology and filtration capsule Greg (Billerica, MA)
useful
2002/6365395 Process for removing protein aggre- Antoniou Millipore Corporation
gates and virus from a protein solu-
tion
2000/6054051 Tangential flow filtration systems van Reis Genentech Inc.(South
Sanfransisco, CA)
1998/5736051 Polyvinylidene fluoride membrane Degen Peter John, Sip- Pall Corporation (East
and method for removing viruses sas Ioannis P, Rapis- Hills, NY)
from solutions arda Gregory C Gregg
Joseph
1998/5731164 Method of Checking the rate of re- Becker Gerhard, Lar- Sanorell Pharma
moval of pyrogenic substances, in son Paul Marcel, Heidl GmbH and Co.
particular viruses, from organic ma- Reiner (Baiersbronn, DE)
terial
1997/5645984 Process for depleting viruses in so- Nader Warner Sanorell Pharma
lutions and determining their deple- GmbH and Co.
tion rate (Baiersbronn, DE)
1991/5017292 Membrane Process and system for DiLeo AJ, Allegrezza Millipore Corporation
isolating virus from solution AE, Burke ET (Bedford, MA)
species of interest in the range 1 to 1000 kDa MWCO (mostly targeting biological fluids)
from mixtures. The flux is found to be dependent on transmembrane pressure until the
pressure reaches a transition point beyond which this dependency drops off. This patented
filter is claimed to be capable of filtering similar sized specimens and from thereon, TFF
is reported to being employed in number of purification stages for high selectivity and
throughput [John et al., 2003]. Later, high performance tangential flow filtration emerged
to yield a high-purity product in protein purification [Robert et al., 2008]. David et al.’s
[David and William, 2001] method employs filtration aid together with low concentra-
tion of metal ions in place of nucleases for commercial scale purification of encapsulated
viruses from cell culture. In these methods, UF plays an important role in separating
viruses from DNA and RNA species present in the cell culture. The filtration is aided not
by enzymatic nucleases but rather by filtration aids such as diatomaceous earth. This offers
advantages for commercial scale purification of encapsulated viruses particularly as spec-
ification tests that would have to be designed to demonstrate the removal (i.e. absence)
of the nuclease(s) in the final product would be eliminated. Similarly, a tangential flow
system with the hollow type UF membrane is recommended as the most preferable and
robust method for virus concentration [Matthew, 2007].
Matthew’s improved method for virus purification from cell culture involves a simple
extraction step in which a detergent is directly added to the cell culture where the resulting
viruses are suitable for clinical administration to mammals, this being a major step towards
cell therapy [Matthew, 2003]. Tullis utilized hollow fiber membranes (200-500 nm pore
size) for virus (110 nm dia) removal from the blood stream [Tullis, 2007]. The retention
level of min 3 LRV of virus removal from protein solutions was achieved using normal flow
or DEF filtration but with one or more UF membranes [Martin et al., 2006; Tkacik and
Kazan, 2006]. Stefan’s virus filtration method for solutions containing macromolecules
suggests that the DEF technique offers economic advantages due to simpler equipment,
simpler operating procedures and reduction in the loss of macromolecules [Stefan, 1996].
It facilitated the virus filtration process in terms of residence time reduction and optimized
yield in comparison with the TFF as DEF involves placing the macromolecule contain-
ing solution in a pressure vessel prior to filtration and pressing the solution through the
membrane with the aid of a pressure source.

The method of using multilayered membranes [Degen et al., 1998a; Tkacik, 2007;
Tkacik and Kazan, 2006] with at least one membrane being oriented tight side down stream
showed satisfactory levels of virus removal but the protein passage and flux are low. Such
multilayered membranes can be UF membranes supported with monomer surface coat-
ing or two anisotropic membranes with one membrane juxtaposed with at least one other
membrane so that substantially all the skin surface of one membrane is in intimate contact
with all of the skin of the other membrane. Thus the membranes made by cocasting or se-
quentially casting plurality of polymer solutions substantially prevent the passage of virus
particles but permit the passage of protein [Tkacik et al., 2010]. Other such membranes
can be comprised of a tight side with porous support which is characterized by having
asymmetric structure free of macro-voids when exposed to protein solution, exhibiting
low protein binding and these are normally used to filter viruses from protein streams. In
order to increase the filtration rate during the UF of viruses, the viruses to be removed
are increased in size by incubation with a high molecular weight receptor, binding prefer-
ably a specific antibody so that the separation effect is improved and large pore diameter
membranes can be chosen [Nowak and Bernhardt, 2002].
From this mini-review of patents, a silver bullet solution was not exactly found but it is
arrived that a suitable membrane and membrane configuration should be selected in tandem
to obtain the required retention performance along with complete protein product recovery.
As such there is a body of knowledge in the patent literature that one ought to consult in
the process of choosing a more suitable filtration setup. It can be seen that there is an art
at play here where the skilled artisan may select through experimental campaigns the opti-
mal combination of membrane, membrane configuration, operating conditions, processing

2.8. Conclusion 50
steps, and so on. Thus well-defined deterministic knowledge for virus filtration by UF is
still outstanding. Having said that, there is a wide scope of research to be done in the area
of virus filtration providing ample opportunities for scientific inventors in this field. For
example, there is a rapidly increasing interest in the development of cost-efficient tech-
nologies for industrial separation of whey protein, a heterogeneous mixture of individual
protein fractions that have their own unique nutritional, functional, physiological and neu-
traceutical properties. Unfortunately, the protein separation / purification is a demanding
process due to the complexity of proteins themselves and their biological environments.
2.8 Conclusion
Although the basic scientific principles behind membrane technology have been around
since the 1950s, it was not until the 1970s that membrane technology was exploited for pu-
rification and or removal of virus particles. In this review, virus filtration is found to be
quite a significant downstream operation faced with challenges including meeting strin-
gent regulations, variability in virus contamination sources and optimal configuration and
operation.
• UF applicable for virus filtration can be operated in both modes, i.e. normal flow
and tangential flow. Two other techniques viz. HPTFF and FFF are largely evolving,
specifically FFF is finding broader applications as it is capable of handling the
streams in native form for characterization. But mostly, in industrial operations,
TFF is the preferred mode because of lower extent of concentration polarization and
filter plugging, high permeability, and operational advantages.

2.8. Conclusion 51
• Quality by Design highlights the importance of particle size as one of the influ-
encing parameters on the final product quality. Survey on particle size study in
membrane filtration, specifically for virus filtration operations, shows that only
limited literature is available on the effect of particle size along with the operating
parameters on the final product quality. With new virus candidates outbreak and
wider range of virus particles, it is important to study the size and its distributions’
role in the final permeate quality which will lead to an indepth understanding to
achieve better efficiency. Hence, it is identified that an urgent focus is needed to
develop a model for prediction of particle size and its distribution.
• Increasing demand of efficient UF virus filters in the bioseparation and water
treatment fields is causing a tremendous growth in UF membrane market. The
challenge lies in optimizing yields and reducing costs with the newer technology
and its development.
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Chapter 3
Experimental Section
Contents
3.1 Membrane Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2 Characterization Techniques . . . . . . . . . . . . . . . . . . . . . . . . 72
3.2.1 Particle concentration measurement by inductively coupled plasma
technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.2.2 Particle morphology studies by transmission electron microscopy . 74
technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Preface
This chapter gives a description of the cross flow rig details and materials used to
carry out the experiments. With a brief theoretical background, the methods employed for
concentration, particle size and morphology studies are also presented in this chapter.
3.1. Membrane Module 69
3.1 Membrane Module

The cross flow module employed in this work is shown in Figure 3.1. The whole
length of the module is approximately 264mm and is constructed in-house from perspex
blocks. The pressure transducer is inserted at the top half of the module. While running
the filtration experiment, bolts and wing nuts are used to fasten the two halves together.
The module also comprises a series of perforated aluminium plates to support the UF
membrane strip.
Figure 3.1: Tangential flow UF experimental setup employed for this study
All the experiments are conducted using this non-commercial experimental prototype
perspex cross flow module of membrane channel dimensions 2 mm*25 mm*210 mm. The
required feed pressure is obtained using the peristaltic feed pump (Master Flex Model
7529-00 by Cole Parmer). The pressure required is obtained by adjusting a valve situated
at the module outlet. A personal computer is used to record the data through the pressure
transducers, flow transducer, a type-T thermocouple located upstream of the module.
Pressure transducers
The pressure transducers employed in the membrane configuration are PDCR 810 from
Druck (Davidsons, Australia). They produce an output voltage of 100 mV for 350 mbar
(i.e. corresponding to 35 kPa) and above, but which could be used for a range by a factor
of 6 (i.e. approximately 200 kPa) for this model with no change in the calibration factor.
The pressure measured and the raw voltage measured by the sensor are related as follows:
C f s Vm
Pressure(measured) = (3.1)
Vex CF
C f s - full scale capacity i.e. max. pressure which the transducer should receive
Vex - excitation voltage - recommended input voltage
Vm - raw voltage measured by the sensor
CF - calibration factor
The maximum deviation in the values obtained will be 0.1% and this means, it could
be ±2.1 kPa deviation in output for a pressure of 35 kPa.
Flow sensors
Flow sensor employed is from Radiospare (RS Stock number: 256-225) which could
measure upto 9.0 L/min and is suited for various liquids. The accuracy level obtained in
this sensor is 1% full scale (FS) and the repeatability is ±0.25 %.

Membrane material
The membranes used for the entire experimental investigation are the poly ether sulfone
(PES) membranes. They are procured from Pall Corp from lots 8220B, 7265G. These UF
membranes are asymmetric in nature. The molecular weight cut off of the UF membranes
is 30,000 and 100,000 Da. Initially, a careful study is carried out with the samples to assess
the membranes so they do not completely reject all the particles as we are interested in
particle size fractionation and hence 100000 Da membrane is employed. Whereas, 30,000
Da membrane is employed just to compare the membrane effect on the filtration efficiency.
Sample material
Silica particles used for this study are procured from Nissan Chemicals, US and the
properties are reported in Table 3.1. The particles obtained are used as purchased condition
and the pH (8.5-10) is quite stable in this range and consistent as well.
Table 3.1: Characteristics of Silica Colloid.

SnowTex ZL SnowTex 20L SnowTex ST-50
Density, kg/m3 1305 1130 1380
Mass, gm 500 525 500
wt% SiO2 40.5 20.5 48
Concn, g/l 522 231.45 662.4
Mass, gm (for 1 1.814 4.08 1.43
g/l)
Particle size, nm 130-140 70-100 20-50
Refractive Index 1.46 1.46 1.46
The methods employed for the analysis are discussed in the following sections of this
chapter.
3.2. Characterization Techniques 72
3.2 Characterization Techniques

A brief outline of the methods employed for particle concentration, size and morphol-
ogy study is sketched here (Fig. 3.2). Particle concentration measurement by inductively
coupled plasma technique, particle morphology study by microscopic technique, whereas
and, particle size measurement by dynamic light scattering technique are discussed in the
following sections of this chapter.
Figure 3.2: An outline of various methods employed for the analysis of silica samples.
3.2.1 Particle concentration measurement by inductively coupled
plasma technique
Inductively Coupled Plasma - Optical Emission Spectroscopy (ICP-OES) is one of
several techniques available for elemental analysis which is capable of multi element de-
termination in solutions. It makes use of the fact that the atoms of elements can take up
energy from an inductively coupled plasma (a plasma is an electrically neutral, highly ion-
ized gas that consists of ions, electrons, and atoms), are thereby excited, and fall back
into their ground state again emitting a characteristic radiation. The identification of this
radiation permits the qualitative analysis of a sample. A quantitative determination takes
place on the basis of the proportionality of radiation intensity and element concentration
in calibration and analysis samples.
In this technique, samples are fed through an auto sampler for unattended operation to
an argon plasma via a nebuliser. The Segmented-array, Charged-coupled-device Detector
(SCD) capable of detecting over 5000 emission lines in the range of 167 to 782nm, quanti-
fies atomic emissions in the plasma and WinLab32 software records the values diligently.
The standards are prepared at 0.5, 1, 10, 100 ppm and the internal standard used for
this sample is Yttrium. The sampler is washed with 2% HNO3 solution (nitric acid) and
the standards are tested. Subsequently, the samples are acquired with the aid of autosam-
pler and are fed to an argon plasma via a nebuliser and spray chamber using the integral
peristaltic pump. Though various nebulisers and spray chambers are available, but the
choice is highly dependent on the nature and quantity of the sample to be analysed. In this
analysis of silica, gemcone high dissolved solid nebuliser with cyclonic spray chamber is
employed. A solid-state radio frequency (RF) generator supplies RF energy at 1300W to a
coil around the horizontally mounted quartz torch to maintain the argon plasma, the (rela-
tively cool) end of which is removed by a shear gas - a thin ’wall’ of high-velocity air (for
samples run at axial view). Light from the plasma and the various atomic emissions therein
passes through windows ’viewing’ the plasma radially into the thermally stabilised, argon-
purged optical compartment housing an Echelle polychromator to separate the light into
its component wavelengths. This polychromator comprises diffraction gratings, mirrors,
lenses and visible and ultra-violet detector sets each comprising arrays of charge-coupled
devices and allows simultaneous quantification of atomic emissions in the plasma.
In this work, Perkin Elmer OPTIMA 7300 DV ICP is employed to measure the concen-
tration of silica samples (feed and permeate). Samples are fed through an S10 auto sampler
to an argon plasma (15 L/min gas flow) via a nebuliser at a gas flowrate of 0.7 L/min. In
order to find out the instrument detection limit, three replicates are run of calibrated blank
and the detection limit is normally three times the standard deviation of 3 replicates. The
software records the values diligently which gives a ready to use concentration values.
3.2.2 Particle morphology studies by transmission electron mi-
croscopy
The transmission electron microscope (TEM), JOEL 1400 with 100 keV accelerating
voltage is used for the image analysis. This imaging study is mainly conducted to see the
morphology of silica particles. The silica sample is prepared by depositing silica nanopar-
ticles on 300 mesh copper grids and left for drying. The dried samples are then mounted
on the sample holder of the TEM to view the images. The TEM images clearly confirm the
silica particles are mostly spherical in nature (Figs. 3.3, 3.4, 3.5) which makes it suitable
for particle size study through light scattering technique.
Figure 3.3: TEM Micrograph of small silica particles at x200K magnification.

Figure 3.4: TEM Micrograph of medium silica particles at x100K magnification.
Figure 3.5: TEM Micrograph of big silica particles at x200K magnification.
technique
Dynamic light scattering - Theory
The dynamic light scattering method is popularly employed to estimate particle size
and its distribution [Berne and Pecora, 1976]. It is also known as photon correlation spec-
troscopy and quasi-elastic light scattering, is non-invasive, highly sensitive and requires
very little sample volume (≤ 400 μl). In this technique, the fluctuations in monochromatic
light scattered from an ensemble of Brownian particles are measured with time. This fluc-
tuation contains information about the dynamics of the scattering particles. The angular
dependence of the scattering intensity arises from different positions on the same particle
and if the particle is large enough to accommodate multiple photon scattering, it is known
as Mie scattering. If the angular dependence of the scattering intensity is lost which hap-
pens when the particle size is smaller than the wavelength of the incident light, multiple
photon scattering is avoided and is known as Rayleigh scattering. Here, the scattered light
intensity I(t) at a time t and I(t+τ) that at a time t+τ are measured. The similarity be-
tween two signals over a period of time is measured by autocorrelator. The autocorrelation
function C(τ) (Equations 3.2 & 3.3), and the normalized autocorrelation function, g’(τ)
(Equation 3.4), are calculated as follows:
C(τ) =< I(t) · I(t + τ) > (3.2)
C(τ) = Ae−2Γ + B (3.3)
C(τ) − C(∞)
g (τ) = (3.4)
C(0) − C(∞)
where Γ is the decay rate or relaxation rate (Equation 3.5) which is due to the transla-
tional, diffusive motion of the particles, D is the diffusion coefficient and q the scattering
wave vector.
Γ = Dq2 (3.5)
The scattering wave factor is given by the Equation 3.6:
q = 4πn/λo sin(θ/2) (3.6)
λo is the wavelength of the incident light, θ is the scattering angle, n is the refractive
index of the solution. Thus if the fluctuation is measured and correlated, the diffusion
coefficient and the particle size can be obtained from the fitted parameter. In the case of
mono-dispersed particles, it is observed that the field-field autocorrelation function (given
in Equation 3.3) decays exponentially with a decay rate proportional to the diffusion coef-
ficient of the particles [Boschel et al., 2003; Frisken, 2001; Patty and Frisken, 2006] and
it produces its own unique autocorrelation function i.e. single exponential decay. In the
case of poly-disperse systems, the autocorrelation function is described by a distribution
function.
∞

g (τ) = G(Γ)exp(−Γτ)dΓ (3.7)
0
where G(Γ) is the normalized distribution function.
The diffusion coefficient depends on the inter-particle interaction, the viscosity (η) of
the solvent and temperature of the solution respectively. The effective diameter (hydrody-
namic or stokes diameter), d, and its speed due to Brownian motion can be simplified to
the Stokes-Einstein relation as follows:
kB T
d= (3.8)
3πηDo
where Do is the free particle diffusion coefficient which is obtained by linear extrapo-
lation of the D values of the diluted samples to zero concentration and kB is Boltzmann’s
constant.
Polydispersity index (PDI) is another useful measure which is obtained from the DLS
data. It is defined as the width of the decay rate and is obtained from Equation 3.9:
PDI = μ2 /Γ2 (3.9)
where μ2 is the second moment and Γ, the decay or relaxation rate, depends on diffusion
coefficient and scattering wave vector (Equation 3.5). A PDI of 1 indicates large variations
in particle size and a reported value of 0 means that there is no variation in size. Thus,
this analysis yields two important values - a mean value for the size and a width parameter
known as the polydispersity index. Apart from the effective diameter and the polydispersity
index, the dynamic light scattering software uses algorithms to extract the decay rates for a
number of size classes to produce a size distribution. The basic measurement thus obtained
is intensity and all other distributions are generated from this using Mie theory.
In essence, the sample materials scatter incoming laser light. Due to the random motion
of these particles, the scattered light intensity fluctuates in time. Processing the fluctuating
signal with a state-of-the-art digital auto-correlator yields the particles diffusion coefficient,
from which the equivalent spherical particle size is calculated using the Stokes-Einstein
equation (Equation 3.8).
Instrumentation
A typical dynamic light scattering (DLS) system comprises six main components as
shown in Fig. 3.6. A laser is used to provide a light source to illuminate the sample
particles within a cell. Most of the laser beam passes straight through the sample, but
some are scattered by the particles within the sample. A photodiode detector or photo
multiplier is used to measure the intensity of the scattered light. As a particle scatters light
in all directions, theoretically its possible to place the detector in any position to detect
the scattering. The intensity of the scattered light must be within a specific range for the
detector to successfully measure it. If too much light is detected then the detector will
become overloaded. To overcome this, an attenuator is used to reduce the intensity of the
laser and hence reduce the intensity of the detected light. In the case of smaller particles or
low concentration, the amount of light striking the detector should be maximized so that
the attenuator will allow more laser light through to the sample. For samples that scatter
more light i.e. larger particles or higher concentration, the amount of light striking the
detector must be decreased. The scattering intensity signal for the detector is passed to a
digital signal processing board called a correlator. The correlator compares the scattering
intensity at successive time intervals to derive the rate at which the intensity is varying.
This correlator information is then passed to a computer where the software analyzes the
data and derive size information.
Figure 3.6: Schematic diagram depicting the main components in a typical dynamic light
scattering setup.
3.3. Concluding Remarks 80
Particle size measurement by dynamic light scattering using Brookhaven particle
sizer
Brookhaven (BIC) 90Plus particle sizer is employed to measure the particle size with a
scattering angle of 90o , laser wavelength of 635 nm and the intensity at 35 mW, TurboCorr
digital autocorrelator. The particle shape was consistently spherical (from TEM images)
and hence the laser diffraction method response can be regarded as reliable. The particle
sizing software generates results in terms of the intensity, number, area and volume size
distributions along with derived statistics namely mean size and PDI. In this work, the data
presented throughout with respect to the particle diameter (average) has a standard error of
less than 3% of the mean of three repeated runs which indicates excellent repeatability.
3.3 Concluding Remarks

This chapter outlined the cross flow experimental setup as well as the silica material
being employed as model virus particles for the experimental investigation. The methods
employed to analyse the results were discussed which included dynamic light scattering
technique for particle size analysis, transmission electron microscopy for particle morpho-
logical and inductively coupled plasma technique for particle concentration. Results and
discussions will be presented in Chapter 4.
Bibliography
Berne, B., Pecora, R., 1976. Dynamic Light Scattering with applications to chemistry,
biology and physics. Wiley. 75
Boschel, D., Janich, M., Roggendorf, H., 2003. Size distribution of colloidal silica in
Bibliography 81
sodium silicate solutions investigated by dynamic light scattering and viscosity mea-
surement. Journal of Colloid and Interface Science 267, 360–368. 77
Frisken, B. J., 2001. Revisiting the method of cumulants for the analysis of dynamic light
scattering data. Applied Optics 40 No.:24, 4087–4091. 77
Patty, P., Frisken, B., 2006. Direct determination of the number weighted mean radius and
polydispersity from dynamic light scattering data. Applied Optics 45 no.10, 2209–2216.
77
Chapter 4
Influence of Particle Size and Size
Distribution on Filtration Efficiency and
Separation Mechanism
Contents
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.3 Cross-Flow Filtration Experimental Details . . . . . . . . . . . . . . . . 89
4.4 Results and Discussion: Filtration Efficiency Study . . . . . . . . . . . . 89
4.4.1 Experimental design and surface response modeling . . . . . . . . 90
4.4.2 Pearson’s correlation analysis . . . . . . . . . . . . . . . . . . . . 92
4.4.3 Model prediction results . . . . . . . . . . . . . . . . . . . . . . . 95
4.4.4 Effect of process parameters and input condition on filtration efficiency 98
4.4.4.1 Effect of TMP on filtration efficiency . . . . . . . . . . . 98
4.4.4.2 Effect of CFR on filtration efficiency . . . . . . . . . . . 99
4.4.4.3 Effect of particle and pore size on filtration efficiency . . 101

83
4.4.5 Concluding remarks for filtration efficiency study . . . . . . . . . . 105
4.5 Results and Discussion - Influence of Particle Size and Size Distribution
on Filtration Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.5.1 Study of particle size characterization based on particle concentration 109
4.5.2 Experimental design and statistical analysis . . . . . . . . . . . . . 112
4.5.3 Model prediction results . . . . . . . . . . . . . . . . . . . . . . . 116
4.5.3.1 Effect of TMP on permeate particle size and polydisper-
sity index . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.5.3.2 Effect of CFR on permeate particle size and polydispersity
index . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.5.4 Particle size distribution (PSD) analysis . . . . . . . . . . . . . . . 119
4.5.4.1 Temporal effect . . . . . . . . . . . . . . . . . . . . . . 119
4.5.4.2 Effect of TMP on particle size distribution in permeate
streams . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.5.4.3 Effect of CFR on particle size distribution in permeate
streams . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.5.4.4 Modeling particle size distribution . . . . . . . . . . . . 125
4.5.4.5 Size polydispersity index and its effects . . . . . . . . . . 126
4.5.5 Concluding remarks for particle size and distribution investigation
section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
84
Preface
There is limited work which analyzes and quantifies the effects of operating parameters
and particle size on final efficiency in general filtration operations, and this lack of work
is specifically more pronounced in tangential flow virus filtration. The effects of feed par-
ticle size, distribution and polydispersity are discussed to investigate this influence on the
filtration process and product property, aiming at filling the knowledge gap. This chapter
is divided into two sections considering the above key discussions, i.e. filtration efficiency
and particle size effects.
Research Questions:
1. How critical are the operating parameters and the allied input conditions in affecting
the filtration efficiency?
2. What is the role of cross interaction of the process parameters and their influence on
the filtration efficiency?
3. Could an empirical model be developed to quantify filtration efficiency via a mathe-
matical relation that takes into account the operating parameters?
The above questions necessitate the investigation of the following which is covered
under the Section 4.5 of this chapter.
1. What is the significance of feed particle size along with the operating parameters on
the permeate particle size and thereby the separation/fractionation?
2. What is the role of polydispersity index of feed on the permeate quality?

Section 4.4 of this chapter has been presented in International Water Association -
Membrane Technology Conference, Beijing, 2009 [vii] and also published in Water Science
and Technology: Water Supply [ii]. Section 4.5 of this chapter is included in manuscript
submitted to (Biotechnology and Bioprocess Engineering [iii]).
4.1 Introduction
Water and wastewater treatment operations must strictly meet various standards to-
wards microbiological, toxicological, organoleptic, and operational regulatory limits. Spe-
cial attention is paid to the removal of microorganisms (bacteria, viruses, protozoa, cysts)
apart from organic and inorganic colloids, for a hygienically safe and microbiologically
stable drinking water [Graveland, 1998; Kitis et al., 2003; Lenes et al., 2010]. Specifi-
cally, virus removal is a critical operation as quantitative risk assessment has become a
mandatory step to project expected health risks of water treatment and distribution sys-
tems. There are various conventional methods employed for virus removal, but the use
of membrane filtration technology is observing a tremendous growth. This is due to the
concern over the quality of drinking water, regulatory pressure, the rapidly declining costs
of membrane systems, and consumer pressures for non-chemical treatment [Bodzek and
Konieczny, 1998; Laine et al., 2000; Pearce, 2007].
UF is recognized by the water industry as a very attractive technology for producing
drinking water especially since it is applicable to treating water sources that are of lower
quality, like ground water [Adham et al., 1998; Arnal et al., 2009]. The removal of virus
particles through UF has proven to be effective as evidenced by the wide acceptance of this
technology in the water industry. Several parameters influence the UF operation includ-
ing the membrane modules and materials, the operating conditions as well as parameters
associated with the water source being treated. For example, tangential flow mode maybe
preferred for dilute feed where in the concentration polarization and cake formation are
expected to be negligible. As the virus particles range from parvovirus (18-24 nm), re-
ovirus (60-80 nm) to pseudorabies virus (120-200 nm) and can be either enveloped or
non-enveloped, the removal efficiency of virus particles is largely influenced by the virus
types as well. Though knowledge of virus particle behaviour in filtration is growing con-
tinuously, the passage/retention of such particles across a membrane is still not completely
understood [Chang et al., 1996, 2008].
Various studies have demonstrated how UF can be efficient in virus removal, with
removal efficiency ranging from 1.5 to 10 log reduction value (LRV) through various
membranes. For example, complete removal of poliovirus is achieved with 30 kDa UF
membranes [Madaeni et al., 1995] whereas MS2 removal with 100 kDa membrane could
achieve more than 6 log removal [Otaki et al., 1998]. In contrast, the removal of coli phage
Qβ and T4 was incomplete through a 20-40 kDa UF membranes showing 2.5 log removal
[Urase et al., 1998]. These works and others aim at just arriving at the log reduction values
but they do not address exactly the relationships between filtration efficiency and operating
parameters such as transmembrane pressure (TMP), membrane pore dimension or opera-
tion mode (dead end or cross flow modes). On the other hand, these works mainly try
to understand the underlying UF mechanisms through various models that are formulated
to describe permeate flux and fouling [Oh et al., 2007; Song and Elimelech, 1995] where
permeate flux is typically based on the different mass transfer mechanisms involved. There
still seems to be a lack of literature works that address the profound influence of virus par-
ticle size (PS) on the mobility and passage of particles across membranes and hence on the
4.2. Materials and Methods 87
final outcome of the filtered product [Han et al., 2003]. In the case of virus filtration where
the removal efficiency is measured in terms of log reduction value (LRV), it is of interest
to understand the influence of particle size along with other operating parameters on this
efficiency indicator.
We study virus removal using surrogate particles through cross flow UF aiming primar-
ily at understanding how model virus particle size influences the LRV, under the Section
4.4 of this chapter. We also examine the interaction of particle size effects with other vari-
able effects like those of TMP and feed flowrate (or otherwise termed cross flowrate in
cross flow mode). A systematic experimental programme based on statistical surface re-
sponse modelling approach is conducted. Two types of viruses are targeted by the model
particles, norovirus and enteric adenovirus. Norovirus has a particle size range of 27-38
nm and is the most common ecological agent for gastroenteritis outbreaks in all ages and
has significant public health impact worldwide. Enteric adenovirus falls in the range of
70-100 nm and is usually detected in the summer seasons.
4.2 Materials and Methods

A schematic diagram of the laboratory scale model of tangential flow filtration unit
used is shown in Fig. 4.1. The feed solution is held in a 10 litres tank and is fed into
the inlet of the membrane module by a peristaltic pump for which the speed can be ad-
justed and the feed suspension passing through the membrane is collected separately as
permeate and the particles which fail to pass through is collected as retentate. From the
pressure transducers, the pressure values are recorded and the transmembrane pressures
are evaluated using Equation 2.4. In this work, there are two response factors measured
i.e. particle diameter and particle concentration. With respect to particle diameter, the data
4.2. Materials and Methods 88
presented throughout corresponds to the average diameter. This is obtained by calculat-
ing the mean of three repeated runs and it is found that the standard error is less than 3%
of the mean of three repeated runs. This indicates excellent reproducibility. With regard
to the concentration measurement (for LRV calculation), the instrument detection limit is
three times the standard deviation of three replicates of calibration blank which is less than
1% demonstrating the reliability of the concentration data. As far as experimental data,
initially number of experiments was performed to identify the range of transmembrane
pressure, and cross flow rate in order to obtain reasonable permeate data. During these ini-
tial runs, experiments were run to check the repeatability and were found that the variation
in the data was less than 3% which demonstrated the repeatability.
Figure 4.1: Schematic diagram of the tangential flow filtration experimental setup.
Silica particles used for this study are procured from Nissan Chemicals, US and the
properties are summarized in Table 3.1 of Chapter 3. The particles obtained are used as
purchased condition. Three different particle size ranges are used for this study: smaller
4.3. Cross-Flow Filtration Experimental Details 89
particles (20-50 nm), medium particles (70-100 nm), and bigger particles (130-140 nm).
4.3 Cross-Flow Filtration Experimental Details

It is common to find UF operations working in the cross flow mode (alternatively
termed tangential flow). This mode of operation is presented in Fig. 4.2 which depicts
the mobility, across the membrane, of particles either permeating through the membrane
or getting retained and being convectively collected as retentate.
Figure 4.2: Cross flow filtration: Sample flowing parallel to the membrane surface.
Cross flow filtration has received attention for its fouling reduction capabilities in re-
moving molecules or particles from the membrane surface by the tangential flow of liquid.
There are mainly three related (input) variables that play an important role in the effective-
ness of filtration namely transmembrane pressure, cross flow, and feed particle size. Key
performance (output) variable considered for this investigation is the log reduction value
which is discussed in the next section.
4.4 Results and Discussion: Filtration Efficiency Study

Various terms have been used to represent the microbial removal efficiency of mem-
brane filters. Initially, Beta ratio was the term used to describe the retention of organisms
from hydraulic fluids of fine particles [Leahy and Sullivan, 1978; Reti, 1977]. Subse-
quently, the term titer reduction T R - the ratio of particles in the influent stream to those in
the effluent, the class definition of filter efficiency was used in membrane filtration [Pall and
4.4. Results and Discussion: Filtration Efficiency Study 90
Kimbauer, 1978]. Currently, the term in common use is log reduction value (LRV) adapted
by Health Industry Manufacturing Association (HIMA) to standardize the terminology.
The LRV is the logarithm to the base of 10 of the ratio of the organisms in the influent
stream to those that emerge in the filtrate/permeate. In order to determine the LRV, it is rec-
ommended that the original influent stream along with the input conditions are chosen such
that it causes some passage of organisms through the filtration system for investigational
purposes. Whereas, few applications, like water purification industry and biotechnology
industry, demand the undesirable particles to be minimum or close to zilch in the permeate
stream. So, these indicators are typically dependent on the type of application, operation
(removal or concentrating), and meeting respective regulatory requirements, Thus to sum-
marize, the efficiency is best described in the filtration system as log reduction value (LRV)
or alternatively as filtration efficiency. Thus LRV being a direct indicator of virus filtration
is defined as:
Cf
LRV = log10 (4.1)
Cp
where C f and C p are the concentrations of the feed and permeate streams respectively.
This equation suggests that for higher LRV values corresponding to higher removal effi-
ciencies, it is desired to have the value of C p much smaller than C f . This term, LRV or
filtration efficiency, is constantly recalled at various sections in the whole of this thesis.
4.4.1 Experimental design and surface response modeling

The experimental design used for this regression modeling of cross flow UF of silica
particles was carried out using three factors viz. transmembrane pressure (TMP), cross
flowrate (CFR) (process variables) and the particle size (FPS) of the feed stream (input
condition). These three factors signify the independent variables that are varied in the
experiments. Statistical experimental design is used in this investigation to map the system
and thus approximate the filtration efficiency by a quadratic model. Such model allows us
to (a) analyse and understand in more detail how the varied factors influence the response
and (b) make predictions of filtration efficiency under different operating conditions. A full
factorial three level experimental design is used to analyse the data. The range of values
actually used for experimentation and their respective coded values are shown in Table 4.1.
The experimental LRVs obtained are also listed in Table 4.1.
Table 4.1: Full Factorial, 3 level experimental design for cross flow UF of silica particles
using 100 kDa MWCO membrane (b : coded variables).
Sample ID Factors (input variables) Response
Transmembrane Pressure Cross flowrate Particle Size
TMP, Levelb CFR, Levelb FPS, nm Levelb LRV
kPa X1 L/min X2 X3
1 20 -1 1 1 Small -1 2.26
2 20 -1 1 1 Medium 0 2.6
3 20 -1 1 1 Big 1 2.71
4 40 0 1 1 Small -1 2.16
5 40 0 1 1 Medium 0 2.41
6 40 0 1 1 Big 1 2.58
7 60 1 1 1 Small -1 1.63
8 60 1 1 1 Medium 0 2.34
9 60 1 1 1 Big 1 2.41
10 20 -1 0.6 0 Small -1 2.24
11 20 -1 0.6 0 Medium 0 2.56
12 20 -1 0.6 0 Big 1 2.66
13 40 0 0.6 0 Small -1 2.12
14 40 0 0.6 0 Medium 0 2.35
15 40 0 0.6 0 Big 1 2.54
16 60 1 0.6 0 Small -1 1.51
17 60 1 0.6 0 Medium 0 2.19
18 60 1 0.6 0 Big 1 2.02
19 20 -1 0.3 -1 Small -1 2.18
20 20 -1 0.3 -1 Medium 0 2.26
21 20 -1 0.3 -1 Big 1 2.4
22 40 0 0.3 -1 Small -1 2.14
23 40 0 0.3 -1 Medium 0 2.2
24 40 0 0.3 -1 Big 1 2.4
25 60 1 0.3 -1 Small -1 1.44
26 60 1 0.3 -1 Medium 0 1.91
27 60 1 0.3 -1 Big 1 1.98
In the surface response modeling exercise of this section, only one response is con-
sidered that being the filtration efficiency i.e. LRV. A multilinear regression model was
fitted to the data considering the three independent variables and the one dependent re-
sponse. This model matched the experimental data well compared to other regression
models. Also, ANOVA analysis indicates that the standard error of estimate is smaller
than the response values and the model predictions are normally distributed, hence this
model is chosen. Thus, the resulting empirical model after eliminating insignificant terms
is given by the Equation 4.2. The equation indicates that X1 (TMP) has a negative influ-
ence whereas X2 and X3 (CFR and FPS) have a positive influence on LRV. It also indicates
that the cross interaction between TMP and FPS significantly affects LRV.
LRV = 2.42 − 0.247X1 + 0.122X2 + 0.223X3 + 0.062X1X3 − 0.139X12 − 0.126X32 (4.2)
4.4.2 Pearson’s correlation analysis

Pearson’s correlation is employed to arrive at the relationship of the independent vari-
ables on LRV (Table 4.2); here increase in transmembrane pressure results in decrease in
LRV (negative correlation: -0.629) whereas increases in the other two factors i.e. cross
flowrate (positive correlation: 0.310) and particle size (positive correlation: 0.569), shows
to have positive influence on the filtration efficiency. The Pearson correlation suggests
that the cross flowrate has a relatively less effect on the LRV (significance value is 0.116)
whereas the TMP and FPS (significance values are less than 0.05) play a significant role
on the control of the filtration efficiency.
The analysis of variance (ANOVA) (Table 4.3) indicates the significance of the multi-
Table 4.2: Pearson’s correlation analysis.

Coefficient Significance
LRV 1 -
TMP -0.629 0
CFR 0.31 0.116
FPS 0.569 0.002
T MP ∗ CFR 0.055 0.783
CFR ∗ FPS 0.109 0.588
T MP ∗ FPS 0.128 0.524
T MP2 -0.204 0.307
CFR2 -0.03 0.881
FPS 2 -0.185 0.356
TMP*CFR*FPS 0.002 0.992
ple regression model. The mean square for the regression model in comparison with the
residual demonstrates that the regression model is highly significant and also supported by
the F test value (19.251) at P=0 (i.e. P is less than 0.001). The summary section gives
the coefficient of determination (R2 = 0.923) which indicates that 92.3% of the variation in
LRV is explained by variation in the independent variables (TMP, CFR, FPS), and the R
value (0.961) indicates a strong correlation between LRV and the factors. The mean value
of LRV (2.23) is much larger than the standard error of estimate (0.115) which clearly
defines the suitability of the multiple regression model for the variance of LRV, within the
tested independent variable ranges.
Table 4.3: Analysis of variance summary.

ANOVA
Source Sum Sq. D.F. Mean Sq. F Prob.
Regression 2.56 10 0.256 19.251 0
Residual 0.213 16 0.013
Total 2.772 26
Summary
R2 R Adj. R2 Standard Error of Estimate
0.923 0.961 0.875 0.115
The quadratic regression model developed based on the experimental design data is
given in Equation 4.2. Through student’s t test, three other terms representing the TMP
and FPS quadratic terms and the TMP-FPS interaction term were, besides the three inde-
Figure 4.3: Log reduction values: predicted vs experimental.
Figure 4.4: Rankit Plot showing the residuals are normally distributed.
pendent factors, identified to be significant.
The model is validated statistically as the predicted R2 (0.923) (shown in parity plot of
Fig. 4.3) is in agreement with the adjusted coefficient of determination (0.875). The Rankit
plot (Fig. 4.4) indicates that the residuals are normally distributed and therefore very low
probability of non-conformance suggesting only minor departure of the residuals from the
normal.
4.4.3 Model prediction results

The model (Equation 4.2) developed based on the surface response method demon-
strates the effect of TMP, CFR and FPS on the filtration efficiency. The model is used to
simulate under different TMP and CFR conditions of 20-60 kPa and 0.3-1.0 L/min range.
Figs. 4.5, 4.6 & 4.7 are shown to demonstrate the model predictions for varying CFR at all
three TMPs.
Figure 4.5: Model prediction showing the effect of cross flowrate at 20 kPa on LRV.
The trend of the predicted plots clearly indicates that model agrees well with experi-
mental results (Fig. 4.8). For comparison purposes, the plots are shown at 0.6 L/min cross
flowrate and for varying TMP. Specifically, it is noted that smaller and bigger particles
agree very well but in the case of medium particles deviation is observed nevertheless it
differs by 10% which is well within the acceptable range. From the statistical point of
view, all statistical estimators (i.e. R2 is in agreement with adj. R2 , F has high value, Prob.
has low value) in our empirical model demonstrate the model’s validity. At the same time,
it is important to note that the standard error of estimate is 0.115 which has led to the
slight deviation. Also, in the model only the interaction effects of significant terms have
been retained (i.e. TMP and FPS) while ignoring the interaction effects between the other
factors (i.e. TMP and CFR, CFR and FPS) according to the results of Pearson’s correlation
analysis. This may also be a reason for the slight deviation. Thus, this model could well
be employed to predict the filtration efficiency for a given set of operating conditions in
the studied experimental range.
Figure 4.8: Model prediction vs experimental trend for varying TMP at 0.6 L/min.
4.4.4 Effect of process parameters and input condition on filtration
efficiency
The present study as mentioned earlier is initiated to investigate the relationship be-
tween selected parameters (TMP, CFR, FPS) and filtration efficiency. The reason behind
the variation of filtration efficiency is due to the partial permeation of particles with UF
membranes. It can be explained based on the following reasons: (i) literature indicates that
UF membranes come with their specified pore size range but normally it does not include
the presence of abnormally large pores [Amirilargani et al., 2009; Sun et al., 2007; Urase
et al., 1998]. At the same time, pore size distribution parameters (like pore size, standard
deviation) actually vary depending upon the zones viz. zone of long tail of big pores, zone
of standard linearization of pore size distribution, and zone of sieving effect. (ii) one other
possible explanation is due to lack of membrane integrity due to the presence of pinholes
[Kitis et al., 2003]. Thus, the interaction between the varying particle sizes and the pore
size is a big challenge in arriving at the final filtration efficiency values. Hence, initiating
a study on the effect of the particle size alongwith the process parameters will be a big
step towards a better understanding of filtration efficiency mechanism. Incorporating the
membrane pore variations and its uncertainties will certainly be a complete compliment in
knowing the separation mechanism but which is not covered in this thesis.
4.4.4.1 Effect of TMP on filtration efficiency
The experimental results of tangential flow UF of silica samples indicate that the UF
polyethersulfone membranes form an efficient or selective barrier depending upon the par-
ticle size and varying operating parameters. Runs were carried out at different TMPs (20,
40, 60 kPa). Increasing TMP results in a reduction in removal efficiency. It is likely that
the higher TMP enhances the membrane pore elongation and thereby allowing more par-
ticles to pass through the membrane thereby decreasing the LRV (Figs. 4.9, 4.10, 4.11 &
4.12).
Figure 4.9: Experimental results showing the effect of TMP at 0.3 L/min on LRV (lines
drawn to indicate trends).
4.4.4.2 Effect of CFR on filtration efficiency
Similarly, runs were carried out at three cross flowrates (0.3, 0.6, 1 L/min). Increasing
the feed flowrates was found to increase the filtration efficiency (Fig. 4.13). It is also
important to note that CFR has less influence on the filtration efficiency than TMP and
FPS. This effect could be attributed to the fact that at higher feed flowrate, the residence
time of particles across the surface of the membrane is reduced limiting the probability
of particle permeation and hence reducing the concentration of particles in the permeate
stream. The improvement in LRV is observed at lower TMP and higher flowrates which
indicates the importance of selection of optimal TMP and flowrate values to achieve better
Figure 4.10: Experimental results showing the effect of TMP at 0.6 L/min on LRV (lines
Figure 4.11: Experimental results showing the effect of TMP at 1 L/min on LRV (lines
Figure 4.12: Model prediction showing the effect of TMP at 0.6 L/min on LRV.
efficiency.
4.4.4.3 Effect of particle and pore size on filtration efficiency
The UF membrane is characterized by the presence of numerous pores of varying sizes
and the final permeate quality is greatly influenced by the physical dimensions of the pore
and the particle size. In UF membranes its conventional to use MWCO wherein the par-
ticles that have molecular weights lesser than the rated membrane pore molecular weight
cutoff (MWCO) point (or dimensions smaller than the pore diameter) is expected to pass
through or fractionate which highly influences the LRV/filtration efficiency.
For a better insight, it is necessary to use a model that relates the filtration efficiency
and particle to pore size (MWCO in the case of UF) ratio. Typically, Ferry-Faxen equa-
tion (Equation 4.3) describes the theoretical relation between the rejection coefficient and
particle diameter to pore rating [Chao and Tojo, 1987; Torras et al., 2006] and is given as:
Figure 4.13: Experimental results showing the effect of feedflow rate at 40 kPa on LRV
(lines drawn to indicate trends).
1−R = (1−PPS R)2 [1−0.104PPS R−5.21PPS R2 +4.19PPS R3 +4.18PPS R4 −3.04PPS R5 ]
(4.3)
where R is the retention coefficient and PPSR is the particle-to-pore size ratio. Combining
the definition of R and LRV, we get

1
LRV = log10 (4.4)
1−R
For comparison studies, Equation 4.3 is modified to suit our study by introducing the
above relation (Equation 4.4). However, it is important to note that Ferry-Faxen model
describes the retention characteristics considering only the particle and pore radius but fails
to include the operating parameters which have a great deal of influence on the retention
mechanism. Hence, Ferry Faxen model though qualitatively agrees with our experimental
results, but quantitatively deviates (Fig. 4.14).
Figure 4.14: A comparison of experimental results (points joined by dotted lines to indicate
the trend) with our model and Ferry-Faxen model.
This necessitates to extend the model to incorporate the effect of TMP and CFR apart
from PPSR on the final filtration efficiency. Our data and statistical investigation have
clearly demonstrated that LRV depends not only on the feed particle size but also on TMP
and CFR as well which has already been discussed. Hence, here we have developed an
empirical model to describe the relationship between log reduction value and the operating
parameters (TMP and CFR) along with the PPSR (Equation 4.5). A multi-linear regression
model thus obtained is based on our experimental results and is given as:
LRV = 2.15 − 0.01T MP + 0.34CFR + 0.67PPS R (4.5)
Thus, in order to validate the above model, a lower MWCO (30,000 Da) membrane
was used in a further test to assess the level of improvement in particle removal. Fil-
tration efficiencies for the two membranes are shown in Table 4.4 for the three different
particle size ranges (small, medium, big) and in Fig. 4.15 as a function of the particle-to-
pore-size ratio (PPSR). For a given membrane, the data shows that LRV increases as the
particle size range being filtered increases. This is expected since UF is a size-exclusion
particle removal process where the membrane would reject the particles more efficiently
if the particles are bigger in size. The interaction between particles and the membrane is
demonstrated in Fig. 4.15 showing the filtration efficiency behavior with PPSR. This figure
clearly shows the improvement in LRV (hence increased particle removal) brought about
by the 30 kDa MWCO membrane, but more significantly, it shows continuity across the
two membranes in the LRV trend under increasing PPSR; both membranes observing very
similar LRV values (2.8 and 2.71) at a PPSR near 0.84. Here we note the value in report-
ing LRV data against PPSR in helping to compare membranes of different pore sizes. It
is also important to note the observed deviation between our model and the experimental
data showing that at higher PPSR values, the interaction between particles and pores are
said to be statistically insignificant but which the model is incapable of defining, hence the
deviation.
Table 4.4: Efficiencies (LRV) for the two MWCO membranes against particles of different
size ranges. Data for lower TMP (20 kPa) and higher flowrate (1 L/min).
Membrane pore size, Da Particle size category LRV

30,000 MWCO Smaller Particles 2.8
Medium Particles 4.1
Bigger Particles 4.6
100,000 MWCO Smaller Particles 2.26
Medium Particles 2.6
Bigger Particles 2.71
From the above study, it drives to an insight that the blocking of pores is statistically
insignificant if PPSR is greater than 1. Thereby, bigger particles efficiently being rejected
Figure 4.15: Efficiencies (LRV) for the two MWCO membranes as a function of the PPSR.
Data for lower TMP (20 kPa) and higher flowrate (1 L/min).
which increases the LRV. Thus, this study is a significant leap proposing a predictive em-
pirical model (Equation 4.5) to achieve a target LRV value for a given TMP and CFR with
a known virus particle size range.
4.4.5 Concluding remarks for filtration efficiency study

The model results (Figs. 4.12 & 4.6) and the experimental results (Figs. 4.10 & 4.13)
indicated that particle size, besides the operating parameters of TMP and flowrate, has a
great deal of influence on surrogate virus particle removal in UF. The proper selection of
operating parameters coupled with proper membrane selection will result in better rejec-
tion and/or separation. Having identified the effect of particle size as a more prominent
factor in the filtration process, a detailed investigation on the feed particle size on the per-
meate quality was carried out which is discussed in the next section. This investigation
showed that among the two operating parameters considered, TMP plays a major role in
controlling the filtration efficiency in comparison with flowrate. In the studied experimen-
tal range, higher LRV values were obtained at lower TMP (20 kPa) and at higher feed
flowrate (1 L/min). Further, the effect on LRV of the interaction between TMP and FPS
seems to be more significant than that of the interaction of flowrate with FPS. The empir-
ical statistical model developed serves as a powerful tool for studying the impact of the
key operating parameters on the permeate quality (Figs. 4.9 & 4.13) and can be employed
for optimisation of operating parameters to achieve maximum efficiency. The above find-
ings are hereby summarized thus answering the research questions posed at the start of the
chapter:
• The feed particle size, transmembrane pressure and cross flowrate have a significant
role in the final filtration efficiency. Among the above three parameters considered,
FPS and TMP are seen to be major key factors (with very high significance based
on the Pearsons correlation analysis) with CFR’s significance being relatively less.
It is very insightful to recognise that the interplay of parameters plays an equally
important role as well. It has been identified that interplay of TMP and FPS are
more influential than TMP and CFR or CFR and FPS. This necessitated a further
investigation on the feed particle size on the separation or fractionation mechanism.
This is presented in Section 4.5
• The effect of above parameters were formulated in an empirical model development.
The model results obtained were compared with the experimental results and found
to be in excellent agreement. Hence, the model obtained from this study can be
employed to predict the filtration efficiency given the TMP and CFR and the feed
particle property within the studied experimental range.

4.5. Results and Discussion - Influence of Particle Size and Size Distribution on
Filtration Mechanism 107
• The above emprirical modeling study demonstrated the dependencies of these pa-
rameters on the filtration efficiency and this serves as an excellent approach/method
to analyse the effects of these parameters on product efficiency for filtration pro-
cesses in general and in the context of virus UF in specific. Moreover, this approach
can be applied for scaled-up processes.
4.5 Results and Discussion - Influence of Particle Size and
Size Distribution on Filtration Mechanism

The efficiency of tangential flow filtration is highly influenced by operating parameters
and the feed particle size as has been demonstrated in the study presented in the previ-
ous section (i.e. Section 4.4). In this section, particle size and distribution are primarily
considered and their effects along with the effects of the operating parameters on the per-
meate particle size are investigated. Model virus particles (silica particles) employed for
this study fall in a similar range of sizes mimicing viruses including reovirus, pseudorabies
virus, adenovirus, herpes simplex viruses, and murine retro viruses. Thus, the use of these
model particles is to give a generic validation for virus separation by UF.
Other than application in the water industry, UF membranes are targeted for purifica-
tion of biological solutions wherein they are employed to fractionate proteins with similar
sizes, achieving almost a complete separation/rejection of virus particles or a particular
protein [Lightfoot and Moscariello, 2004; Saxena et al., 2009]. Hence, it is considered to
be a complex mechanism and a combination of fractionation (i.e. similar sized particles
getting separated) and/or rejection (smaller particles being separated from bigger ones).
Previously, it was assumed to be difficult to separate particles if they differ by less than
an order of magnitude of similar sizes. Whereas recent studies clearly indicate that by
carefully choosing the operating conditions, suitable mechanism (i.e. protein fractionation
or viral clearance) could be achieved [van de Ven et al., 2009; van Reis et al., 1997]. For
example, fractionation of proteins is efficiently achieved by carefully selecting the ionic
strength and pH [Arkhangelsky et al., 2008; Saksena and Zydney, 1994]. Similarly, elec-
trostatic and electrokinetic interactions have been shown to have a great deal of influence
on the fractionation mechanism of proteins up to 100 fold [Pujar and Zydney, 1994]. As
far as the authors know, very limited literature is available to explain or understand frac-
tionation and separation mechanism considering virus particle size and distribution as well
as virus particle interactions with the UF operating parameters. Specifically, the study with
respect to the filtrate stream is still lacking. Hence, it is very vital to bridge the knowledge
gap in order to meet the regulation requirements of the end products [Fischer and Raasch,
1985; Lu and Hwang, 1995; Zhou et al., 2008]. Number of literatures address the flux de-
cline behavior considering particle characteristics of the feed [Bakhshayeshi and Zydney,
2008; Foley et al., 1995b; Hwang et al., 1998; Lu and Ju, 1989; Mackley and Sherman,
1992b; Saxena et al., 2009]. Whereas, the relationship between the particle properties of
feed and permeate alongwith the filtration efficiency (LRV) is still not adequately estab-
lished. Hence, the approach of correlating these factors will help to initially observe the
dependencies and then to understand the underlying mechanisms.
This section targets to cover the following objectives (1) to examine the size distri-
bution of the model virus particles: pre-filtration and post-filtration by the dynamic light
scattering technique (2) to investigate the impact of the size distribution on the filtration
mechanism and (3) empirical model development for permeate particle size (PS p ) and
polydispersity index (PDIP ) which aids in identifying specific dependencies of the UF
efficiency on feed particle size as well as on operational conditions.
4.5.1 Study of particle size characterization based on particle concen-
tration
Initially, a careful study is conducted with the silica samples (recall Table 3.1) to es-
tablish the most appropriate concentration for the measurement of particle size and the
distribution of the pre-filtration and post-filtration samples. According to the manufac-
turer’s specification (Brookhaven (BIC) 90 Plus Particle Sizer), one of the indicators of
the quality of the data being collected is the average count rate which is a measure of the
signal intensity in kilo counts per second (kcps). The auto-correlation plot along with the
average count rate guides the analysis as they are the closest parameters to the raw/actual
data.
The particle sizing software is capable of producing results in terms of multimodal
distribution and log normal distribution (Fig. 4.16). The log normal distribution has been
widely used until the multimodal algorithm was available and lognormal distribution offers
a simplified representation. On the contrary, multimodal size distribution gives more infor-
mation like the groups of particles of varying sizes and BIC employs the Non-Negatively
Constrained Least Squares (NNLS) algorithm for data representation.
In this section, the data presented throughout refers to the effective diameter with re-
spect to the particle size and the multimodal size distribution with respect to the particle
size distribution. The standard error of the average diameter is less than 3% of the mean
of the three repeated runs which indicates excellent repeatability. The correlation function
plot immediately gives an idea about the sensitivity to the range of particles and applicabil-
Figure 4.16: Two different representation of particle size distribution of the original feed
sample: multimodal size distribution (top) and log normal distribution (bottom).
ity for the test sample (Fig. 4.17). In order to maintain the uniformity and consistency for
the analysis, the particle size distributions are presented in terms of intensity weighted dis-
tributions as it forms the basic measurement technique. The intensity weighted distribution
is defined as the intensity of light scattered by the suspension of particles with diameter
(say d) which depends on size, scattering angle, index of refraction, and wavelength.
The characterization of silica particles at various concentrations is carried out to find
out the suitability and reliability for the experimental runs. It is observed that at very high
concentrations, the instrument reported a very high count rate (of Mcps order) which indi-
cates those values are unreliable for all the three particle size ranges. From the measured
values of reliable signal intensity, it is concluded that the recommended concentration for
the measurement is to be less than 5 g/L for smaller particles, 10 g/L for bigger particles
and 50 g/L for medium particles (Fig. 4.18).

Figure 4.17: Illustration of the correlation function - Exponential decay of auto-correlation

function.
Figure 4.18: Counts per second versus concentration plot for three different particles. This
shows the range of suitable concentration below 800 kcps threshold as specified by the
BIC particle sizer.
4.5.2 Experimental design and statistical analysis

The experimental design used for this regression modeling of cross flow UF of silica
particles is carried out using three process variables viz. transmembrane pressure (TMP),
cross flowrate (CFR), feed particle size (FPS). These three factors represent the indepen-
dent variables that are controlled in the experiments. Statistical experimental design is used
in this investigation to map the system and thus approximate the responses by a regression
model. Such a model will allow us to (a) analyze and understand in more detail how the
factors influence the permeate particle size and polydispersity index, (b) make predictions
of permeate particle size and polydispersity index under different operating conditions in
the studied experimental range. The range of values actually used for experimentation and
their respective coded values are shown in Table 4.5. In this study, the responses are the
permeate particle size and polydispersity index. A multiple regression model is used to
establish an empirical relationship between the factors and the responses. The cross flow
unit is operated in the range of 20-60 kPa transmembrane pressure, with cross flowrate in
the range of 0.3-1.0 L/min for the feed particle size range 35.6-142.9nm.
In the surface response modeling exercise of this study, the resulting multiple regres-
sion model giving the relationship of permeate particle size (PS p ) and the permeate PDI
(PDI p ) with the operating parameters (TMP, CFR and FPS) after eliminating insignificant
terms are given as follows (Equations 4.6 & 4.7):
PS p = a0 + a1 T MP + a2CFR + a3 FPS + a4CFR2 + a5 FPS (4.6)

Table 4.5: Full Factorial, 3 level experimental design for cross flow UF of three particle
size ranges of silica colloid.
Factors (input parameters) Responses (Permeate stream)
Transmembrane Pressure (TMP) Cross Flowrate (CFR) Feed Particle Size (FPS) Observed
Expt. TMP, kPa Levelb X1 CFR, Levelb X2 FPS, nm Levelb X3 Dia, nm PDI
No. L/min
1 20 -1 1 1 46.5 -1 42.4 0.27
2 40 0 1 1 46.5 -1 37.7 0.22
3 60 1 1 1 46.5 -1 36.3 0.22
4 20 -1 0.6 0 46.5 -1 42.2 0.28
5 40 0 0.6 0 46.5 -1 38.6 0.24
6 60 1 0.6 0 46.5 -1 35.1 0.17
7 20 -1 0.3 -1 46.5 -1 38.5 0.22
8 40 0 0.3 -1 46.5 -1 37 0.22
9 60 1 0.3 -1 46.5 -1 29.8 0.22
10 20 -1 1 1 92.4 0 88.4 0.18
11 40 0 1 1 92.4 0 86.6 0.16
12 60 1 1 1 92.4 0 83.8 0.14
13 20 -1 0.6 0 92.4 0 86.2 0.15
14 40 0 0.6 0 92.4 0 85.9 0.14
15 60 1 0.6 0 92.4 0 82 0.13
16 20 -1 0.3 -1 92.4 0 82.9 0.10
17 40 0 0.3 -1 92.4 0 81.2 0.10
18 60 1 0.3 -1 92.4 0 76.9 0.06
19 20 -1 1 1 144.6 1 146.8 0.07
20 40 0 1 1 144.6 1 141.4 0.01
21 60 1 1 1 144.6 1 139.7 0.01
22 20 -1 0.6 0 144.6 1 143.4 0.05
23 40 0 0.6 0 144.6 1 142.4 0.03
24 60 1 0.6 0 144.6 1 139.5 0.05
25 20 -1 0.3 -1 144.6 1 142.9 0.02
26 40 0 0.3 -1 144.6 1 139.5 0.01
27 60 1 0.3 -1 144.6 1 135.7 0.002
PDI p = a0 + a1 T MP + a2CFR + a3 FPS (4.7)
The coefficients (ai ) of the regression model are listed in Table 4.6 which indicates for
permeate particle size, the resulting model is a quadratic regression model, whereas PDI
is linearly dependent on the operating parameters. The p-values in Table 4.6 also provide
a check on the significance of each of the coefficients where a coefficient with a smaller
p-value is more significant. The analysis of variance (ANOVA) test results are also shown
in Table 4.6 indicating the goodness of the model as the value of standard deviation (SD) of
regression is much larger than the standard deviation of the residuals. The sum of square
(SS) and mean squares (MS) for the regression models in comparison with the residual
demonstrates that the regression correlation is highly significant. The F ratio for permeate
particle size (6639.74) and permeate PDI (133.73) are very much higher than Fcritical at
95% confidence value (i.e. 98.88 for permeate particle size and 0.25 for permeate PDI),
thereby, confirming the significance of the regression correlation (Table 4.6).
Table 4.6: Summary of Coefficients, Analysis of variance (ANOVA).

Permeate Particle Size Permeate PDI
Coefficients Values SE p-value Values SE p-value
Constant (a0 ) 87.863 0.532 3.42*10−34 0.127 0.004 5.28*10−20
TMP (a1 ) -3.053 0.286 6.10*10−10 -0.019 0.005 0.001
CFR (a2 ) 2.148 0.286 2.23*10−07 0.016 0.005 0.004
FPS(a3 ) 51.876 0.286 4.86*10−35 -0.099 0.005 9.67*10−16
CFR*CFR(a4 ) -1.625 0.507 0.004 – – –
FPS*FPS (a5 ) 2.720 0.497 1.99*10−05 – – –
ANOVA - Permeate Particle Size
Parameters DF SS MS F SD p-value
Total Corrected 26 48920.5 1881.56 43.377
Regression 5 48889.6 9777.92 6639.74 98.883 0
Residual 21 30.925 1.473 1.214
ANOVA - Permeate PDI
Parameters DF SS MS F SD p-value
Total Corrected 26 0.202 0.008 0.088
Regression 3 0.191 0.0637 133.173 0.252 0
Residual 23 0.0110 0.001 0.0219
The model predictions under the conditions of experiments as given in the experimental
design in Table 4.5 is plotted with observed values for both the responses (Figs. 4.19 &
4.20). From these parity plots, the correlation coefficient (R2 ) of the model is determined
to be in the range of 0.95 (permeate PDI) and 0.99 (permeate particle size) indicating that
the model is suitable for representing the factor-response relationships within the range
studied.
Figure 4.19: Observed vs Predicted: Permeate Particle Size.
Based on the surface response methodology, the correlation established by the estima-
tion of coefficients (Table 4.6) leads to the following insights: It is found that TMP (p
= 6.1*10−10 ) is more significant in comparison with CFR (p = 2.23*10−07 ) for permeate
particle size whereas TMP & CFR (p = 0.005) are equally significant for permeate PDI.
Table 4.6 also demonstrates, in all cases, the standard error (SE) is small and TMP, CFR
and FPS play a significant role along with interplay of these factors on the final product
Figure 4.20: Observed vs Predicted: Permeate PDI.
permeate particle size. Whereas for permeate PDI, TMP, CFR and FPS play a crucial role
and are linearly related.
4.5.3 Model prediction results

The empirical model (Equations 4.6 & 4.7) developed based on the surface response
method is employed to predict the behavior of changes on the final permeate particle size
and PDI. The following sections discuss the model prediction at varying TMP (20-60 kPa)
and CFR (0.3-1 L/min) for the three particles considered. It is quite important to note that
the empirical model developed agrees well with the experimental results thereby clearly
becoming an efficient tool for predicting the responses.
4.5.3.1 Effect of TMP on permeate particle size and polydispersity index
From the model results, it is found that with increase in TMP, the permeate particle
size decreases and there is a decline in permeate PDI as well (Figs. 4.21 & 4.22). At
higher TMP, more driving force and hence there is likely to be more of particle permeation
through the membrane. But at the same time, because of the combination of hydrodynamic
and colloidal forces among the particles, more of bigger particles fail to pass through the
membrane. Also, mixture of particles occupies the site of membrane pores which highly
hinders the bigger particles permeation. But interestingly, particles of slightly smaller sizes
pass through the membrane with ease.
Figure 4.21: Experimental (points joined by dashed lines to indicate the trend) Vs Model
Prediction: Effect of TMP at the middle CFR (0.6 L/min) on the permeate particle size.
4.5.3.2 Effect of CFR on permeate particle size and polydispersity index
In the case of increase in CFR, both the permeate particle size and PDI increases (Figs.
4.23 & 4.24). At higher rate of flow, pore narrowing of larger pores is less thereby making
bigger particles to pass through, but because of the lesser residence time particle concen-
tration is less.
Thus, from the above model predictions, it is clearly evident that the empirical model
developed could be used as a predictive tool to estimate the permeate particle property.
Prediction: Effect of TMP at the middle CFR (0.6 L/min) on the permeate PDI.
Prediction: Effect of CFR at the middle TMP (40 kPa) on the permeate particle size.
Prediction: Effect of CFR at the middle TMP (40 kPa) on the permeate PDI.
4.5.4 Particle size distribution (PSD) analysis

The experimental results of tangential flow UF of silica particles demonstrate the mech-
anism of separation and or fractionation of particles through UF polyether sulfone mem-
branes operated at varying system parameters. Experimental runs reported here are car-
ried out in the range of 20-60 kPa transmembrane pressure (TMP) and 0.3-1 L/min cross
flowrate (CFR). In Fig. 4.25, the particle size distribution of the original silica particles
employed for this study is depicted. The effective diameters of these particles are found to
lie between 35.6 nm to 142.9 nm range with standard errors of maximum 4.2nm.
4.5.4.1 Temporal effect
During tangential flow of particles along the membrane, multiple particles arrive at the
pore entrance, so initially the flow happens instantly and consequently the particle size
distribution decreases (Figs. 4.26 & 4.27). Thus, in general, particle permeation over time
is found to shift to left, however the dynamic change is not very rapid. The particle size
Figure 4.25: Representative distribution of feed stream sample of all three particles.
distribution evolution behavior is due to the fact with time the particle blocking the pores
maybe possible but it is insignificant which is quite evident from the plots. This particle
size distribution evolution can be related and agrees well with the available literatures of
the particle size distribution evolution on the membrane surface [Belfort and Nagata, 1985;
Chang et al., 2008].
4.5.4.2 Effect of TMP on particle size distribution in permeate streams
To study the influence of transmembrane pressure on the particle permeation, bigger
particles ultrafiltered in cross flow mode at 20-60 kPa pressure range are analysed for
particle size distribution. Based on the above study, particle permeation study can be
explained as two mechanisms depending on the transmembrane pressure change (Figs.
4.28 & 4.29):
• At lower and medium transmembrane pressures (i.e. 20, 40 kPa), it is found that
the particles are more towards separation mechanism i.e. smaller particle size range
are found to be present in the permeate stream and bigger particles in the retentate
Figure 4.26: An illustration of the particle size distribution evolution in the permeate
stream at TMP=20 kPa.
Figure 4.27: An illustration of the particle size distribution evolution in the permeate
stream at TMP=60 kPa.
stream.
• At higher transmembrane pressure (i.e. 60 kPa), the particles of similar sizes (frac-
tionation) are found in both the streams (permeate and retentate), along with smaller
particles permeate through the membrane. Thus at higher TMP, both separation and
fractionation are taking place.
Figure 4.28: Particle size distribution of the permeate stream at various TMPs (20-60 kPa),
time=9 mins, CFR=0.6 L/min.
These observations are in line with the results of the filtration efficiency results reported
in the previous section. It shows that at lower TMP, the log reduction value is high which
means that less of particle concentration in the permeate stream. It is also confirmed that
as summarised above, at lower and medium TMPs (LRV = 2.66, 2.54 respectively), the
trend is similar but with slight increase in the distribution which is clearly evidenced in
the minor drop in LRV value (0.12 LRV). Whereas, at higher TMP the distribution in-
tensity is greater and a mixture of small and similar sized particles are observed. This is
directly related to more of permeate concentration and hence there is a higher decline in
Figure 4.29: Particle size distribution of the retentate stream at various TMPs (20-60 kPa),
time=9 mins, CFR=0.6 L/min.
the filtration efficiency values (0.5 LRV). Thus, at higher TMP particles experience frac-
tionation and separation which has a direct influence of decreasing the filtration efficiency
values. Whereas, at lower TMP, particles experience more of rejection mechanism and
hence better filtration efficiency values.
4.5.4.3 Effect of CFR on particle size distribution in permeate streams
In order to understand the effect of cross flowrate on the final permeate particle size
distribution, cross flow experiments are analysed at three different rates ranging 0.3 -1
L/min. It is found that with change in cross flowrate, the particle size distributions change
is equally pronounced in the permeate stream. But there is a clear indication of particles
separating than fractionating when there is a change in the cross flow velocity.
With increase in cross flowrate, smaller particle permeation and bigger particle rejec-
tion is quite distinctively evident from the particle size distribution (Fig. 4.31). Thus, in
the case of change in cross flowrate, the filtration mechanism is more influenced by the
Figure 4.30: Particle size distribution of the permeate stream at various CFRs (0.3-1
L/min), time=9 mins, TMP=40 kPa.
Figure 4.31: Particle size distribution of the retentate stream at various CFRs (0.3-1 L/min),
time=9 mins, TMP=40 kPa.
separation mechanism than the fractionation in the studied cross flowrate range.
The above particle size distribution based study agrees well with the filtration effi-
ciency studies as indicated in the previous section. It is reported that with increase in cross
flowrate (from 0.3 to 1 L/min), the log reduction value increases from 2.4 to 2.58 LRV
which account only for a minor improvement which is evidenced in the small change in
PSD as well. This filtration efficiency increase is well supported by the results of Ra-
machandran and Fogler [Ramachandran and Fogler, 1999] where with increasing flow the
efficiency of particle retention has increased which has been attributed to the hydrodynamic
bridging effect.
4.5.4.4 Modeling particle size distribution
In this section, particle size distribution is modeled from the predicted values of perme-
ate particle size and PDI. The particle size distribution is best described by the following
equation:

1 (x − x̄)2
I(x) = √ exp − (4.8)
2πσ2 2σ2
where
I(x) is the intensity distribution at x, σ is the standard deviation which is ob-
tained from Equation 4.9 and x̄ is average particle size.
σ
PDI = (4.9)
x̄
The modeled particle size distribution is plotted against the experimental value for
comparison. It is evident that the predicted PSD, is overly consistent with the experimental
PSDs (Figs. 4.32, 4.33, 4.34). Thus, for a set of input conditions (TMP, CFR, FPS), using
Equations 4.6 & 4.7 permeate particle size and PDI could be computed. And further,
Equation 4.8 could be employed to plot the PSD of the permeate stream. Thus, in this way
the permeate PSD could be predicted within the range of TMP, CFR and FPS studied.
4.5.4.5 Size polydispersity index and its effects
The polydispersity index (PDI) is a measure of the nonuniformities that exist in the
particle size distribution. Three feed particle size ranges used for the experimental runs are
analysed for the polydispersity index effect. It is observed that smaller particles have higher
PDI (0.208) than the medium particle (0.151) and bigger particles (0.053). Further, the
tangential flow UF study of these particles clearly indicated that smaller particles exhibited
lower filtration efficiency (Fig. 4.35). Therefore the higher the polydispersity index of
original suspension the lower the filtration efficiencies. In order to demonstrate the effect
of PDI on the filtered product, LRV values at 40kPa TMP and 0.6 L/min crossflow velocity
are reported here (Fig. 4.35). It is also worth noting down that permeate PDI shows the
same effect as well on the filtration efficiency.
In the case of medium and smaller particles with a drop of 0.05 in PDI, the increase in
LRV is 0.23. Whereas in the bigger particles with less than 0.1 PDI (i.e. PDI=0.053), there
is an increase of 0.42 LRV in comparison with the smaller particles. This trend indicates
that if the original feed suspension has particles with broader distribution of sizes, the
potential barrier between particles is more prevalent and these inter-particle interactions
play a significant role in fractionation of particles, especially if particle size is less than
100 nm and the PDI is more than 0.1. Thus, the lower the feed and permeate PDIs, the
higher the LRV. However, it is found that feed PDI is statistically highly significant on LRV
(with p-value less than 10−4 ) whereas permeate PDI has slightly less influence on LRV (p-
Filtration Mechanism
Figure 4.32: Representative particle size distribution of observed (points joined by dotted lines to show the trend) and predicted
127
plots for smaller permeate particles at (a)-(c) at TMP=20,40,60 kPa and 0.3 L/min (d)-(f)at TMP=20,40,60 kPa and 0.6 L/min
(g)-(i)at TMP=20,40,60 kPa and 1 L/min.
plots for medium permeate particles at (a)-(c) at TMP=20,40,60 kPa and 0.3 L/min (d)-(f)at TMP=20,40,60 kPa and 0.6 L/min
128

129
plots for bigger permeate particles at (a)-(c) at TMP=20,40,60 kPa and 0.3 L/min (d)-(f)at TMP=20,40,60 kPa and 0.6 L/min
Figure 4.35: Effect of feed and permeate PDIs on filtration efficiency at middle TMP (40
kPa) and CFR (0.6 L/min).
value = 0.033). Thus, it is deduced from the above study that increase in feed PDI and
permeate PDI, both, lead to decrease in filtration efficiency. The permeate PDI is highly
influenced and or controlled by the operating conditions and input parameters which is
governed by Equation 4.7. This reflects on the complex interactions existing between the
operating conditions.
4.5.5 Concluding remarks for particle size and distribution investiga-
tion section
Tangential flow UF of three size ranges of silica particles (as model virus particles) was
investigated to understand the rejection and or fractionation of particles considering the ef-
fects of feed particle size and transmembrane pressure and cross flowrate. For fractionation
of particles, suitable transmembrane pressure is to be chosen whereas if the particles are
to be separated, cross flow velocity is the key factor for better efficiency of membrane fil-
tration. From the experimental range employed in this study, it was found that the lower
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dependency on the operating parameters whereas permeate PDI varies linearly with the
operating parameters. The particle level study covered in this article offers opportunities
for academic and industrial researchers to gain fundamental insights into how particle size
properties are linked to permeate quality.
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Chapter 5
Modeling and Simulation
Contents
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
5.2 Earlier Modeling patterns . . . . . . . . . . . . . . . . . . . . . . . . . . 142
5.2.1 Macro-scale modeling: A quick review . . . . . . . . . . . . . . . 143
5.2.1.1 Flux decline prediction models . . . . . . . . . . . . . . 145
5.2.2 Particle property models . . . . . . . . . . . . . . . . . . . . . . . 147
5.3 Novel Model Formulation: Population Balance Approach . . . . . . . . 150
5.3.1 Statement of the problem . . . . . . . . . . . . . . . . . . . . . . . 151
5.3.2 Modeling of size distribution of suspended particles through a cross-
flow UF process . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
5.3.2.1 Input and output streams . . . . . . . . . . . . . . . . . 154
5.3.2.2 Constitutive equations . . . . . . . . . . . . . . . . . . . 155
5.3.2.3 Permeation probability . . . . . . . . . . . . . . . . . . 156
5.3.2.4 Expression of solid permeate flow . . . . . . . . . . . . . 157
5.3.2.5 Balance equations . . . . . . . . . . . . . . . . . . . . . 157
5.4 Model Solution and Simulation Results . . . . . . . . . . . . . . . . . . 160

138
5.4.1 Discretisation of the model . . . . . . . . . . . . . . . . . . . . . . 161
5.4.1.1 Discretisation issues . . . . . . . . . . . . . . . . . . . . 161
5.4.1.2 The internal coordinate and its significance . . . . . . . . 163
5.4.1.3 The method of moments . . . . . . . . . . . . . . . . . . 166
5.4.1.3.1 Different mean sizes . . . . . . . . . . . . . . . 169
5.4.1.4 Number and volume distributions . . . . . . . . . . . . . 171
5.4.1.5 Useful quantity predicted from the above model . . . . . 171
5.4.2 Simulation using gPROMS . . . . . . . . . . . . . . . . . . . . . . 172
5.4.2.1 Model entities of the entire process . . . . . . . . . . . . 173
5.4.2.2 Parameters and variables employed in the model develop-
ment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
5.4.3 Simulation results . . . . . . . . . . . . . . . . . . . . . . . . . . 174
5.4.3.1 Representative particle size distribution in output streams 174
5.4.3.2 Effect of feed particle size on particle size distribution . . 177
5.4.3.3 Effect of transmembrane pressure drop on the particle size
distribution . . . . . . . . . . . . . . . . . . . . . . . . . 178
5.4.3.4 Effect of cross flow rate on particle size distribution . . . 178
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
139
Preface
The ultimate purpose of modeling filtration processes is the mathematical understand-
ing of the dynamic behavior of the process. A validated model can be implemented in
simulation, optimization and control of the process, overall helping to achieve desired
product characteristics. Typical filtration models found in literature focus on flux predic-
tions and fouling mechanisms. Our comprehensive filtration model, based on population
balances, is novel approach in the field of virus filtration. It takes into account individual
particles and their particle size property, equations of conservation, and kinetic submodel.
The model developed is capable of successfully predicting the permeate particle size and
size distribution as well as final filtration efficiency. This represents a big leap in under-
standing filtration mechanisms and process behaviour and importantly can be applied in
scale-up studies. This novel model demonstrates a promising step to optimize and control
the process to achieve the required regulations. This chapter details the model formulation,
model solution and the model parametric analysis.
Research Questions:
1. Is the population balance framework, which has a wide spectrum of applications in
other fields of chemical engineering problems like coagulation, precipitation, crys-
tallisation, a suitable approach for modeling the filtration process?
2. Is it suitable to describe particle permeation by a stochastic kinetic model?
The content of this chapter has been presented in Engineering With Membranes, Portu-
gal [v] and International Congress on Membranes and Membrane Processes, Hawaii [vi].
Manuscript under review for submission in Chemical Engineering Science journal [iv].
5.1 Introduction
Process modeling is an important avenue towards understanding and optimizing design
and operations of an unit operation [Ahmad et al., 2006; Bodzek and Konieczny, 1994;
Mohammadi et al., 2005]. While there is a constant evolution in mathematical model
formulations to predict the performance of membrane filtration processes in terms of per-
meate flux and fouling mechanisms, the same cannot be said about models for prediction
of permeate particle size and filtration efficiency. While others have attempted modelling
systems such as deep bed filtration [Santos and Bedrikovetsky, 2004], this work, we be-
lieve, is a maiden attempt to illustrate the application of the population balance framework
to predict the final product efficiency in the context of tangential flow ultrafiltration (UF).
The main objective of this research is to develop a novel model for the TFF based on
the population balance theory. Such predictive modelling will serve as a promising tool
for optimizing and controlling the TFF process. The innovation of this work lies in the
above fact, whereas previous ultrafiltration models have been dominated by flux modeling
techniques, i.e. at the macroscale.
The framework of population balances to model particulate processes was proposed
in the seminal work by Hulburt, Katz [Hulburt and Katz, 1964] and has since penetrating
into various engineering problems and applications. Randolph [Randolph, 1964; Randolph
and White, 1977] give a comprehensive insight on population balance theory. The use of
population balance models (PBM) has seen a significant growth in the last 15 years or so.
The population balance equation (PBE) offers its utility for describing the complex nature
of particle systems distributed along the particle size domain. Its applicability to a wider
range of systems is well explained by Ramakrishna [Ramakrishna, 2000]. These applica-
tions include crystallisation, [Gerstlauer et al., 2006; Rawlings et al., 1992] polymerisation
[Alvarez et al., 1994], fluidized bed [Tan et al., 2005] to name a few. The uniqueness of
the population balance method lies in the fact that the processes are described in terms
of internal coordinates and external coordinates; the internal coordinate referring to size,
chemical composition whereas the external coordinate describes the position in space of
the entity concerned. For the model to mimic the exact way as the process, the right choice
of phase coordinates is a primary factor. The internal coordinate, hence, is defined in terms
of any size related variable (diameter or volume) and, say, if volume is considered as the
internal coordinate the accumulation also is given in terms of the volume range.
The solution of PBEs can be obtained by any one of the methods, viz. analytical meth-
ods (including analytical solutions and similarity solutions), method of moments, method
of weighted residuals and methods such as finite element method or direct discretisation.
There is an extensive literature on the solution and dynamical analysis of particulate pro-
cesses [Gelbard and Seinfeld, 1978; Ramakrishna, 2000; Randolph, 1964].
The detailed investigation carried out in studying the influential parameters on the fil-
tration mechanism (which is already presented in Chapter 4), clearly indicates that apart
from various operating parameters like, cross flow velocity, and transmembrane pressure,
particle size plays an important role in challenging the filtration efficiency. It is more com-
mon to find typical UF operations working in the cross-flow mode (alternatively termed
tangential flow) for bio-technological applications in comparison with the dead-end fil-
tration mode (Fig. 5.1) [Meltzer and Jornitz, 2007]. Cross-flow filtration has received
attention for its fouling reduction capabilities in removing molecules or particles from the
5.2. Earlier Modeling patterns 142
membrane surface by the tangential flow of liquid. Currently, various theories and mod-
els exist to describe the transport mechanism (mass/fluid/momentum) for tangential flow
filtration mechanism [Lee and Clark, 1998; Mohammadi et al., 2005], but none have ad-
dressed the particle size distribution (PSD) and further still none have attempted describing
the transport mechanism as a stochastic one. This work aims at extending the population
balance modeling concept to UF processes thereby describing the evolution of the particle
size distribution in the filtration product streams. The status-quo in modeling, i.e. flux
predictions, would be completed by size distribution modeling.
Figure 5.1: Illustrations of (a) dead-end filtration and (b) Tangential flow filtration.
5.2 Earlier Modeling patterns

Numerous process models have been presented in the literatures that describe flux de-
cline, fluid dynamics, and fouling characteristics. The key performance (output) variables
in UF include the rejection coefficient, log reduction value, the permeate flux and the
transmission characteristics. There are mainly three related (input/operational) variables

that play a significant role in the effectiveness of filtration; change in the pressure drop
across the layer, fluid flow rate along the membrane, and the feed suspension properties.
With respect to the modeling objective, the UF models can be classified into two cate-
gories [Driscoll, 2001]. The first category includes models that are related to the filtration
process design and equipment including the membrane. In this category, models are pre-
dominantly targeted at flux decline prediction under the effect of various membrane and
process parameters. Process models that are members of the second category are aimed
at particle suspension properties, more specifically, at particle polydispersity parameters.
For the second category of models, the prediction of flux decline is a secondary modeling
objective and so the first (more conventional) category of models is in effect a subset of
the second category. But the first category of models is predominantly found in the litera-
ture whereas the second category is still in its infancy demanding more attention. Hence,
this work tries to address the modeling technique at the particle scale which will help in
optimisation and controlling the process. Before presenting our model, let us have a quick
review of already available models in order to capture the essence of existing modeling
techniques to highlight the uniqueness of the current work.
5.2.1 Macro-scale modeling: A quick review

Many realistic models for UF are available in the literature which are macroscopic in
nature, predicting flux and fouling phenomenon, based on cake layer formation, concen-
tration polarization, shear induced diffusion, and adsorption [Belfort et al., 1994; Bowen
and Sharif, 2002; Ghosh, 2008; Lee and Clark, 1998; Richard Bowen and Williams, 2001].
These models, viz. analytical, semi-analytical, and numerical comprehensively predict the
flux decline behavior and thereby membrane performance. In membrane separation pro-
cesses, another promising modeling technique is based on fluid dynamics which is well
addressed by Belfort [Belfort, 1989; Belfort and Nagata, 1985]. The fluid flow through
membranes is a combination of free flow and flow through porous media where the free
flow is best described by Navier-Stokes equation and the flow through the porous media
is represented by Darcy’s law for non-inertial, incompressible flows with small porosity.
Development of these models have found broader applicability and computational fluid
dynamics (CFD) is the most sought after technique. These models are chiefly categorized
as either solubilisation diffusion models or concentration polarization models [Ghidossi
et al., 2006; Lee et al., 1996] and normally coupled with mass transfer equations. Lee
et al. [Lee et al., 1996] modeled cross-flow UF through CFD technique where the ef-
fects of the particle size, concentration and membrane dimension on the flux decline were
explained. Huang and Morrissey’s [Huang and Morrissey, 1999] work used a numeri-
cal model to reproduce the process of concentration polarization and to model the solute
concentrations on the membrane surface. Nassehi [Nassehi, 1998] used the finite element
method and presented a more robust simulation technique compared to other previous
works. Pak et al. [Pak et al., 2008] used the finite volume method and studied the ef-
fect of various physical parameters on the growth of concentration polarization layer in
a two-dimensional convective-diffusion equations coupled with resistance-in-series model
for permeation transport. Damak et al. [Damak et al., 2004] used finite difference method
to simulate a laminar, incompressible and isothermal flow in a cylindrical tube with a per-
meable wall. Though improvement of these models is in a continuous trend with regard
to understanding the flux decline and fouling, debate still exists in understanding the ba-
sics of polarization phenomenon. With better understanding, the processes become more
complex and the complexity of these models is considerably reduced by the use of CFD
technique which in turn helps minimizing the number of experiments. These models are
usually solved using commercially available packages (eg. Fluent, CFX) and has the con-
venience to analyze the effects of operating parameters on the membrane performance and
many authors confirm the benefits of using CFD to optimize membrane systems and pro-
cesses [Belfort and Nagata, 1985; Ghidossi et al., 2006; Kleinstreuer and Belfort, 1984].
The handicap in these models is that the permeate particle size and its distribution deter-
mination are not accounted for. In reality, particles interact with each other and with the
membrane pores impacting on the filtration efficiency and mechanism.
5.2.1.1 Flux decline prediction models
It is well established that flux declines with time during an UF process, such decline
being attributed to membrane fouling (see [Song, 1998] and citations within). There has
been a number of works that reviewed flux prediction models over the years [Chudacek
and Fane, 1984; Kim, 2007; Lee et al., 1996; Sarkar et al., 2009]. Three main theories
have been used in these modeling techniques leading to three types of models; (1)
Gel-polarization model [Rautenbach and Schock, 1988; Wakeman and Akay, 1994;
Wijmans et al., 1984], (2) Osmotic pressure model [Ahmad et al., 2006; Kim, 2007] and
(3) the resistance model [Sarkar et al., 2009; Zaamouche et al., 2009]. The description of
the flux in the first type of models is in terms of the bulk, gel, and permeate concentrations
(Cb , Cg and Cp ):
J = f (Re, S c, Cb , Cp , Cg ) (5.1)
Unknown parameters within the flux equation are experimentally determined. In the
second type of flux models, the flux is a function of transmembrane (ΔPTM ) and osmotic
pressures (ΔΠ) as well as concentration at the membrane (Cm ):
J = f (ΔPTM , ΔΠ, Cm ) (5.2)
This equation is popularly written in the modified Hagen-Poiseuille form (original refer-
ence deriving the H-P equation, perhaps Bird et al. [Bird et al., 2006]) and gives the flux
as superficial permeate velocity. Viscosity is also considered which can be corrected for
non-water permeates.
Lastly, the third type of flux model describes the flux as being dependent on resistances
against the pressure driving force. Typically three resistances in series are used, being the
resistances across the gel (Rg ), the fouling (Rf ) and the membrane (Rm ) layers:
J = f (ΔPTM , Ri ) (5.3)
While the above three types of models for describing flux have been prevalent in the liter-
ature, other approaches have also been used to an extent including the film model, those
models that use a combination of the above three flux modeling methods [Bolton et al.,
2006; Mohammadi et al., 2005; Zhang and Song, 2000] and those that extend the descrip-
tion to other flux dependencies, such as on pH and electrostatic forces.

5.2.2 Particle property models

In the previous section, we looked at past UF models, those aimed at flux prediction.
Flux modeling has received much attention, presumably because this is a macroscale vari-
able that is easily measurable and understood. We now look at a second category of UF
models, those with descriptions extended to particle properties particularly particle size
and size distribution. Particle size distribution (PSD) is an important variable in UF be-
cause this is directly responsible for a greater impact on filterability [Roorda et al., 2004].
The emphasis on particle size leads to descriptions at the particle scale, as such it is prod-
uct property focused which is important to ensure product quality, purity and successful
operation of an UF system. Moreover PSD prediction can provide information about the
numbers of particles in the permeate and retentate which is of significance in the UF of
virus suspensions, where quantification of virus numbers is directly required [Alspach and
Allgeier, 2003; Brandenburg and Zhuang, 2007]. Future UF operations may well become
routinely characterized by alternative variables like PSD. Unlike flux, PSD was previously
not easy to measure particularly at the ultra size range wherein UF processes operate, but
with advances in analytical instrumentation, it is nowadays possible to measure PSD us-
ing techniques like dynamic light scattering (DLS). In spite of the importance of PSD, UF
models have not addressed nor incorporated this variable sufficiently. The following is a
review of previous related works.
Chang and Hwang [Chang and Hwang, 1995] developed a mathematical model which
predicts the unsteady state permeate flux of crossflow MF of polydispersed solution. The
PSD of the cake formation was obtained and they have observed that the permeate flux
increases with an increase in the mean particle size of the cake. Whereas, mean particle size
of the cake increases with increase in the mean particle size of the original solution. Vyas
et al. [Vyas et al., 2000] estimated permeate flux, internal and surface fouling, porosity and
the PSD of the feed particles of lactalbumin. Special emphasis was given to the influence
of the feed properties on the performance of the microfiltration process. Wakeman’s work
[Wakeman, 1994] discussed the effect of parameters such as particle size, feed suspension
concentration, pH, transmembrane pressure and crossflow velocity on the development of
the particle layer and the corresponding permeate flux. His subsequent work states that it is
qualitatively and quantitatively difficult to predict the effect of particle shape. Barauh et al.
[Baruah et al., 2005] framed a global model that accounts for pH, ionic strength, sieving
through membrane cake, effect of hydrodynamics to predict the performance of crossflow
UF processes. They also highlighted the difficulty in using molecular dynamics to solve
the MF/UF process because of the complexity of the mechanism of the process. They
attributed the complexity involved in modeling MF to the complex nature of the fluids
and the difficulty in specifying the behavior of their suspended and dissolved components.
Benjamin McCoy’s work [McCoy, 1995] dealt with the membrane and mixture parameters
and showed how these parameters influence the sieving coefficient. For a steady-state UF
process with a well-mixed upstream concentration distribution, the theory predicts how
much the downstream concentration distribution shifts toward smaller solutes as larger
solutes are hindered or rejected by the membrane.
The modeling approaches, considering particle size as one of the factors, can be sum-
marized as (i) force or torque balance models describing the particle size and its deposition
rate on the membrane surface based on variety of forces acting on the particle [Chang and
Hwang, 1995; Hwang et al., 1998; Lu and Hwang, 1995] (ii) hydrodynamic models de-
tailing the mechanisms of particle deposition and cake formation in the microfiltration of
micron particles [Belfort and Nagata, 1985] (Table 5.1). The major limitation of such
models formulated, so far, are not capable of predicting the permeate quality say in terms
of number density or concentration values [Stoller, 2009]. It is very important and cru-
cial to note that the models incorporating the particle size and its distribution is limited to
handling the input/feed suspension [Chang et al., 1996, 2008; Foley et al., 1995b].
Table 5.1: Summary of various models considering feed particle size as a factor to study
the permeate flux.
Trajectory Analysis (Force or Torque)

Brownian diffusion [Porter, Cross flow rate promote back transport alongwith
1990] shear induced radial migration of particles.
Shear induced diffusion Local particle concentration by balancing the
[Romero and Davis, 1990] convective motion of particles with the shear-
induced diffusion.
Inertial lift [Belfort et al., 1994; Lateral migration theory to explain flux paradox.
Green and Belfort, 1980]
Dean vortices [Winzeler and Higher performance with introduction of Dean
Belfort, 1993] vortices.
Frictional drag, Brownian force Cake porosity, specific cake resistance, effect of
[Tarabara et al., 2002] cross-flow rate and transmembrane pressure on fil-
trate rate.
Lateral migration velocity Permeation drag, inertial lift and sedimentation
[Chellam and Wiesner, 1992] dominate lateral migration in far field region
whereas van der Waals force is important near the
membrane wall.
In the current presentation, we discuss and present an extended particle population
UF model for virus filtration. The interesting aspect of virus filtration is: in the case of
virus purification, the degree of recovery or total removal of virus is less critical. Viruses
purified by UF include Aedes aegypti, Minute Virus of Mice, Autographa californica M
nucleopolyhedrovirus which are industrially important vectors [Guo et al., 2009; Hensgen
et al., 2010; Michalsky et al., 2009]. Whereas, in the case of virus removal or protein
5.3. Novel Model Formulation: Population Balance Approach 150
separation studies, the main aim is to achieve complete removal of viruses with recovery of
protein product. Typically, these feed suspensions are very dilute and hence cake formation
and concentration polarization are assumed to be negligible. Thus this modeling is based
on the condition where such negligible concentration polarization and cake formation are
expected.
The particle population focus founding this proposed model is statistical in its nature
and is consequently more informative about the particle phase and specifically the evolu-
tion of the PSD in the product streams. Such description which is extensible to particle
properties other than PSD has been lacking in the literature where previous works have
been more focused on flux determinations [Mohammadi et al., 2005; Song, 1998]. The
details of our model are presented and discussed in the subsequent sections.
5.3 Novel Model Formulation: Population Balance Ap-
proach
The process of UF, can be classified as a particulate one with the solid particulate
phase suspended in the carrier liquid phase. A population balance allows one to describe
the evolution of individuals (particles in the UF case) in the entire population, which is
generally defined with the forces driving the population dynamics. The evolution of each
individual is described by internal coordinates such as size, age or velocity. Size is a
typical property used to describe the variable changes to individual particles. For purposes
of statistical simplification, we describe particles in classes rather than individual particles,
where each particle class has a range of sizes carrying the particles that fall in that size
range. Consequently the output of such models is distributions across the time and particle
size domains providing continuous and clear picture of the particle state. Thus, a detailed
mechanistic model is proposed which utilizes the population balance background along
with the stochastic behavior of particles, in the following sections. This developed model
has a promising future, as a tool for the optimization, control and design of UF processes
for virus removal and purification operations. This model is quite unique as it is capable
of predicting precisely the virus population in the output streams and the final filtration
efficiency as well.
5.3.1 Statement of the problem

In cross-flow UF process, the suspension (i.e. feed stream) while passing through the
membrane will have these random phenomena: (i) particles permeate through the mem-
brane if the particle size (pa) is smaller than the pore size (po) (ii) not all particles smaller
than the pore size pass through, (iii) particles bigger than the pores (and a fraction of
smaller particles as well) are dragged by the shear rate in the tangential mode as reten-
tate. The above mechanism is based on the fact that the UF is size exclusion i.e. sieving
mechanism.
To model the UF process, the UF system is described physically as the one which has
the feed suspension as input stream and the permeate and retentate as the output streams,
each has liquid and particles and the separation boundary is the membrane as shown in
Fig. 5.2. The focus of the PBE model in predicting size properties in the filtration process
is shown in Fig. 5.3.
The modeling problem is addressed via the following steps:
• The formulated model and the accompanying allied equations are presented along-
with the initial conditions.

Figure 5.2: A schematic diagram of the tangential flow filtration: Particles sieving through
the membrane (permeate) and those being excluded (retentate).
Figure 5.3: A schematic diagram of the membrane filtration and its relationship with the
proposed PBE model.
• The operating parameters and the input conditions of the tangential flow filtration
process are defined.
• The model is solved and simulated using gPROMS (Process Systems Enterprise,
UK).
Figure 5.4 is a summary of the model and its interplay with other equations and phe-
nomena.
Figure 5.4: An overview of the model encapsulating the set of balances involved in the
model formulation.
5.3.2 Modeling of size distribution of suspended particles through a
cross-flow UF process
The population balance equation (PBE) is a continuity statement written in terms of
the number density function. The macroscopic population balance of tangential flow UF is
modeled based on the approach proposed by Randolph and Larson [Randolph and Larson,
1988]:
∂n
= B−G+ Fk nk /V (5.4)
∂t k
where n and nk are the number distributions (no./m3 ), F is the flow rate of either the
input or output streams (L/min), G is the growth term, B is the nucleation term, V is the
volume of the original suspension (L).
In the tangential flow UF process, it is assumed that there is no aggregation, growth
and/or nucleation taking place hence the first two terms of the right hand side of the equa-
tion are ignored. Whereas the driving force being transmembrane pressure is defined in
terms of filtration kinetics. Incorporating these, the above equation reduces to
∂nr
=F+ Fk nk /V (5.5)
∂t k
where nr is the number density in the retentate stream, F is the filtration kinetics, Fk ,
nk are the flow rate and number density respectively, which is defined in the following
section. It is quite evident that the number density in retentate stream is zero initially i.e.
at time t=0.
5.3.2.1 Input and output streams
In the cross-flow UF process, the cross flow rate (otherwise feed flow rate) is chosen
as the reference flow and hence the total flow streams in the system could be well thought
of as input streams with feed flow rate (F f ) and the outlet streams being the permeate and
retentate streams (F p , Fr ) and each streams are proportional to the distribution function
[Randolph and Larson, 1988].
Inlet (i) and outlet (o) streams are thus defined by the respective equations as follows:

Fk nk pa = Fi ni pa − Fo no pa (5.6)
pa pa pa
where Fi & Fo are the flow rates of the inlet and outlet streams respectively. And ni &
no are the number densities of the inlet and outlet streams respectively. The inlet and outlet
streams are given as:

Fi ni pa = F f n f pa (5.7)
pa pa

Fo no pa = F p n p pa + Fr nr pa (5.8)
pa pa pa
where n p & nr are the number densities of the permeate and retentate streams respec-
tively.
The above equation is a modeling simplification that has a profound effect in reducing
the complexity of the set of equations that satisfactorily describe the system. Physically,
this modeling simplification indicates that particle classification occurs as size split at the
output streams and thus this modeling simplification is well justified [Randolph, 1964;
Randolph and White, 1977].
The particle number density of the feed suspension is defined as:
exp(−((L pa − L¯pa )2 /2σ2 ))

n f pa = √ (5.9)
2πσV
where L pa & L¯pa are the particle size and average particle size respectively, σ is the
standard deviation.
5.3.2.2 Constitutive equations
It is a well established fact that the driving force of the UF process is the transmem-
brane pressure drop and therefore, the kinetics of the model is defined as power law which
includes the stochastic behavior of particles as well:

k
F= k1 T MPk2 P pa (5.10)
pa=i
where TMP is the transmembrane pressure drop, F is the filtration kinetics, k1 and k2
are kinetic parameters, and P pa is the stochastic behavior of particles which is defined in
the next section. Thus, this approach of defining kinetic model is quite novel as it is capable
of capturing the random behavior of the particles influenced by the driving force. Initially,
the model is simulated with guess values to verify it is solving well [gPROMS, 2009a] and
finally, for validation purposes the kinetic parameters are obtained via the experimental
results.
5.3.2.3 Permeation probability
The filtration mechanism, using stochastic behavior, can be conceived of random par-
ticle behavior in the system: if the particles of a range of size class (in the suspension)
approaching the membrane (particle size lesser than the pore size) has a diameter lesser
than the pore, then it is very likely that the particle will permeate through the pore. In
this way each individual particle smaller than the membrane pore has a probability (P pa )
of passing through the pore. This stochastic behavior has a significant role in the above
defined filtration kinetics. The probability distribution is defined as follows:
La 2 La 2
P pa = [ ] 2
+ [ ]2 (5.11)
La + L pa
2 2
La + L pa L pa−1
2
This relationship holds valid if the particle size is smaller than the pore, otherwise, the
probability approaches zero. There are occasions where the data for particle size distri-
bution is limited, in those cases, the average particle size of the feed stream is considered
to explain the mechanism. The same assumption maybe extended for the pore size of the
membrane, and where the pore size distribution is unavailable, the average pore size maybe
considered and this approach is employed in this work.
Now, the above discussed stochastic behavior of the particles permeating is demon-
strated here for model depiction. From Figure (Fig. 5.5), it is evident that with increasing
particle size the probability with which the particles permeate decreases and when it ex-
ceeds the average pore size, it approaches zero.
5.3.2.4 Expression of solid permeate flow
An extension of the stochastic behavior coupled with the kinetic rate definition as de-
fined above, the solid permeate flow is typically solved with number density of the perme-
ate particles. And if L pa is smaller than L po , then the particle permeation is correlated with
the number density distribution and thus, giving a good indication of the model behavior of
the permeate particles. This has an explicit bounding condition of no permeation when the
particles approaching the pore are larger which is seen in the simulation result of particle
permeate flow (Fig. 5.6).
5.3.2.5 Balance equations
The overall conservation in terms of overall and component mass balance equations
are written as follows with the assumption that the density of all the streams is constant,
true for dilute systems.
F f n f = F p n p + Fr nr (5.12)
Figure 5.5: Plot showing the behavior of the probability of particles permeating through
the membrane: Smaller the particles higher the probability and vice-versa. Also the plot
shows that the particles approaching membrane pore reflects zero probability of particle
permeation.
Figure 5.6: Solid permeate flow indicating the bounding condition of no particle flow of
bigger particles.
F f = F p + Fr (5.13)
The number conservation is thought of as the total number of particles of a given size
entering-in (n f pa ) either permeates through the membrane (n p pa ) and the rest are excluded
as retentate (nr pa ) which is given as:
n p pa = n f pa − nr pa (5.14)
Finally, the permeate concentration of the particles in the suspension is obtained as
particle permeation through the total permeate rate.
Particle and pore dimension
The size domain is defined after the generalization method given in [Abbas and Ro-
magnoli, 2007], where the general equation describing the size of a particle (Equation
5.15) is given by:

5.4. Model Solution and Simulation Results 160
L pa = ab pa (5.15)
where a is the minimum detectable size and b is the geometric constant obtained based
on the maximum particle size present in the system. The pore size of the membrane L po is
assumed to be constant in the model formulated and the solute permeate flow is obtained
when the particle to pore size ratio is less than one. Thus to consolidate, the original pop-
ulation balance equation (Equation 5.5) coupled with the allied equations, i.e. Equations
5.6, 5.12, 5.13, 5.10 & 5.14) are solved to simulate the model and the approach to solve
the model is presented in the following section.
5.4 Model Solution and Simulation Results

The novel model formulated which is based on the population balance framework cou-
pled with the stochastic behavior of particles is, in its raw form, a partial differential equa-
tion in time and size. The solution methods for such models broadly include analytical
solution and numerical technique. But analytical solutions exist only for few cases, and
thus numerical techniques are resorted to. In order to ease the solution of population bal-
ance equations, various numerical techniques are developed. These include include Monte
Carlo method [Ramakrishna, 2000; Rosner et al., 2003], method of characteristics [Ku-
mar and Ramakrishna, 1996], discretised methods like finite volume or finite element or
finite difference [Marchisio et al., 2003], standard method of moments [Hulburt and Katz,
1964], quadrature method of moments [Gimbun et al., 2009] and direct quadrature method
of moments [Su et al., 2008].

5.4.1 Discretisation of the model

The discretisation of a typical population balance equation is well covered by several
researchers as mentioned above. In this work, a discretisation numerical method is chosen
which needs to a relatively low computational charge as the complex balance equation is
solved as a set of ordinary differential equations. Furthermore, this method also allows to
calculate the particle size distribution with a great precision when using a large number of
discretised classes. Thus, the original model equation (Equation 5.5) is discretised with
respect to the size domain into a normal differential equation. This method overcomes the
disadvantage of other methods as it allows the kinetic rate to be discretised along the size
domain as well.
5.4.1.1 Discretisation issues
Discretisation of the population balance equation transforms it into a family of linear
differential equations by discretising along the size domain. Thus the original PBE results
into an equation with reference to the independent size variable (L pa ):
∂nr pa
= F pa + Fk nk pa /V, pa = 1, 2, 3, ......n (5.16)
∂t k
The above equation becomes a system of ordinary differential equation as the time
derivative is only an ordinary one. Hence,
dnr pa
= F pa + Fk nk pa /V, pa = 1, 2, 3, ......n (5.17)
dt k
which is the discretised form of Equation 5.16. Here, since the density function is
along the size axis and is defined as classes, the width of the class is just the difference
between the immediate adjacent sizes of that interval and is defined as follows:
φ pa = L pa − L pa−1 (5.18)
The scale of the size axis showing the relationship between the class, density, is shown
in Fig. 5.7. Thus the discretisation of such size axis is chosen or defined according to
the input conditions. In this work, the change in size axis is kept constant (i.e. ΔL pa =
constant).
Figure 5.7: The discretisation of the density function and the relationship between size
class, and density.
Thus, Equation 5.17 takes the form as follows:
dnr1
= F1 + Fk nk1 /V (5.19)
dt k
dnri
= Fi + Fk nki /V (5.20)
dt k
for i = 2,3...n-1
dnrn
= Fn + Fk nkn /V (5.21)
dt k
The permeate flow is given as solid permeate flow which is obtained from the kinetics
and the liquid permeate flow is obtained from the transmembrane pressure drop equation.
Thus, the total permeate flow rate is the sum of solid and liquid permeate flowrates.
5.4.1.2 The internal coordinate and its significance
The internal coordinate which is the size axis plays a significant role in the simulation
aspect. Proper choice of the particle size range dictates the sensitivity and the step size
gives a broader and or narrower type of the simulation results, at the same time it gives the
finer discretization of the problem in itself.
The general equation describing the particle size could well be expressed as either
arithmetic (Equation 5.22) or geometric progression (Equation 5.23) as:
L pa = L1 + (n − 1)b∀pa = 1, 2, ....n (5.22)
where L1 is the lowest size of the particle which the measurement could be obtained,
b is the arithmetic constant to be determined and L pa is the size of the respective path size
range. Ln is the largest particle size present in the system.
L pa = ab pa ∀pa = 1, 2, ....n (5.23)
where a is the lowest size of the particle which the measurement could be obtained, b
is the geometric constant to be determined and L pa is the size of the respective path size
range. Ln is the largest particle size present in the system. Therefore, the smallest size is
given by the following equation as per the above equation:

L0 = a (5.24)
In this work, it is realized that the geometric progression suited appropriate to the
measurement, hence Equation 5.23 is employed throughout the simulation results. Also,
geometric series is found to be the most suitable for determining the sequence of size
ranges according to number of researchers [Abbas and Romagnoli, 2007].
Thus the size characterization of the chosen range is given as:
φ pa = ab pa − ab pa−1 (5.25)
In other words, the midpoint of the discrete particle size range (S pa ) is given by the
following equation (Equation 5.26) and which gives the average of the particle size:
ab pa − ab pa−1
S pa = (5.26)
2
As shown in the Equation 5.23, the discrete particle size range could well be adjusted
based on two parameters viz. geometric constant, b, and the number of discrete particle
size range, n. Increasing the geometric constant will influence two factors viz. computa-
tional time and the accuracy of the results. But it is found that changing the discretisation
size of the particle affects the simulation time and also has a considerable impact on the
accuracy of the results. Hence, enough care is taken while choosing the constant bearing
the fact of maximum particle size available in the suspension.
Varying the number of size ranges has a direct effect on the upper limit of the particle
size range. This is achieved by choosing the lower and upper limit of the particle size
expected or known. Accordingly, the value of n is obtained and higher the number of
particle size classes will slightly increase the simulation time whereas it also has a less
effect on the accuracy.
To illustrate the above, let us consider the following cases:
Case 1 : Calculating the number of particle size class:
Considering the minimum detectable particle size is 10 nm, maximum particle size
range is 200 nm with geometric constant 1.032, then the number of particle size class is
obtained from Equation 5.23 as:

200
bn = (5.27)
10
1.032n = 20 (5.28)
leading to n = 95.
Case 2 : Calculating the geometric constant:
In order to vary the particle sizes in itself, then accordingly the geometric constant is
adjusted. For example, considering the number of particle size classes as say 100, with the
min. particle size being 10 nm, but for 2 max. particle sizes i.e. 150 nm and 300 nm, we
get,
100
1
150
b= = 1.0275 (5.29)
10
100
1
300
b= = 1.0346 (5.30)
10
Based on the above examples, the two parameters are fixed depending upon the input
suspension stream conditions. In total, this method generalizes the particle size discretisa-
tion to any system desired.
Now, the role of particle size class and the geometric constant is demonstrated in Fig.
5.8 which clearly indicates the significant effect on the size distribution. It is recalled that
increase in both size class range and the geometric constant increases the simulation time.
Figs. 5.8 (a-c) show the distribution behavior with increasing geometric constant whereas
Figs. 5.8 (d-f) show the effect of number of size class ranges. These plots demonstrate
that the geometric progression has a major role than the number of size classes chosen.
Hence, proper choice of both the parameters is an important criteria in discretising the
model formulation and a particle size distribution is shown to depict the relation of the
particle size and the number of size classes of the original suspension (Fig. 5.9).
5.4.1.3 The method of moments
The time evolution of population balance models is generally computational expensive,
depending upon the complexity and nature of the problem, a suitable method is chosen.
The method of moments proposed by Hulburt et al. [Hulburt and Katz, 1964] is a popu-
lar technique which is used extensively by number of researchers. This method converts
the partial differential equation that is population balance equation into a set of coupled
ordinary differential equations. This method is chosen as only low order moments of the
number density function is of interest.
The population density is a function of particle sizes present in the suspension and the
number density function is used to express the number of particles, n pa in a size class L pa
to L pa+1 per unit volume of the suspension. The jth moment of the number density function
5.4. Model Solution and Simulation Results
Figure 5.8: Plots indicating the significance of the geometric constant (a-c) and number of size classes (d-f) for the model discreti-
sation.
167
5.4. Model Solution and Simulation Results
Figure 5.9: Representative plot indicating the relation of particle size and the number of size classes.
168
(Equation 5.31) is described as follows:

μj = L pa j ndL pa (5.31)
Thus, the zeroth moment, μ0 of the distribution at j=0 is expressed as (Equation 5.32):

μ0 = L pa 0 ndL pa (5.32)
The first, second and third moments (Equations 5.33, 5.34 & 5.35) also provide infor-
mation about the distribution and are as follows:

μ1 = L pa 1 ndL pa (5.33)

μ2 = L pa 2 ndL pa (5.34)

μ3 = L pa 3 ndL pa (5.35)
Thus, the above equations give the various moments of number density functions which
could well be employed to evaluate further information which is discussed in the following
section.
5.4.1.3.1 Different mean sizes The moments obtained as explained in the previous sec-
tion leads to various definitions of mean size of particles. Generally, three types of means
are employed viz. number weighted mean diameter (arithmetic mean), Ln (Equation 5.36),
the surface area weighted mean diameter (Sauter mean diameter), La (Equation 5.37), and
the volume weighted mean diameter (De Brouckere mean diameter), Lw (Equation 5.38).
μ2
Ln = (5.36)
μ1
μ3
La = (5.37)
μ2
μ4
Lw = (5.38)
μ3
It is important that the modeling takes into account the type of measurement being
used where validation is a subsequent step to modeling. Number of techniques are avail-
able to measure particle sizes in various forms as mentioned above i.e. based on number,
length, volume. Thus, depending upon the requirement, one can compute particle size
respectively. To consolidate, the generalized form is given as:
j−k1
μj
L j,k = (5.39)
μk
It is important to select the correct mean size to represent the samples of particles
for the particular application. In this work, measurements are made by the Brookhaven
Particle Size Analyser (BIC 90 Plus) which gives broad range of measurements including
intensity weighted, number, area and volume weighted particle sizes. For validation study
which will be discussed in Chapter 6, the number based distribution is employed as per the
definition given (Equation 5.40):

μ2 L2pa n j dL pa
Ln = = (5.40)
μ1 L pa n j dL pa
5.4.1.4 Number and volume distributions
The previous section gives a glimpse of various methods of representing the particle
sizes, but it is crucial to choose the right way of presenting the data especially when the
model validation is the key aim. The discretised population balance equation given above
represents the number density of particles (n pa ) appearing in the particle size range φ pa .
Considering S as the characteristic size of the particles in the size range and αv the volu-
metric shape factor (kv ) then the volume of particles is:
kv πS pa
ΔV pa = Δn pa (5.41)
6
Thus, having known one candidate, it is easier to calculate the other distribution. Thus
this model depending upon the actual data available, it has the advantage of converting into
the required distribution form.
5.4.1.5 Useful quantity predicted from the above model
The above formulated model clearly determines the size and size distribution profiles
which has never been attempted to date for tangential flow filtration considering the basic
balance equations and transmembrane as the driving force. But it will be very useful and
applicable to translate the above data to a measureable and or useful quantity. This work
being on virus filtration, hence the useful quantity reported here is the log reduction value
which is the critical deciding factor of accept/reject of final product. Primarily are the
synonymous terms Log Reduction Value (LRV) or Log Titer Reduction (LTR) which refer
to the virus reduction factor (Equation 5.42) defined as the logarithmic10 of the ratio of
the virus concentration (titer) in the feed (C f ) and the virus concentration (titer) in the
permeate stream (C p ). In some literature, LRV is also referred to as reduction factor.

Cf
LRV = log10 (5.42)
Cp
5.4.2 Simulation using gPROMS

gPROMS (General PROcess Modeling System) is a numerical modeling package pro-
duced by Process Systems Enterprise Limited. gPROMS finds extensive applications with
process systems involving systems of algebraic, differential and partial differential equa-
tions, and is optimised for these systems. gPROMS uses a modular structure for the devel-
opment of entities including model, process, estimation and experimental entities. Model
entities represent the inner working of the process and can be built in a single entity or in
a modular form. Process entities provide the operating conditions for the system, such as
in initial conditions, control parameters, and other solver information. Estimation and ex-
periment entities are used for parameter estimation and optimisation procedures, with the
estimation entity containing the guesses and solution parameters for the procedure and the
experiment entity containing the experimental data. Flows of data between model entities
are represented using streams which simply carry values from one model to another. The
gPROMS user guide is a good reference point for the descriptions of the modeling process
and a number of useful examples [gPROMS, 2004].
gPROMS is employed to solve the set of differential and algebraic equations for the
formulated model of filtration. The choice of this software is obvious because of its capa-
bility of solving distributed parameter systems and being a simple and friendly language
with strong numerical capabilities.
5.4.2.1 Model entities of the entire process
Each model entity has the equations that govern the behavior of the model, and incase
if more than one model is used, the streams used to connect the models are described as
well. The model entity also has the information on the solution method for the partial
differential equations if the model has partial differential equations (PDEs). The sections
used in the simulation of the models are Parameter, Distribution Domain, Unit, Variable,
Stream, Set, Boundary and Equation.
The Parameter sections lists any global parameters that are referred to in the models
that are defined in the Process entity. The Distribution Domain section defines any domains
required for the distributed variable and PDEs in the model and gives the upper and lower
limits on this domain. The Unit section defines any submodels that will be used within
the model and gives these models an identifying name that will be used within the model
equations. Units can either by singular or an array of units. The variable section lays out all
the variables that will be used within the model. The Stream section of the model defines
any Streams entering or leaving the model entity, and states the variables that are contained
within the Stream. The Set section of the model entity defines the approximation method
to be used for any distribution domains used in the model. This includes the method
of approximation for the derivatives (eg. forward difference method), the order of the
solution (first, second order derivatives, etc.) and the number of solution points to use. The
Equation section of the model entity has all the algebraic, differential or PDEs equations
and it has the flexibility to solve simultaneous equations [gPROMS, 2009a,b].
5.4.2.2 Parameters and variables employed in the model development
Various types of parameters and variables could be defined in gPROMS and are glob-
ally defined that do not change during the entire simulation of the process. Parameters
are defined as being either real numbers, integers or logical values. Parameters can also
be defined as arrays of values. Variables in gPROMS can be grouped as controlled vari-
ables which are defined by the process entity and describe variables that can be directly
controlled or process variables which can vary as the simulation proceeds. All variables in
gPROMS are defined using a unit type which are defined by the user in the variable type
entities section. Variables can also be defined as arrays of values, or as a distributed vari-
able for use in PDEs. Distributed variables are variables that vary continuously along a de-
fined domain and use distributed domains described in the relevant section of the gPROMS
model [gPROMS, 2009a,b]. Table 5.2 contains a summary of the partitioned sections avail-
able for the model entity. Table 5.3 contains simplified summary of the partitioned sections
available for the process entity.
5.4.3 Simulation results

Values used for model simulation are tabulated in Table 5.4 and the parameters used in
combination for the analysis of results is summarised as three cases in Table 5.5.
5.4.3.1 Representative particle size distribution in output streams
The distributions reported (both permeate and retentate streams) throughout this chap-
ter represents the number distribution percentage normalised to the original feed stream.
Table 5.2: Summary of the partitioned sections available for the model entity.
PARAMETER
Parameter declarations
VARIABLE
Variable declarations
DISTRIBUTION DOMAIN
Domain declarations
BOUNDARY
Boundary equations
TOPOLOGY
Unit connection equations
EQUATION
Model equations
INITIAL
Equations
Table 5.3: Simplified summary of the partitioned sections available for the process entity.
UNIT
Process equipment declarations
SET
Parameter value setting
INITIAL
Initial condition specfications
SOLUTION PARAMETERS
Model based activities specifications
SCHEDULE
Operating policy specifications
Table 5.4: Input data for the simulation of the model.

Parameters Values
Membrane pore size (average) 100 nm
Membrane area 0.0018 m2
Particle size of feed (average) 80-120 nm
Standard deviation of feed particle size 30 nm
Minimum feed particle size 10 nm
Weight percent concentration of particles to suspension in feed 0.1%
Feed flow rate 0.03 - 0.06 L/min
Transmembrane pressure drop 60-120 kPa
Table 5.5: Various input conditions utilised for the model simulations.
TMP, kPa CFR, L/min FPS, nm
Case 1 60 0.045 80,100,120
Case 2 60,90,120 0.06 100
Case 3 60 0.03,0.045,0.06 100
Figs. 5.10 & 5.11 show the permeate and retentate particle size distributions simulated for
the input conditions reported as in Table 5.5 with crossflow rate at 0.03 L/min (case 3). It is
clearly evident that during the initial stages of filtration particles easily permeate and over
time, particles are more of fractionating than separating. The effect of particle transport
across and or through the membrane is investigated with changing operating parameters
which is discussed as case studies in the next sections.
Figure 5.10: Representative particle size distribution in the permeate at TMP=90 kPa and
CFR=0.045 L/min.
Figure 5.11: Representative particle size distribution in the retentate at TMP=90 kPa and
CFR=0.045 L/min.
5.4.3.2 Effect of feed particle size on particle size distribution
Case 1: With increase in average particle size, the distribution peak shifts right (Fig.
5.12). For a given pore size, if the particle size increases, the amount of particles permeat-
ing through the membrane is getting restricted relatively and vice versa.
Figure 5.12: Particle size distribution of three range of feed particle sizes.
Figure 5.13: Particle size distribution of permeate for three feed particle size ranges.
5.4.3.3 Effect of transmembrane pressure drop on the particle size distribution
Case 2: At higher transmembrane pressures, it is found that peak of the number distri-
bution shifts upwards in the permeate stream (Fig. 5.14) and decreases in retentate stream
(Fig. 5.15). When there is an increase in TMP, there is a possibility for the pore elongation
during which more of the particles permeate which is leading to the increase in the peak.
This has a direct influence on the final filtration efficiency. It is observed that with increase
in TMP, there is a decrease in the LRV values (Table 5.6). At higher TMP, as can been
seen from the permeate particle size distribution, more particle permeation hence higher
the permeate concentration which decreases the log reduction value (Table 5.6).
Figure 5.14: Permeate particle size distribution with changing transmembrane pressure at
CFR=0.045 L/min.
5.4.3.4 Effect of cross flow rate on particle size distribution
Case 3: With increase in crossflow rate, there is a drop in the peak value of permeate
streams whereas increase in the retentate streams (Figs. 5.16, 5.17). Under conditions of
low cross flow velocity, residence time of the particles on the membrane surface is high
Figure 5.15: Retentate particle size distribution with changing transmembrane pressure at
CFR=0.045 L/min.
and it is found that mixture of particles permeate easily. But with increase in cross flow
rate, the more fraction of particles get convected in the retentate - this is attributed to the
fact that back transport of finer particles occur from the membrane surface on to the cross
flow. Also, at high cross flow rate, less of residence time leading to less of permeation
drag through the membrane. The previous literatures also indicate that there is a paradox
of both increase and decrease of particle drag along and across the membrane when there
is an increase in the cross flow velocity in tangential flow filtration [Foley et al., 1995a].
Nevertheless, in this study it is observed that with increase in the cross flow velocity,
the particles are tangentially carried along the membrane as retentate resulting in a better
rejection of bigger particles. This is reflected in the log reduction value with increase in
the cross flow rate (Table 5.6), there is an increase in the log reduction value meaning less
of particle permeation which is confirmed in the permeate particle size distribution.

Figure 5.16: Permeate particle size distribution with changing crossflow rate at TMP=90
kPa.
Figure 5.17: Retentate particle size distribution with changing crossflow rate at TMP=90
kPa.
Table 5.6: LRV at various TMPs and crossflow rates of smaller particles (80 nm avg.).
TMP, kPa LRV CFR, L/min LRV
60 1.16 0.03 0.99
90 1.03 0.045 1.03
120 0.86 0.06 1.21

Literature suggests several models are available for flux prediction and most models
do not take into account the influence of feed particle size and size distribution. We have
shown in this and previous chapters that feed particle size has a great deal of influence on
the final quality of the filtered product. A novel model formulation based on population
balances was developed to predict the particle size and its distribution in the filtered
product streams as well as the prediction of filtration flux. This modeling effort was
solved by a discretisation method and facilitated the understanding of the behaviour of
the filtration process under the effects of operating parameters such as cross flow rate,
pressure, and feed particle size. The validation of the model will be the subject of the
following chapter, in a parameter estimation exercise that uses data presented in Chapter
4.
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Chapter 6
Optimization-based Parameter
Estimation
Contents
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
6.2 Overview of Parameter Estimation . . . . . . . . . . . . . . . . . . . . . 196
6.3 Filtration Kinetic Parameter Estimation . . . . . . . . . . . . . . . . . . 197
6.4 Estimation Results and Discussion . . . . . . . . . . . . . . . . . . . . . 200
6.4.1 Parameter estimation summary . . . . . . . . . . . . . . . . . . . . 200
6.4.2 Overlay plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.5 Model Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.5.1 Validation against literature data . . . . . . . . . . . . . . . . . . . 208
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Preface
Kinetics of filtration flux together with associated mechanisms are well covered in the
literature. Whereas the kinetics related to the stochastic behavior of particle permeation
has not received much attention. The unknown parameters that emerge from the filtration
model presented in Chapter 5 are estimated using maximum likelihood theory in the cur-
rent chapter. Such method is an optimization-based approach that utilizes the non-linear
model and experimental data of the filtration and particle states. First, an overview of
parameter estimation is given before presenting details of the estimation problem. The
results of the parameter estimation exercise are then presented and finally the model with
its estimated parameters is validated against data from literature.
Research Questions:
1. What are the feasibility of estimation of filtration kinetic parameters via
optimization-based parameter estimation techniques?
2. Can the model be validated with (i) our experimental results and (ii) available data
from others’ works?
3. What potential does this kinetic parameter estimation approach have for filtration
kinetics identification in general?
6.1 Introduction
Several membrane transport models have been proposed for flux decline and fouling
mechanisms based on film theory, solution diffusion model, concentration polarization
models to name a few. All these models invariably employ simple linear or nonlinear meth-
ods or trial and error to estimate the parameters that emerge from those models (among
others [Cassano et al., 2007; Denisov, 1994; Murthy and Chaudhari, 2008]). For example,
the kinetics of cake layer formation during cross flow filtration is studied by Knyaz’kova
and the pore blocking model is solved by linear dependence of flux-volume [Knyaz’kova,
1995]. Similar studies have been reported elsewhere for cross flow filtration [Baruah et al.,
2005; Jiao and Sharma, 1994; Mackley and Sherman, 1992a]. The simple models used in
filtration kinetic studies do not capture the true kinetics and are limited because temporal
data of particle size are rarely used. Our model presented in Chapter 5 defines the filtration
kinetics relating to the stochastic behavior of particle permeation. The model is itself part
of the parameter estimation formulation and along with size and concentration temporal
data is used to estimate unknown parameters. This approach is unique, providing a sys-
tematic way to determine kinetic parameters and thus bridges a gap towards more accurate
estimation of filtration kinetics.
Parameter estimation, also called data regression is an important step involved in the
formulation and validation of mathematical models. The model kinetic system contains
parameters (recall Chapter 5) that need to be identified via a parameter estimation step,
the latter being an important prerequisite step for simulation, dynamic optimization, and
model-based control. Essentially, the parameter estimation problem is an optimization one
where the model is fitted to the experimental data. Once the parameters are estimated, the
identified model maybe used for predictive simulation analysis where the various process
aspects may be elucidated, and used for optimization and control scheme development. A
number of efficient and robust methods have been developed depending upon the structure
of objective functions. Various criteria relating to parameter estimation problems include:

• Model framework: defined for algebraic and differential equation models as well as
for linear or nonlinear models.
• Objective function selection: refers to a scalar function with dependence to the cho-
sen parameters. The choice of the objective function dictates the value of the esti-
mates as well as the statistical properties.
• Solution techniques: developed for specific algorithms and methods to minimize or
maximize the objective function. Generally, the parameter estimation problem is
solved by any one of the five optimisation problems (Table 6.1).
• Statistical properties: uncertainty in model parameters as well as in calculated val-
ues.
• Model adequacy: validations to how good the model responds to the system.
Table 6.1: The types of parameter estimation problems.

Parameter Estimation Methods Description
Choosing the best function to min- The sum of the differences between the model and experi-
imise mental data is minimised. This is known as method of least
squares.
Minimisation of the chosen func- Minimisation of the chosen function with respect to the pa-
tion rameters where the value(s) of estimated parameter(s) at the
minimum represents the optimum.
Optimal design of experiments I Optimal design of experiments to obtain the best parameter
estimates. Usually the model structure is known.
Optimal design of experiments II Optimal design of experiments to choose a model structure
from several competing models
Infinite number of model structures Determination of the model structure when there is a lack
of data which is considered to be the toughest parameter
estimation methods. This is known as system identification
problem.
The key feature of our approach of parameters estimation is using maximum likeli-
hood function. It determines values for the unknown parameters in order to maximize the
probability that the mathematical model will predict the values obtained from the exper-
iments. The complete details of this technique are available in [gPROMS, 2004, 2009a].
The overall scheme employed in a typical parameter estimation strategy is shown in Fig.
6.1.
Figure 6.1: Flowchart showing the various steps involved in parameter estimation: pre-
estimation and post-estimation checks in arriving at the accurate values.
It is a known fact that the parameter estimation is extremely sensitive to initial guesses
6.2. Overview of Parameter Estimation 196
provided for the unknown parameters. If the initial guesses are not close to the final solu-
tion, the estimation procedure fails. It is thus suggested that first the model parameters are
determined crudely such that simulated trends follow the experimental results. In the rest
of this chapter, optimization-based parameter estimation is presented with specific focus
on our filtration kinetics problem.
6.2 Overview of Parameter Estimation

The model developed and described in Chapter 5 has unknown parameters (k1 ,k2 ,k3 )
to be estimated. Generally, models are greatly improved and understood by experimental
studies. The data obtained from the experimental investigation discussed in Chapter 4 are
employed for this estimation exercise.
The following details are specified in a parameter estimation exercise:
• The initial condition in the experimental run
For the model developed, the number density of the particles at time t=0 is zero.
• The duration of the experimental run
The experiment is carried out for 600 secs
• Values of time-independent quantity
The operating parameters i.e. transmembrane pressure (range 20-60 kPa), cross-
flow velocity (0.3-1 L/min)
The input parameters i.e. feed particle size (average particle size of 3 different
ranges lying 35.6-142.9nm.)
• Time variant output quantities

6.3. Filtration Kinetic Parameter Estimation 197
Permeate concentration and particle size
Parameter estimation problem is solved using gEST tool of gPROMS package. The
MXLKHD solver, an indirect solver, is used where the global optimum is found by ap-
plying sequential quadratic programming method. This solver takes advantage of the DA-
SOLV solver for solution of the model equations.
6.3 Filtration Kinetic Parameter Estimation

The filtration model described in Chapter 5 and the equations constituting the model
can be represented by Y(t) = [D(t), C(t)], U = [TMP, CFR] as the manipulated variables
and θ representing the vector of parameters to be identified which in this case is [k1 , k2 ,
k3 ]. The problem can be formulated as follows:
f (Y(t), U, θ) = 0 (6.1)
Equation 6.1 is accompanied by a set of initial conditions for the differential equation
of the model and these can be collated into the vector Y(0) = y, where y is a set of values
in this case representing [D(0), C(0)]. For each experiment the measured variables are
denoted as follows:
Ŷ(t) = [D(t),
ˆ C(t)]
ˆ (6.2)
Two criteria are generally used in parameter estimation, namely the least squares or
maximum likelihood. Maximum likelihood is a mathematical optimization technique that
attempts to find a best fit to a set of data by minimizing the error between the experimen-
tal measurements and predicted values. The least squares is effectively a special case of
maximum likelihood method.
Considering the problem specified the maximum likelihood criterion that describes the
highest probability of the model predicting the real data is given by the following objective
function:
⎧ ⎡ ⎤⎫
N 1 ⎪ NE NVi N Mi j ⎢⎢
⎨ ⎢ (Yî jk − Yi jk )2 ⎥⎥⎥⎪⎬
Φ(θ) = ln(2π) + min
. ⎪
⎩ ⎢⎣ln(σ i jk ) +
2 ⎥⎦⎪
⎭ (6.3)
2 2 i=1 j=1 k=1 σ i jk
2
where N is the number of measurements taken, NE is the number of experiments, NVi
is the number of variables measured in the ith experiment, N Mi j is the number of measure-
ments of the jth variable in the ith experiment, σ2i jk is the variance of the kth measurement of
variable j in experiment i. The variance σ2i jk can be described by any of a number of models
available when the error structure of the data is known [gPROMS, 2009b]. These are ei-
ther homoscedastic where the variance error is assumed to be constant, or heteroscedastic
where the variance error is a function of the measured and predicted values and may be
proportional to Ŷ(t)γ/2 and Y(t)γ/2 , where γ is a parameter determined in the optimisation
range which is between 0 and 1.
In the homoscedastic case, used in this work, the variance error may be determined by
analyzing similar sets of experimental data. The value of the variance can be specified in
the estimation as:
σ2i jk = ω2i jk (6.4)
where ω is the constant standard deviation of the measurement error and the objective
function is given as follows:
⎧ ⎡ ⎤⎫
⎪
⎨ NVi N Mi j ⎢⎢⎢ (Yî jk − Yi jk )2 ⎥⎥⎥⎪⎬
Φ(θ) = min ⎩ j=1 k=1 ⎢⎣ln(ω i jk ) + ⎥⎦⎪
2
. ⎪ ω2 i jk ⎭ (6.5)
For some other cases, more information is available about the data, and the error struc-
ture may be known to depend on the magnitude of the measured or predicted values. For
example, it may be known that Ŷ(t) increases with increase in the variance of Ŷ(t). For
these cases, the heteroscedastic model of variance is employed:
σ2i jk = ω2i jk Ŷi2jk (6.6)
In the above context, the maximum likelihood criterion becomes a weighted least
squares problem as follows:
⎧ ⎡ ⎤⎫
⎪
⎨NVi N Mi j ⎢⎢⎢⎢
⎪ (Yî jk − Yi jk )2 ⎥⎥⎥⎪ ⎪
Φ(θ) = min
. ⎪ ⎢⎣ln(ω i jk Ŷi jk ) +
2 2 ⎥⎥⎦⎬
⎪ (6.7)
⎪
⎩ j=1 k=1 ω2 i jk Ŷi2jk ⎪ ⎭
By this definition, the error weights, ωi jk , are chosen to reflect the relative level of
confidence in the measurements. This leads to the minimisation of the objective function
with more weight given to the data that has less error. For pure heteroscedastic variance
models, the structure of the error takes the more complex form as
σ2i jk = ω2i jk [Ŷi2jk ]γ (6.8)
If γ has the value of 0, Equation 6.8 reduces to Equation 6.4 and at the other extreme,
where γ has the value 1, Equation 6.8 becomes equal to Equation 6.6.
6.4. Estimation Results and Discussion 200
For our problem, the measured variables D(t) and C(t) are described by constant rel-
ative variance model on the measured values. The choice of this model is justified as the
measurement errors in both variables are constant and do not depend on the magnitude of
the measurement. It is possible using the gEST parameter estimation facility in gPROMS
to specify the parameter estimation problem according to any one type of variance models
mentioned above. gEST also allows for the determination of ω along with θ as part of the
optimisation of the estimation. The specification of the variance model is a simple pro-
cedure in gPROMS and can typically be shown as in Table 6.2 for the above mentioned
measurements of parameter estimation problem.
Table 6.2: Specification of the variance model in the gEST file.

Measure
Permeate.Mean2 [i.e. D(t)]
CONSTANT-RELATIVE-VARIANCE (1 : 0.01 : 3)
Measure
Permeate.Cp [i.e. C(t)]
CONSTANT-RELATIVE-VARIANCE (1 : 0.01 : 3)
6.4 Estimation Results and Discussion
6.4.1 Parameter estimation summary

Table 6.3 shows the summary of the parameter estimation stage indicating the constant
relative variance model being employed for the measured variables (D(t) and C(t)). The
objective function contributions to each experimental variable associated in the kinetic
parameter estimation is also listed. It shows that the objective function of both D(t) and
C(t) are of the same order for the measured experimental data.
Table 6.4 summarizes the estimated values of the model parameters. As mentioned
earlier, constant relative variance model is employed for the parameters estimates along
6.4. Estimation Results and Discussion 201
Table 6.3: Parameter estimation summary showing the number of experiments used for
estimating the parameters, the variables D(t) and C(t) measured, variance model employed
and the objective function contribution.
Variables Measured Variance Model Objective Function Contribution
Experiment 1: PEB1
PERMEATE.MEAN2 CONSTANT RELATIVE VARIANCE -64.442
PERMEATE.CP CONSTANT RELATIVE VARIANCE -51.841
Experiment 2: PEB2
Experiment 3: PEB3
Experiment 4: PEM2
Experiment 5: PEM3
Experiment 6: PES1
Experiment 7: PES2
Experiment 8: PES3
with with initial guess of ω = 0.1 and lower and upper bounds are 0.01 and 3.0 respectively.
Table 6.4: Model parameter estimates.

Parameter Optimal Estimate
k1 75.9
k2 -4.20
k3 2.91*10−3
Table 6.5 shows the correlation matrix, R, which is calculated from the variance co-
variance matrix (Vi j ) as follows:
Vi j
Ri j = ,i j (6.9)
Vii V j j
Ri j = 1, i = j (6.10)
6.5. Model Validation 202
Values close to 1 in the off diagonals indicate a high correlation of the corresponding
factors and vice versa. So, in our work, it is found that k1 , k2 , and k3 are not very highly
correlated.
Table 6.5: Correlation matrix.

Parameter Optimal Value Parameter Number 1 2 3
k1 75.9 1 1.00 -.999 0.516
k2 -4.2 2 -.999 1.00 -0.520
k3 2.91*10−3 3 0.516 -0.520 1.00
6.4.2 Overlay plots

As explained earlier, the estimation problem is solved by the gEST facility and the
optimal solution points of the parameters are recorded in Table 6.4. The estimated values
obtained are found to fit the experimental values satisfactorily which is reflected in Table
6.6, i.e. weighted residual value is much less than χ2 -value.
Table 6.6: Lack of fit test.
Weighted Residual χ2 value (95%) Comment

34.5 66.339 Good fit: weighted residual less than χ2 -value
6.5 Model Validation

The optimal estimated parameters thus obtained (Table 6.4) are employed for model
prediction as shown in Figs. 6.2 & 6.3. The model fit is satisfactory whereas the mismatch
observed between the measured values and the predicted values are attributed to experi-
mental and characterization errors, but unmodelled mechanisms could also contribute to
this mismatch.
The model is now validated with the optimally estimated parameters, and simulation
analysis can be carried out. First, the model simulations of the output streams are shown
6.5. Model Validation
203
Figure 6.2: Comparison of measured values and predicted values during the parameter estimation step.
6.5. Model Validation
204
Figure 6.3: Comparison of measured values and predicted values during the parameter estimation step.
for three different particle sizes (Figs. 6.4, 6.5, 6.6 & 6.7). It is important to note that such
model simulation results in the area of filtration, mechanistic understanding of particle size
distribution considering the operating parameters (TMP and CFR) and the input conditions
(FPS) are unique to this study. Experimental results discussed in Chapter 4 are employed
for validation purposes. Specifically, the model predictions of filtration efficiency and
permeate particle size distribution are tested. The results of this validation are presented
next.
Figure 6.4: Simulation results of permeate particle size distribution for bigger sized parti-
cles (140nm avg.) at TMP=40 kPa and CFR=0.6 L/min.
Figure 6.5: Simulation results of retentate particle size distribution for bigger sized parti-
cles (140nm avg.) at TMP=40 kPa and CFR=0.6 L/min.
Figure 6.6: Simulation results of permeate particle size distribution for medium sized par-
ticles (80nm avg.) at TMP=40 kPa and CFR=0.6 L/min.
Figure 6.7: Simulation results of retentate particle size distribution medium sized particles
(80nm avg.) at TMP=40 kPa and CFR=0.6 L/min.
The filtration efficiency values are plotted to compare the model predictions with the
experimental values. It is clearly evident that model predictions agree well qualitatively
with the experimental results but it is important to put forth the fact that variation is ob-
served in the bigger particles (Fig. 6.8). The deviation in the results may be attributed to
the fact that the model suffers in calculating the solid permeate concentration as it is more
dependent on the estimated parameters. More experiments will help to resolve this.
Figure 6.8: Model predictions vs. experimental results of filtration efficiency at TMP=40
kPa and CFR=0.6 L/min.
Now, the mean diameter obtained from the model and the experimental results are
compared and it is quite evident that both agree well (Fig. 6.9). The deviation is more
pronounced in the case of smaller particles in comparison with the medium and bigger
particles owing to the fact that the smaller particles easily pass through the membrane
wherein particle sizes are represented in a distributed form but pore size is not defined
as a distribution but in the actual scenario it has a distribution. Literature also reports
that the membrane has bigger pores which are not covered in the average pore calculation
[Amirilargani et al., 2009; Sun et al., 2007]. The model suffers this limitation of non
inclusion of pore size distribution which is not addressed in this thesis.
Figure 6.9: Model predictions vs. experimental results of permeate particle size for all
three particle size ranges at TMP=40 kPa and CFR=0.6 L/min.
The model’s ability to predict particle size distribution is also validated (Figs. 6.10,
6.11 & 6.12). The model demonstrates a fair ability in predicting the permeate particle
size distribution. The variation observed in the particle size distribution of the smaller
particles is due to model’s inability to predict the multimodal distribution. This result
represents a milestone in UF modeling and answers the research questions posed at the start
of this chapter, that is, that the optimisation-based estimation can be a feasible approach
for filtration kinetics calculations towards validated UF models.
6.5.1 Validation against literature data

In this section, the model is validated with other researchers’ literature data. Though
a number of research works are available in virus filtration, as mentioned earlier, very
limited data is available with respect to the particle size focus. Here, Sano et al.’s [Sano
et al., 2006] experimental results are used to validate the model for the filtration efficiency
prediction. Sano’s work does not deal with the particle size distribution but it is exploited
Figure 6.10: Model predictions vs. experimental results of permeate particle size distribu-
tion at TMP=40 kPa and CFR=0.6 L/min of bigger sized particles.
tion at TMP=40 kPa and CFR=0.6 L/min of medium sized particles.
tion at TMP=40 kPa and CFR=0.6 L/min of smaller sized particles.
for the log reduction value prediction results only. In their work, removal of indigenous
noroviruses through microfiltration and UF membranes of sewage sludge and waste water
were studied. Table 6.7 gives the data used for predicting the filtration efficiency.
Table 6.7: Validation of the model with the Sano et al.’s [Sano et al., 2006] results.
Sample Sample Membrane Norovirus size, nm TMP, kPa LRV (Sano et al.) LRV (Our model)
Case 1 Sewage Sludge MF (0.1 μ) 20-30 50.00 4.00 4.05
Case 2 Waste water MF (0.1 μ) 20-30 50.00 2.17 2.00
Case 3 Sewage Sludge UF (100 kDa) 20-30 100.00 5.07 4.60
Case 4 Waste water UF (100 kDa) 20-30 100.00 3.19 3.90
It clearly shows that the model predicts quite precisely in two cases (i.e. Case 1 and 2)
whereas, it differs by close to 10 % in Cases 3 & 4. These results are a clear indication of
the viability of the model for filtration efficiency predictions knowing the input condition
(original particle sizes of the feed stream) and for any operating parameters. The particle
size distribution validation becomes difficult because of the lack of literature data.
To conclude, the current novel model successfully predicts the more vital indicator of
filtration, i.e. log reduction value of virus filtration, the permeate particle size and distri-
bution which have a significant role especially during complete virus removal operation
and/or fractionation of proteins during the filtration mechanism. A number of limitations
in the current form of the model needs to be highlighted: (i) pore size distribution is not
modelled and (ii) multimodal size distribution prediction is lacking. From the above vali-
dation studies, it is very clear that the model can serve as promising tool for optimization
studies which are capable of predicting the optimum operating parameters to maximize the
filtration efficiency and to obtain the optimum permeate particle size.

This chapter has successfully estimated the unknown parameters of the model pre-
sented in Chapter 5. This estimation was based on maximum likelihood theory and repre-
sents a unique approach in the field of filtration kinetic identification. The model predic-
tions are shown to be in very good agreement with the experimental results, thus verifying
the validity of the model for log reduction value and the permeate particle size prediction.
The model was also found to be in close agreement with filtration efficiency literature data.
The validated model demonstrated its prediction capability through simulations of the per-
meate particle size distribution under various TMP and CFR conditions. The model is thus
proposed as a key tool for scale-up of UF operations.
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Chapter 7
Conclusions and Future Directions
Contents
7.2 Recommendations for Future Directions . . . . . . . . . . . . . . . . . . 218
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Tangential flow UF of silica particles (model virus particles) was investigated to iden-
tify the significant factors that affect the filtration efficiency. Factors considered were feed
particle size and the operating parameters transmembrane pressure and cross flowrate. Ex-
perimental results indicate that feed particle size has a great deal of influence on surrogate
virus particle log reduction efficiency in UF. The proper selection of operating parameters
coupled with proper membrane selection will result in better filtration efficiency. Hav-
ing identified the feed particle size as a prominent factor for the filtration mechanism, a
detailed investigation on the feed particle size on the permeate quality was then carried
out. This filtration efficiency investigation showed that among the two operating param-
eters considered, transmembrane pressure plays a major role in controlling the filtration
efficiency in comparison with flowrate. In the studied experimental range, higher LRV
values are obtained at lower TMP (20 kPa) and at higher feed flowrate (1 L/min). Further,
the effect on LRV of the interaction between TMP and FPS seems to be more significant
than that of the interaction of flowrate with FPS. The empirical statistical model developed
serves as a powerful tool for studying the impact of the key operating parameters on the
permeate quality (Figs. 4.9 & 4.13) and can be employed for optimisation of operating
parameters to achieve maximum efficiency. The above findings are hereby summarized:
• The feed particle size, transmembrane pressure and cross flowrate have a significant
role in the final filtration efficiency. Among the above three parameters considered,
FPS and TMP are seen to be major key factors with CFR’s significance being rel-
atively less. It is very insightful to note that the interplay of parameters plays an
equally important role as well. It has been identified that interplay of TMP and FPS
are more influential than TMP and CFR or CFR and FPS. This necessitates a further
investigation on the feed particle size on the separation or fractionation mechanism.
• The effect of above factors was quantified in an empirical model development. The
model results obtained were compared with experimental results and found to be
in in excellent agreement. Hence, the model obtained from this study could, given
TMP and CFR, be employed to predict the filtration efficiency and the feed particle
property within the studied experimental range.
• The above study provided clear indications about the dependencies of filtration effi-
ciency on these factors. This will serve as an excellent approach/method to analyze
the effects of influential parameters on product efficiency for larger scale operations.
In a second experimental investigation, the impact of size effect on the filtration mech-
anism (i.e. rejection and/or fractionation) was considered. For fractionation of particles,
suitable transmembrane pressure should to be chosen whereas if the particles are to be
separated, cross flow velocity is the key factor for better efficiency of membrane filtration.
From the experimental range employed in this study, it was found that the lower the trans-
membrane pressure, the more of separation, while the higher the transmembrane pressure,
a combination of separation and fractionation occurs. Whereas with change in cross flow
velocity, separation mechanism dominates and has good control over filtration efficiency.
Also the polydispersity study showed that the lower the dispersion of particles, the higher
the filtration efficiency attributed to the lower levels of inter particle forces. Detailed sta-
tistical investigation indicated that permeate particle size follow a polynomial dependency
on the operating parameters whereas permeate PDI varies linearly with the operating pa-
rameters. The particle level study covered in this article offers opportunities for academic
and industrial researchers to gain fundamental insights into how particle size properties are
linked to permeate quality.
The ultimate purpose of modeling filtration processes is the mathematical understand-
ing of the dynamic behavior of the process. A validated model can be implemented in sim-
ulation, optimization and control of the process, overall helping to achieve desired product
characteristics. Typical filtration models found in literature focus on flux predictions and
fouling mechanisms. Our comprehensive filtration model, based on population balances,
is novel approach in the field of virus filtration. It takes into account individual parti-
cles and their particle size property, equations of conservation, and kinetic submodel. The
model developed is capable of successfully predicting the permeate particle size and size
distribution as well as final filtration efficiency. This represents a big leap in understanding
filtration mechanisms and process behaviour and importantly can be applied in scale-up
studies. It is also important to note down the limitations of the model when applied for
industrial settings:
• The model assumes that fouling is negligible which may be a limiting factor in in-
dustrial applications.
• The input parameters are to be characterized precisely which are the input conditions
in the model which have a direct influence on the accuracy of final predicted values.
For example, measuring particle size in the feed is a challenge and therefore it limits
the use of this methodology in the industrial setting.
• Another issue with regard to the feed particle size, in industrial setting, there may
be some particles which are beyond the defined range of particle size in the model.
This may affect the final results.
• Information technology issue is another challenge in implementing the model in the
industrial setting. It needs to be overcome as the model will need to receive data
from the process on the fly (i.e. in real time). The speed of the model here becomes
a limitation; it needs to be recorded in a faster algorithm.
• During inline testing in industrial settings, intermittent manual monitoring may be
required to check the particle size measurement and concentration in order to avoid
some erratic behavior.
Finally, the last part of this study successfully estimates the unknown filtration kinetic
parameters based on maximum likelihood theory. This optimisation-based approach uses

7.2. Recommendations for Future Directions 218
the developed non-linear model. The model was shown to provide a good fit to the experi-
mental data. Minor mismatch between model output and experimental data was observed
in quantitative behavior of the filtration efficiency values for the bigger feed particles. The
model predictions in the form of particle size distribution agree well with the experimental
results for medium and bigger particles, whereas smaller particles present some mismatch.
This was attributed to experimental errors as well as unmodelled filtration mechanisms and
highlights the need for more experimental data as well as the importance of incorporating
multimodal distribution forms in the model. The model was then shown to be closely
matching with literature data on LRV. The optimisation-based parameter estimation ap-
proach is regarded as a milestone in filtration kinetic parameter estimation which lends a
stong hand in developing validated fitlration models more complex than those that solely
depend on filtration flux kinetics.
7.2 Recommendations for Future Directions

The first part of this study in Chapter 4 involving the filtration of three different particles
can be extended to include:
• A more extensive investigation in which bigger and smaller feed particles are mixed.
This will provide more understanding about the effect of particle-particle interaction
on the final filtration efficiency.
• The role played by the membrane pore size distribution on particle-pore interactions
and the impact this has on the mechanism of separation and/or fractionation.
The second part in Chapter 4 elucidating the role of transmembrane pressure and the
cross flow velocity on the separation and fractionation mechanisms respectively can be
7.2. Recommendations for Future Directions 219
further improvised:
• It will be insightful to conduct similar set of experiments with other types of mem-
branes.
• In this work, model virus particles were employed for the investigation, but it will
be useful to employ other model viruses like bacteriophage to examine the effect of
virus characteristics on the filtration efficiency behavior.
• Virus surface charge characteristics can be studied to analyse for any effect on filtra-
tion efficiency.
Chapter 5 demonstrates that the novel model developed successfully predicts the parti-
cle size distribution of the output streams. But the mismatch between the model simulation
output and the experimental results indicates that other factors need to be accounted in the
modeling which includes:
• Pore size distribution of the membrane plays a role in affecting the final filtrate par-
ticle size distribution and hence inclusion of pore size distribution may have a sig-
nificant impact in arriving at exact predictions.
• Defining multi-modal distributions as well pore blockage mechanisms may improve
the model significantly.
• Virus particle aggregation and breakage are not considered in our current model,
hence it is highly recommended to incorporate these factors in the future models.

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NOVEL MODEL FORMULATION FOR

VIRUS REMOVAL BY TANGENTIAL FLOW

Angayar Kumari Pavanasam

A thesis submitted in fulﬁlment of the requirement for the

School of Chemical Engineering

The University of New South Wales

Sydney 2052 AUSTRALIA

it contains no materials previously published or written by another person, or substantial

I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also

presentation and linguistic expression is acknowledged.’

Dissertation Abstract International. I have either used no substantial portions of copyright

copy of my thesis or dissertation’.

Signed: .......................... Date: .........

Mom - Gnanam, Dad - Pavanasam, Sister - Selvi, & Brother - Prabhu

critical time helped me a lot to push myself to complete on time.

opportunity to learn and prosper.

timely reminders regarding the reviews and courses.

thanks for those (silent) words of wisdom and memories.

with all my tantrums.

ANGAYAR KUMARI PAVANASAM

perienced small start-up ﬁrms. Membrane ﬁltration, being non-invasive, non-destructive

traﬁltration being an eﬃcient process for removing macromolecules, colloids, endotoxins,

viruses, and bacteria.

model outputs. A novel approach in the form of optimization-based parameter estimation

scale-up of ultraﬁltration processes.

Keywords: Ultraﬁltration, population balance, virus ﬁltration

Publications based on this work

Refereed Journal Articles

view of Recent Patents", Recent Patents on Chemical Engineering, 151-156, 1(2),

ating Parameters on Virus Ultraﬁltration Eﬃciency", Water Science and Technology:

Water Supply, 31-38, 11(1), 2011.

Size and Polydispersity of Model Virus Filtration", Biotechnology and Bioprocess

Tangential Flow Ultraﬁltration: A Novel Approach", Chemical Engineering Science

(Under review for submission).

Balance Model for Ultraﬁltration Processes", Engineering With Membranes (EWM

2008), Portugal, May 2008.

ultraﬁltration processes: A population balance approach, International Congress on

Membranes and Membrane Processes (ICOM 2008),Hawaii, July 2008.

ating parameters on virus ultraﬁltration eﬃciency, International Water Association -

Membrane Technology Conference (IWA-MTC 2009), Beijing, September 2009.

tration, EuroMembrane (EM 2009), France, September 2009.

dents Conference, Wollongong, Australia, February 2010.

Virus Removal by Tangential Flow Ultraﬁltration", Dean’s Award for Excellence in

Postgraduate Research, University of New South Wales, Sydney, November 2008.

cellence in Postgraduate Research, University of New South Wales, Sydney, October

AeDNV Aedes aegypti DensoNucleosisVirus

AF4 Asymmetric Flow Field Flow Fractionation

ALV Avian Leukemia Virus

ATCC American Type Culture Collection

BHK Born Hamster Kidney cells

BVDV Bovine Viral Diarrhea Virus

B19 Human Parvovirus

CFR Cross Flow Rate

CHO Chinese Hamser Ovary

CJD CreutzfeldJacob disease

CMR Comprehensive Microbial Resource

CPV Canine Parvo Virus

DEF/NFF Dead End Filtration/Normal Flow Filtration

DLS/MALS Dynamic Light Scattering/MultiAngle Light Scattering

EBV Epstein-Barr Virus (also known human herpesvirus 4)

EMC Encephalo MyoCarditis

FCS Fetal Calf Serum

FDA Food and Drug Administration