New hydrogen-bonding organocatalysts: Chiral cyclophosphazanes and phosphorus amides as catalysts for asymmetric Michael additions

Article abstract of DOI:10.3762/bjoc.10.18

Ten novel hydrogen-bonding catalysts based on open-chain PV-amides of BINOL and chinchona alkaloids as well as three catalysts based on rigid cis-PV-cyclodiphosphazane amides of N1,N1-dimethylcyclohexane-1,2-diamine have been developed. Employed in the asymmetric Michael addition of 2-hydroxynaphthoquinone to β-nitrostyrene, the open-chain 9-epi-aminochinchona-based phosphorus amides show a high catalytic activity with almost quantitative yields of up to 98% and enantiomeric excesses of up to 51%. The cyclodiphosphazane catalysts show the same high activity and give improved enantiomeric excesses of up to 75%, thus representing the first successful application of a cyclodiphosphazane in enantioselective organocatalysis. DFT computations reveal high hydrogen-bonding strengths of cyclodiphosphazane PV-amides compared to urea-based catalysts. Experimental results and computations on the enantiodetermining step with cis-cyclodiphosphazane 14a suggest a strong bidentate H-bond activation of the nitrostyrene substrate by the catalyst.

Full text of DOI:10.3762/bjoc.10.18

PROCESS SCALE
PURIFICATION OF
ANTIBODIES
Edited by
UWE GOTTSCHALK
Group Vice President
Purification Technologies
Sartorius Stedim Biotech
A JOHN WILEY & SONS, INC., PUBLICATION

PROCESS SCALE
PURIFICATION OF
ANTIBODIES

PROCESS SCALE
PURIFICATION OF
ANTIBODIES
Edited by
UWE GOTTSCHALK
Group Vice President
Purification Technologies
Sartorius Stedim Biotech
A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2009 by John Wiley & Sons, Inc. All rights reserved.
Published by John Wiley & Sons, Inc., Hoboken, New Jersey.
Published simultaneously in Canada.
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in
any form or by any means, electronic, mechanical, photocopying, recording, scanning, or
otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright
Act, without either the prior written permission of the Publisher, or authorization through
payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222
Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at
www.copyright.com. Requests to the Publisher for permission should be addressed to the
Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030,
(201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission.
Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best
efforts in preparing this book, they make no representations or warranties with respect to the
accuracy or completeness of the contents of this book and specifically disclaim any implied
warranties of merchantability or fitness for a particular purpose. No warranty may be created
or extended by sales representatives or written sales materials. The advice and strategies
contained herein may not be suitable for your situation. You should consult with a professional
where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any
other commercial damages, including but not limited to special, incidental, consequential, or
other damages.
For general information on our other products and services or for technical support, please
contact our Customer Care Department within the United States at (800) 762-2974, outside the
United States at (317) 572-3993 or fax (317) 572-4002.
Wiley also publishes its books in a variety of electronic formats. Some content that appears in
print may not be available in electronic formats. For more information about Wiley products,
visit our web site at www.wiley.com.
Library of Congress Cataloging-in-Publication Data
Process scale purification of antibodies / [edited by] Uwe Gottschalk.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-0-470-20962-2 (cloth)
1. Monoclonal antibodies—Purification. 2. Biochemical engineering. I. Gottschalk, Uwe.
[DNLM: 1. Antibodies, Monoclonal—isolation & purification. 2. Antibodies,
Monoclonal—therapeutic use. 3. Chromatography—methods. 4. Technology,
Pharmaceutical—methods. QW 575.5.A6 P963 2009]
TP248.65.M65P76 2009
615′.19—dc22
2008038592
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1

CONTENTS
Preface
xvii
xxi
About the Author
Contributors
xxiii
1
Downstream Processing of Monoclonal Antibodies:
Current Practices and Future Opportunities
Brian Kelley, Greg Blank, and Ann Lee
1
1.1
1.2
Introduction, 1
A Brief History of cGMP mAb and Intravenous
Immunoglobulin (IgIV) Purification, 2
Current Approaches in Purification Process Development:
Impact of Platform Processes, 4
Typical Unit Operations and Processing Alternatives, 8
VLS Processes: Ton-Scale Production and Beyond, 11
Process Validation, 13
Product Life Cycle Management, 15
Future Opportunities, 17
Conclusions, 20
1.3
1.4
1.5
1.6
1.7
1.8
1.9
1.10
1.11
Acknowledgments, 21
References, 21
v

vi
CONTENTS
2
The Development of Antibody Purification Technologies
25
John Curling
2.1
2.2
Introduction, 25
Chromatographic Purification of Antibodies before
Protein A, 27
2.3
2.4
2.5
Antibody Purification after 1975, 31
Additional Technologies for Antibody Purification, 32
Purification of mAbS Approved in North America and
in Europe, 37
2.6
2.7
Acknowledgments, 44
References, 45
3
Harvest and Recovery of Monoclonal Antibodies: Cell Removal
and Clarification
53
Abhinav A. Shukla and Jagannadha Rao Kandula
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
Introduction, 53
Centrifugation, 56
Microfiltration, 58
Depth Filtration, 64
Flocculation, 68
Absolute Filtration, 70
Expanded-Bed Chromatography, 72
Comparison of Harvest and Clarification Unit Operations, 73
Acknowledgments, 74
References, 75
4
Protein A-Based Affinity Chromatography
79
Suresh Vunnum, Ganesh Vedantham, and Brian Hubbard
4.1
4.2
Introduction, 79
Properties of Protein A and Commercially Available
Protein A Resins, 80
4.2.1
4.2.2
4.2.3
4.2.4
4.2.5
4.2.6
4.2.7
4.2.8
4.2.9
Protein A Structure, 80
Protein A–Immunoglobulin G (IgG) Interaction, 80
Stoichiometry of Protein A–IgG Binding, 81
Protein A Stability, 81
Commercial Protein A Resins, 81
Static Capacity, 83
DBC, 83
Leaching, 84
Production Rates, 85
4.3
Protein A Chromatography Step Development, 85
4.3.1
4.3.2
Loading/Binding, 86
Wash Development, 87

CONTENTS
vii
4.3.3
4.3.4
4.3.5
Elution, 88
Stripping, 89
Regeneration and CIP, 89
4.4
4.5
4.6
Additional Considerations During Development and
Scale-Up, 90
4.4.1
4.4.2
4.4.3
Controlling HMW Formation, 91
Removal of Soluble HMW Contaminants, 91
Turbidity, 92
Virus Removal/Inactivation, 94
4.5.1
4.5.2
4.5.3
Virus Removal, 95
Low-pH Inactivation, 95
Bovine/Transmissible Spongiform Encephalopathy
(BSE/TSE) Clearance, 96
Validation and Robustness, 96
4.6.1
4.6.2
Validation, 96
Robustness, 97
4.7
4.8
4.9
Conclusions, 97
Acknowledgments, 98
References, 99
5
Purification of Human Monoclonal Antibodies: Non-Protein A
Strategies
103
Alahari Arunakumari and Jue Wang
5.1
5.2
Introduction, 103
Integrated Process Designs for Human Monoclonal
Antibody (HuMab) Production, 105
Purification Process Designs for HuMabs, 106
5.3
5.3.1
5.3.2
5.3.3
Protein A Purification Schemes, 106
Non-Protein A Purification Schemes, 107
Host Cell Protein (HCP) Exclusion Approach for
Ion-Exchange Purification Schemes, 108
5.4
5.5
5.6
Conclusions, 119
Acknowledgments, 121
References, 121
6
Purification of Monoclonal Antibodies by Mixed-Mode
Chromatography
125
Pete Gagnon
6.1
6.2
6.3
6.4
6.5
Introduction, 125
A Brief History, 126
Prerequisites for Industrial Implementation, 127
Mechanisms, Screening, and Method Development, 129
Capture Applications, 137

viii
CONTENTS
6.6
6.7
6.8
6.9
6.10
Polishing Applications, 138
Sequential Capture/Polishing Applications, 139
The Future, 139
Acknowledgments, 140
References, 140
7
Integrated Polishing Steps for Monoclonal Antibody Purification
145
Sanchayita Ghose, Mi Jin, Jia Liu, and John Hickey
7.1
7.2
Introduction, 145
Polishing Steps in Antibody Purification, 146
7.2.1
7.2.2
7.2.3
7.2.4
Ion-Exchange (IEX) Chromatography, 146
HIC, 151
HA Chromatography, 155
Mixed-Mode and Other Modes of
Chromatography, 156
7.2.5
Dedicated Virus Removal Steps, 158
7.3
Integration of Polishing Steps, 159
7.3.1
7.3.2
Case Study I: Selection and Placement of
Polishing Steps, 159
Case Study II: Selection of Operational Mode and
Influence of the Previous Polishing Step, 161
7.4
7.5
7.6
Conclusions, 163
Acknowledgments, 163
References, 164
8
Orthogonal Virus Clearance Applications in Monoclonal
Antibody Production
169
Joe X. Zhou
8.1
8.2
8.3
Introduction, 169
Model Viruses and Virus Assays, 170
Virus Clearance Strategies from First in Human (FIH) to
Biological License Application (BLA) Filing, 172
Orthogonal Viral Clearance in mAb Production, 173
8.4
8.4.1
Major Viral Clearance Steps: Capture, Low-pH
Viral Inactivation, and Polishing, 173
Disposable Systems, 173
8.4.2
8.5
8.6
8.7
Conclusions and Future Perspectives, 183
Acknowledgments, 184
References, 184

CONTENTS
ix
9
Development of A Platform Process for the Purification of
Therapeutic Monoclonal Antibodies
187
Yuling Li, David W. Kahn, Olga Galperina, Erich Blatter, Robert Luo,
Yaling Wu , and Guihang Zhang
9.1
9.2
Introduction, 187
Chromatography Steps in the Platform Process, 189
9.2.1
9.2.2
Capture Step: General Considerations, 189
Intermediate/Polishing Steps, 191
9.3
9.4
Virus Inactivation, 193
UF/DF Platform Development, 194
9.4.1
9.4.2
9.4.3
Use in Platform Processes, 194
Challenges, 194
Application Examples, 195
9.5
9.6
Platform Development: Virus Filtration and Bulk Fill, 195
9.5.1
9.5.2
Virus Filtration in Platform Processes, 195
Bulk Fill in Platform Processes, 196
Examples of Platform Processes, 196
9.6.1
9.6.2
9.6.3
Example 1: Three-Column Protein A-Based
Process, 196
Example 2: Three-Column HCIC-Based
Process, 197
Example 3: Two- or One-Column Process without
Protein A, 198
9.7
Developing a Viral Clearance Database Using a Platform
Process, 199
9.8
9.9
Summary, 200
References, 201
10 Advances in Technology and Process Development for
Industrial-Scale Monoclonal Antibody Purification
Nuno Fontes and Robert van Reis
203
10.1
10.2
Introduction, 203
Affinity Purification Platform, 204
10.2.1
10.2.2
10.2.3
Overview, 204
Standard Purification Sequence, 205
Challenges and Opportunities, 205
10.3
Advances in CEX Purification of mAbs, 206
10.3.1
10.3.2
10.3.3
Overview, 206
High-Capacity CEX, 207
An Exclusion Mechanism in Ion-Exchange
Chromatography, 207
10.3.4
10.3.5
Factors Affecting the Critical Conductivity, 210
Advances in mAb CEX Process Development, 213

x
CONTENTS
10.4
HPTFF, 215
10.4.1
10.4.2
Overview, 215
Advances in HPTFF, 217
10.5
10.6
A New Nonaffinity Platform, 218
References, 219
11 Continuous Chromatography for the Purification of
Monoclonal Antibodies
223
Thomas Müller-Späth and Massimo Morbidelli
11.1
11.2
11.3
11.4
11.5
Introduction, 223
Product Variants and the Separation Problem, 224
Definition of Performance Parameters, 225
Gradient Chromatography for Biomolecules, 225
Continuous Chromatography to Increase Productivity, 226
11.5.1
11.5.2
11.5.3
The Concept of Continuous Chromatography, 226
The Simulated Moving Bed (SMB) Process, 226
Advantages and Disadvantages of Batch and
SMB Chromatography, 227
11.6
The MCSGP Process, 229
11.6.1
11.6.2
MCSGP Process Design, 229
MCSGP for the Capture of mAbs from Clarified
Cell Culture Supernatants, 230
11.6.3
MCSGP for the Separation of mAb Variants, 232
11.7
11.8
11.9
Upgrades for Continuous Processes to Improve Stability, 233
Impact of Increasing Fermentation Titers, 233
Outlook, 236
11.10 Acknowledgments, 237
11.11 References, 237
12 Process Economic Drivers in Industrial Monoclonal
Antibody Manufacture
239
Suzanne S. Farid
12.1
12.2
Introduction, 239
Challenges When Striving for the Cost-Effective
Manufacture of mAbs, 240
12.2.1
12.2.2
Constraints, 240
Uncertainties, 241
12.3
Cost Definitions and Benchmark Values, 242
12.3.1
12.3.2
Capital Investment, 242
Cost of Goods (COG) per Gram, 243
12.4
12.5
Economies of Scale, 246
Overall Process Economic Drivers, 247
12.5.1
12.5.2
Titer, 247
Overall DSP Yield, 248

CONTENTS
xi
12.5.3
12.5.4
12.5.5
Batch Duration, 250
Batch Success Rate, 250
Logistics, 251
12.6
12.7
DSP Drivers at High Titers, 251
12.6.1
12.6.2
12.6.3
Material Reuse and Lifetime, 252
Buffer/WFI Demands, 253
Chromatography Capacity, 254
Process Economic Trade-Offs for DSP Bottlenecks, 254
12.7.1
Chromatography Resin Dynamic Binding
Capacity, 254
12.7.2
12.7.3
12.7.4
12.7.5
Chromatography Flow Rates, 255
Chromatography Resin Cycle Limits, 255
Platform Processes, 255
Alternatives to Chromatography, 256
12.8
12.9
Summary and Outlook, 256
References, 257
13 Design and Optimization of Manufacturing
263
Andrew Sinclair
13.1
13.2
13.3
Introduction, 263
Process Design and Optimization, 265
Modeling Approaches, 267
13.3.1
13.3.2
Cost Modeling for mAb Manufacture, 267
Process Simulation for mAb Manufacture, 274
13.4
13.5
Cost Models in Practice, 279
13.4.1
13.4.2
Manufacturing Strategies—Disposables vs.
Continuous, 280
Manufacturing Technologies—Single-Use
Systems, 281
Simulation in Practice, 286
13.5.1
13.5.2
Managing Multiproduct Manufacture, 286
Resource Management, 288
13.6
13.7
Acknowledgments, 290
References, 290
14 Alternatives to Packed-Bed Chromatography for Antibody
Extraction and Purification
293
Jörg Thömmes and Uwe Gottschalk
14.1
14.2
Introduction, 293
Increasing the Selectivity of Harvest Procedures:
Flocculation of Particulate and Nonparticulate
Impurities, 294
14.3
Solutions for Antibody Extraction, Concentration, and
Purification, 295

xii
CONTENTS
14.3.1
14.3.2
Extraction and Concentration by Precipitation, 295
Extraction and Concentration by Liquid-Phase
Partitioning, 297
14.3.3
Concentration by Evaporation, 298
14.4
Nonchromatographic Solutions for Antibody Purification
and Formulation, 298
14.4.1
14.4.2
14.4.3
Crystallization, 298
Controlled Freeze–Thaw, 300
Lyophilization, 301
14.5
14.6
14.7
14.8
Membrane Adsorbers, 302
Conclusions, 304
Acknowledgments, 305
References, 305
15 Process-Scale Precipitation of Impurities in Mammalian
Cell Culture Broth
309
Judy Glynn
15.1
15.2
Introduction, 309
Precipitation of DNA and Protein—Other
Applications, 311
15.3
A Comprehensive Evaluation of Potential Precipitants
for Impurity Removal, 312
15.3.1
15.3.2
15.3.3
15.3.4
15.3.5
15.3.6
15.3.7
Protocol, 312
Ammonium Sulfate Precipitation, 314
Polymer Precipitation, 315
Precipitation with Ionic Liquids, 315
Precipitation with Cationic Detergents, 315
Ethacridine Precipitation, 316
Caprylic Acid Precipitation, 317
15.4
15.5
15.6
15.7
15.8
Industrial-Scale Precipitation, 319
Cost of Goods Comparison, 320
Summary, 321
Acknowledgments, 321
References, 322
16 Charged Ultrafiltration and Microfiltration Membranes
in Antibody Purification
325
Mark R. Etzel
16.1
16.2
16.3
16.4
16.5
Introduction, 325
Charged Ultrafiltration Membranes, 326
Concentration Polarization and Permeate Flux, 327
Stagnant Film Model (SFM), 328
Osmotic Pressure Model, 328

CONTENTS
xiii
16.6
16.7
16.8
16.9
Mass Transfer Coefficient, 329
Sieving Coefficient, 329
Diffusion–Convection Model, 330
Scale-up Strategies and the Constant Wall
Concentration (Cw) Approach, 331
16.10 Protein Fractionation Using Charged Ultrafiltration
Membranes, 333
16.11 Case Study, 333
16.11.1 Methods, 334
16.11.2 Results, 334
16.11.3 Discussion, 337
16.12 Membrane Cascades, 337
16.13 Charged Microfiltration Membranes, 339
16.14 Virus Clearance, 339
16.15 Salt Tolerance, 341
16.16 Conclusions, 343
16.17 Acknowledgments, 344
16.18 References, 345
17 Downstream Processing of Monoclonal Antibody Fragments
349
Mariangela Spitali
17.1
17.2
Introduction, 349
Production of Antibody Fragments for Therapeutic
Use, 351
17.3
Downstream Processing, 351
17.3.1
17.3.2
17.3.3
17.3.4
Primary Recovery, 352
Capture, 356
Expanded-Bed Chromatography, 361
Further Purification and Polishing, 362
17.4
Improving the Pharmacological Characteristics of Antibody
Fragments, 365
17.5
17.6
17.7
Conclusions, 367
Acknowledgments, 368
References, 369
18 Purification of Antibodies Other Than IgG: The Case of
IgM and IgA
373
Charlotte Cabanne and Xavier Santarelli
18.1
18.2
Introduction, 373
Purification of Immunoglobulin M (IgM), 374
18.2.1
18.2.2
18.2.3
IgM Structure and Properties, 374
IgM Purification Technologies, 374
Affinity and Pseudoaffinity Matrices, 376

xiv
CONTENTS
18.3
Purification of IgA, 378
18.3.1
18.3.2
IgA Structure and Properties, 378
Affinity and Pseudoaffinity Matrices, 378
18.4
18.5
18.6
Conclusion, 380
Acknowledgments, 380
References, 380
19 Purification of Antibodies From Transgenic Plants
387
Zivko L. Nikolov, Jeffrey T. Regan, Lynn F. Dickey, and Susan L. Woodard
19.1
19.2
Introduction, 387
Antibody Production in Transgenic Plants, 388
19.2.1
19.2.2
Subcellular Localization and Glycosylation, 388
Other Factors Affecting mAb Accumulation, 390
19.3
Downstream Processing of Antibodies Produced in
Transgenic Plants, 391
19.3.1
19.3.2
19.3.3
Tissue Disintegration, 393
Solid–Liquid Separation, 394
Clarification and Pretreatment of Crude
Extracts, 395
19.4
19.5
Purification of Plant-Derived mAbs Using Protein A, 396
Purification of Plant-Derived mAbs Using Nonprotein A
Media, 397
19.6
19.7
19.8
19.9
Polishing Steps, 398
Conclusions, 399
Acknowledgments, 401
References, 401
20 Antibody Purification: Drivers of Change
407
Narahari Pujar, Duncan Low, and Rhona O’Leary
20.1
20.2
Introduction, 407
The Changing Regulatory Environment—Pharmaceutical
Manufacturing for the Twenty-First Century, 410
20.2.1
20.2.2
Using Design Space to Enable Change, 412
High-Throughput and Microscale Approaches to
Process Development and Characterization, 415
20.3
20.4
Technology Drivers—Advances and Innovations, 416
20.3.1
20.3.2
PAT, 416
Process Control Technology, 417
Economic Drivers, 418
20.4.1
20.4.2
20.4.3
20.4.4
Cost of Goods (COG), 418
Single-Use Disposable Components, 418
Globalization, 419
Follow-on Biologics (FOBs) or Biosimilars, 420

CONTENTS
xv
20.5
20.6
20.7
Conclusions, 421
Acknowledgments, 422
References, 422
Index
427

PREFACE
Monoclonal antibodies are an important component of the biopharmaceutical
industry.Within the burgeoning market of protein-based therapeutics, they are
the market leaders in terms of volume sales and the most common class of
product. Almost all commercial antibodies are produced in cultured mamma-
lian cells, and an entire subindustry has grown up around downstream process-
ing to ensure that manufacturing processes generate safe and pure products
suitable for administration to humans.This is an industry in which I have been
involved for many years, and one that is facing exciting and difficult
challenges.
I first came into contact with the world of monoclonal antibodies in 1986,
when their production in cultured mammalian cells was still in its infancy.This
was at the Cancer Research Campaign Laboratories in Nottingham, UK. I was
a PhD student from Germany, and one of my main tasks was to purify anti-
bodies from mouse ascites, a horrible process for obvious reasons.A milligram
of antibodies was worth far, far more than its weight in gold.
At the time, my colleagues and I had visions of curing cancer by drug tar-
geting, and we linked all sorts of cytotoxic agents to the antibodies we pro-
duced. Unfortunately, some of the expectations surrounding the medical use
of antibodies turned out to be premature and unrealistic. Our awareness of
this coincided with the first real downturn in the biotechnology sector, but
antibodies survived in niche markets for diagnostics and research reagents.
Years later, new life has been breathed into therapeutic antibodies and they
are now back, stronger than ever. Indeed, they represent the fastest-growing
area in biotherapeutics with 21 products on the US market (as of 2007) and
hundreds in clinical and preclinical development (1).
At the end of the 1980s, antibodies were produced commercially using
mammalian cells cultured in perfusion fermenters, but yields rarely exceeded
xvii

xviii
PREFACE
100mg/L. Huge volumes of culture broth needed to be processed, and
the easiest way to bring the volume down was polyethylene glycol (PEG)
precipitation with tons of material and endless centrifugation cycles. The
yields were poor and difficult to reproduce, but there were no alternatives.
Since that time, the productivity of cell cultures has increased significantly,
with 1–5g/L titers now routine and the real prospect of 10–20g/L yields in
the next decade. How far we have come since the early days of
biomanufacturing!
The increase in titers has heaped pressure on the downstream processes
that we use to extract and purify antibodies from cell culture broth, and the
technologies used in downstream processing have been forced to modernize
and improve in the face of this increasing challenge. There is little doubt that
packed-bed chromatography is the workhorse of current downstream process-
ing, its high resolution and relative simplicity making it the central enabling
technology in modern bioseparation processes (2). As productivity increases,
however, doubts have been cast on the ability of column chromatography to
cope with the dramatically increasing product titers in fermentation (3).Unlike
fermentation, capturing steps in downstream processing have hardly any
economy of scale. Bind-and-elute cycles in chromatography are driven by mass
rather than by volume, and this means that increasing batch sizes translate into
increasing costs in a near linear fashion. This phenomenon particularly affects
the first column, where all of the product must be captured. This initial recov-
ery step has therefore been identified as the most serious potential bottleneck,
with knock-on effects throughout the processing facility, e.g., in terms of
column sizes, buffer preparation, and hold. However, this opinion is not shared
by everyone, and debate continues as to whether or not packed-bed chroma-
tography is here to stay (4, 5).
These challenges and their surrounding issues set the scene for this exciting
book, in which I have compiled a selection of chapters from top-tier industrial
and academic experts providing up-to-date accounts of current best practice
in the manufacture of monoclonal antibodies. Opinions on the suitability
of packed-bed chromatography in today’s manufacturing environment differ,
particularly in the light of emerging competitive technologies, and the first
chapter by Ann Lee and colleagues captures that debate and puts the case for
and against the continuing reliance on traditional chromatography methods.
The second chapter by John Curling provides an informative historical over-
view of the development of antibody purification technologies, providing the
basis for the next five chapters, which consider some of today’s major steps in
antibody processing—harvesting and recovery (Abhinav Shukla and col-
leagues), Protein A chromatography (Suresh Vunnum and colleagues), non-
Protein A strategies (Alahari Arunakumari and Jue Wang), mixed mode
chromatography (Pete Gagnon), and integrated polishing (Sanchayita Ghose
and colleagues).
Looking closer, the pace at which fermentation is guiding the way is not
the only challenge for modern downstream processing. The regulatory frame-

PREFACE
xix
work, particularly current good manufacturing practice (cGMP) is a moving
target, and quality requirements are constantly leading to tighter specifications
and higher safety margins, e.g., with regard to small, nonenveloped viruses.The
chapter by Joe Zhou therefore deals with orthogonal methods for virus
removal, before we consider platform technologies that integrate virus clear-
ance with capture and purification (Yuling Li and colleagues). Nuno Fontes
and Robert van Reis then consider the important aspects of scaling up anti-
body purification to industrial levels with a platform of methods that offer the
potential to set a new standard in antibody manufacture. Finally in this section,
Thomas Müller-Späth and Massimo Morbidelli consider the use of continuous
chromatography for the high-resolution separation of antibodies, based on a
laboratory-scale strategy they developed.
The next two chapters look at the economic perspectives of antibody manu-
facture, one from the standpoint of process economics (Suzanne Farid) and
the other from the standpoint of process design and optimization (Andrew
Sinclair). We then turn to the consideration of emerging technologies, which
may replace, augment, or supplement traditional chromatography: floccula-
tion, precipitation, and membrane adsorbers for antibody purification (Jörg
Thömmes and Uwe Gottschalk);precipitation for the elimination of impurities
(Judy Glynn); and charged filtration membranes (Mark Etzel).
While most of the book focuses on the purification of typical, full-size IgG
molecules produced in fermenters, the final section deals with noncanonical
antibody varieties and novel sources. There are chapters dealing with the
purification of antibody fragments (Mariangela Spitali) and non-IgG mono-
clonals (IgM and IgA; Charlotte Cabanne and Xavier Santarelli), followed by
a chapter considering the promising use of plant-based systems for antibody
manufacture, and the particular challenges faced when isolating antibodies
and other biopharmaceuticals from plant sources (Zivko Nikolov and
colleagues).
The final chapter wraps up the book by looking to the future and consider-
ing what drives change in the industry, particularly what factors are likely to
influence the techniques and technologies that will be adopted for antibody
purification in the decade to come. This concluding chapter is written by Hari
Pujar, Duncan Low, and Rhona O’Leary, three distinguished authors repre-
senting the top-tier companies in the sector.
In all likelihood, we will not see a revolution in downstream processing like
the one that has galvanized upstream process development over the last 20
years. The chapters in this book are, however, evidence that the future of
antibody purification holds great promise, underlining the progress that has
been made in closing the performance gap between upstream production and
downstream processing.
All the contributors to this book live and die for the production of anti-
bodies. Some of us have been there from the very first day, while others have
joined more recently, but we all passionately believe that technological
advances and innovation can help to break through the current ceiling in

xx
PREFACE
antibody processing and can lead to affordable, high-quality pharmaceutical
products in the future.
U we Gottschalk
REFERENCES
1. Frost & Sullivan (2008). US Biotechnology—Therapeutic Monoclonal Antibodies
Market. Frost & Sullivan, Palo Alto, CA.
2. Curling, J., Gottschalk, U. (2007). Process chromatography: Five decades of innova-
tion. BioPharm International 20, 70–94.
3. Langer, E.S. (2008). Fifth Annual Report and Survey of Biopharmaceutical Manufac-
turing Capacity and Production. BioPlan Associates Inc., Rockville MD.
4. Kelley, B. (2007). Very large scale monoclonal antibody purification: The case for
conventional unit operations. Biotechnology Progress 23, 995–1008.
5. Low, D., O’Leary, R., Pujar, N.S. (2007). Future of antibody purification. Journal of
Chromatography B 848, 48–63.

ABOUT THE AUTHOR
Dr. Uwe Gottschalk
Group Vice President
Purification Technologies
Sartorius Stedim Biotech
D-37079 Göttingen
Tel: (0551) 308-2016
Telefax: (0551) 308-3705
Email: Uwe.Gottschalk@Sartorius-Stedim.com
Dr. Gottschalk is Group Vice President, Purification Technologies, at Sartorius
Stedim Biotech. He worked in different capacities for Bayer Health Care from
1991 to 2004, overseeing the purification of monoclonal antibodies and recom-
binant proteins produced in various expression systems.
Dr. Gottschalk holds a PhD in Chemistry from the University of Münster.
He is a member of BioPharm International’s editorial advisory board and has
written extensively in the areas of biotechnology and somatic gene therapy. In
academia, Dr. Gottschalk is Head Lecturer at the University of Duisburg–
Essen (Germany) and also lectures at the Ecole Polytechnique Fédérale de
Lausanne (EPFL) in Lausanne (Switzerland).
Dr. Gottschalk is a board member of the VBU Association of German
Biotech Companies.
xxi

CONTRIBUTORS
Alahari Arunakumari, PhD, Senior Director, Process Development, Medarex,
Inc., 519 Route 173 West, Bloomsbury, NJ 08804; Email: aarunakumari@
medarex.com
Greg Blank, PhD, Director, Late Stage Purification, Genentech, Inc.
Erich Blatter, PhD, Senior Scientist, Purification Sciences-BioPharmaceutical
Development, Human Genome Sciences, Inc.
Charlotte Cabanne, PhD, Associate Professor, EA 4135, ESTBB, Université
Victor Segalen Bordeaux 2
John Curling, CEO, John Curling Consulting AB, Swedenborgsgatan 3-5, SE-
753 34 Uppsala, Sweden; Email: john@consultcurling.se
Lynn F. Dickey, PhD, VP of Research and Technology Development, Biolex
Therapeutics
Mark R. Etzel, PhD, Professor, University of Wisconsin, 1605 Linden Drive,
Madison, WI 53706; Email: etzel@engr.wisc.edu
Suzanne S. Farid, PhD, Senior Lecturer/Associate Professor, The Advanced
Centre for Biochemical Engineering, Department of Biochemical Engi-
neering, University College London, Torrington Place, London WC1E 7JE,
UK; Email: s.farid@ucl.ac.uk
Nuno Fontes, PhD, Late Stage Purification—Process Research & Develop-
ment, Genentech, Inc., 1 DNA Way South, San Francisco, CA 94080; Email:
fontes.nuno@gene.com
xxiii

xxiv
CONTRIBUTORS
Pete Gagnon, Chief Scientific Officer, Validated Biosystems, 240 Av Vista
Montana, Ste 7F, San Clemente, CA 92672; Email: pete@validated.com
Olga Galperina, PhD, Associate Director, Purification Sciences-
BioPharmaceutical Development, Human Genome Sciences, Inc.
Sanchayita Ghose, PhD, Manager, Process Sciences—Downstream, Bristol-
Myers Squibb, Co., East Syracuse NY 13057; Email: sanchayita.ghose@bms.
com
Judy Glynn, PhD, Senior Principal Scientist, Pfizer, Inc., 700 Chesterfield
Parkway West, Chesterfield, MO, 63017; Email: judy.k.glynn@pfizer.com
Uwe Gottschalk, PhD, Group Vice President Purification Technologies,
Sartorius Stedim Biotech, Göttingen, Germany; Email: uwe.gottschalk@
sartorius-stedim.com
John Hickey, Associate Director, Process Sciences—Downstream & Project
Management, Bristol-Myers Squibb, Co.
Brian Hubbard, PhD, Executive Scientific Director, Process & Product Devel-
opment, Amgen Inc.
Mi Jin, PhD, Scientist, Process Sciences—Downstream, Bristol-Myers Squibb,
Co.
David W. Kahn, PhD, Director, Purification Sciences-BioPharmaceutical
Development, Human Genome Sciences, Inc.
Jagannadha Rao Kandula, Manufacturing Sciences, Bristol-Myers
Squibb, Co.
Brian Kelley, PhD, Senior Director, Bioprocess Development, Genentech,
Inc., Inc., 1 DNA Way, South San Francisco, CA 94080; Email: kelley.brian@
gene.com
Ann Lee, PhD,Vice President, Process Research & Development, Genentech,
Inc.
Yuling Li, PhD, Senior Director, Purification Sciences-BioPharmaceutical
Development, Human Genome Sciences, Inc., 14200 Shady Grove Road,
Rockville, MD 20850; Email: Yuling_Li@hgsi.com
Jia Liu, PhD, Engineer, Process Sciences—Downstream, Bristol-Myers
Squibb, Co.
Duncan Low, PhD, Process Development, Amgen
Robert Luo, PhD, Senior Scientist, Purification Sciences-BioPharmaceutical
Development, Human Genome Sciences, Inc.

CONTRIBUTORS
xxv
Massimo Morbidelli, PhD, Institute for Chemical and Bioengineering, ETH
Zürich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland; Email: massimo.
morbidelli@chem.ethz.ch
Thomas Müller-Späth, Dipl.-Ing., Institute for Chemical and Bioengineering,
ETH Zürich
Zivko L. Nikolov, PhD, PE, Dow Professor, Biological and Agricultural Engi-
neering, Texas A&M University, College Station, Texas 77843-2117; Email:
znikolov@tamu.edu
Rhona O’Leary, PhD, Early Stage Purification, Genentech
Narahari Pujar, PhD, Bioprocess R&D, Merck & Co., Inc., West Point, PA;
Email: hari_pujar@merck.com
Jeffrey T. Regan, Biolex Therapeutics
Xavier Santarelli, PhD,Professor,EA 4135,ESTBB,UniversitéVictor Segalen
Bordeaux 2, 146 rue Léo Saignat, Bordeaux cedex, France; Email: xavier.
santarelli@estbb.u-bordeaux2.fr
Abhinav A. Shukla, Manufacturing Sciences, Bristol-Myers Squibb, Co., East
Syracuse, NY 13057; Email: abhinav.shukla@bms.com
Andrew Sinclair, CEO and Founder, BioPharm Services, Lancer House,
Chesham, HP5 1RD, UK; Email: a.sinclair@biopharmservices.com
Mariangela Spitali, PhD, Senior Group Leader, Protein Purification Research,
Bioprocess R&D,UCB Celltech,UCB Pharma Ltd.,208 Bath Road,Slough,
Berkshire, SL1 3WE, UK; Email: Mari.Spitali@celltech.UCB-group.com
Jörg Thömmes, PhD, Senior Director Purification Process Development,
Biogen Idec, San Diego, USA
Robert van Reis, Late Stage Purification—Process Research & Development,
Genentech, Inc.
Ganesh Vedantham, PhD, Director, Process & Product Development,Amgen
Inc.
Suresh Vunnum, PhD, Scientific Director, Process & Product Development,
Amgen Inc., 1201 Amgen Court West, Seattle,WA 98006; Email: vvunnum@
amgen.com
JueWang, PhD,Senior Manager,Purification Process Development,Medarex,
Inc.
Susan L. Woodard, PhD, Department of Biological and Agricultural Engi-
neering, Borlaug Center/TAMU 2123, Texas A&M University
Yaling Wu, PhD, Senior Scientist, Purification Sciences-BioPharmaceutical
Development, Human Genome Sciences, Inc.

xxvi
CONTRIBUTORS
Guihang
Zhang, PhD,
Senior
Scientist,
Purification
Sciences-
BioPharmaceutical Development, Human Genome Sciences, Inc.
Joe X. Zhou, PhD, Scientific Director, Process Development Department,
Amgen, Inc., CEO of Wison Bio-engineering Company, 1399 Zhangheng
Road, Zhangjiang, Pudong, Shanghai 201203, China; Email: xhz_1999@
yahoo.com or joezhou@wison.com

1
DOWNSTREAM PROCESSING OF
MONOCLONAL ANTIBODIES:
CURRENT PRACTICES AND
FUTURE OPPORTUNITIES
Brian Kelley, Greg Blank, and Ann Lee
1.1 INTRODUCTION
Monoclonal antibodies (mAbs) are now established as the most prevalent
class of recombinant protein therapeutics.They can be expressed at high levels
in cell culture, are typically very soluble, and are relatively stable during pro-
cessing. The nearly universal use of mammalian cell expression systems for
mAb synthesis, combined with the selection of homologous, humanized mAb
framework protein sequences, provides opportunities to harmonize manufac-
turing processes around base platforms that can then be used with only slight
variations from product to product. In addition, by using a platform process,
manufacturing plants designed for the production of one mAb can usually be
readily adapted to produce others.
For these reasons, mAbs represent a unique group of biological products.
They accommodate rapid process development time lines, can be produced in
large quantities, and may be manufactured in multiple facilities during their
lifecycle as a result of their common process flowsheets. As a result, they have
relatively low manufacturing costs and benefit from the flexibility of produc-
tion at either in-house or contract production facilities. Although mAbs are
not commodity products that are substitutable in the clinical setting, they have
Process Scale Purification of Antibodies, Edited by Uwe Gottschalk
Copyright © 2009 John Wiley & Sons, Inc.
1

2
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
distinct advantages in production scale and cost, as well as in product develop-
ment speed and convenience, when compared to other recombinant protein
therapeutics.
This introductory chapter attempts to set the context for the following
chapters, which cover many aspects of mAb purification in detail. A typical
mAb purification process flow sheet is described and used to illustrate the
impact of purification platforms on mAb production. Factors to consider with
respect to the various process alternatives or new technologies described in
upcoming chapters are addressed, emphasizing the integration of unit opera-
tions and process design principles into an optimized, holistic process. Both
current practices and controversial topics are introduced, among them the
challenges of very large-scale (VLS) production, issues related to facility fit,
the maturation of process purification technology for mAb processing, the
need for innovations in mAb downstream processing, and the impact of the
evolving regulatory environment. It is hoped that this backdrop will stimulate
critical thinking and comprehensive analysis when the processing options
described in the following chapters are being considered.
1.2 A BRIEF HISTORY OF cGMP mAb AND INTRAVENOUS
IMMUNOGLOBULIN (IgIV) PURIFICATION
The processes used for production of IgIV from human plasma differ from
those used for recombinant mAbs. Figure 1.1 shows a consensus processing
scheme, based on many published process flow sheets, for the purification of
IgIV. Most IgIV processes lack chromatographic steps and instead rely on
multiple fractional precipitation steps based on the Cohn process developed
in the 1950s (1). Some recently developed processes include chromatographic
Plasma
Fraction 1
precipitation
Fractions 2 + 3
precipitation
1–2 Virus inactivation steps
(solvent/detergent, filtration,
caprylate, low pH)
+/– 1 or 2 Chromatography
steps
UF/DF
FIGURE 1.1 Cohn-based IgIV consensus process.

A BRIEF HISTORY OF cGMP mAb AND INTRAVENOUS
3
steps, but this is used to a limited extent and still in combination with upstream
steps based on the Cohn process (2, 3). The processes used for recombinant
mAb purification have borrowed very little from plasma fractionation technol-
ogy, other than ultrafiltration to formulate and to concentrate the drug sub-
stance.The low cost of manufacturing IgIV and the very large production scale
have led to debate on the value of going “back to the future” and applying
IgIV processing technologies to the production of recombinant mAbs. A
review of current mAb processing platforms will put this proposal into
context.
The first cGMP for mAb purification reflected the state of the art in the
1980s and early 1990s, prior to the accumulation of substantial process knowl-
edge and the introduction of improved separation media that made today’s
more efficient and scalable processes possible. Examples of the diversity of
early processes include the use of various microfiltration or depth-filtration
media for harvest; affinity chromatography with Protein G in addition to
Protein A; conventional capture columns to protect the Protein A resin; incor-
poration of challenging separation methods for large-scale production, such
as size exclusion chromatography (SEC); solvent/detergent virus-inactivation
methods; and the requirement for four or even more chromatography steps
(Figure 1.2). In addition, downstream processing was sometimes performed in
the cold. Chromatographic media often provided relatively low loading capac-
ities, which were not a significant issue when cell culture titers were measured
in hundreds of milligrams per liter. To address the need for kilogram-scale
production, very large bioreactors were used; the focus for capture resin selec-
tion was based on maximizing volumetric productivity and on the ability to
Cell culture
Centrifuge
Microfiltration
+/– UF/DF
Protein A
IEC or HIC
Protein G
Protein A or G
+/– Intermediate UF/DF
+/– Solvent/detergent
2–3 Chrom steps
Virus filtration
UF/DF
FIGURE 1.2 Early mAb purification schemes. IEC = ion-exchange chromatography;
HIC = hydrophobic interaction chromatography.

4
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
process large volumes of feed rapidly, rather than on the handling of large
batches (greater than 20kg of product). Many of these early mAb products
were also derived from a more diverse set of framework protein sequences,
reflecting the historical progression from murine and chimeric mAbs to today’s
fully humanized antibodies, which gave rise to a more varied set of process
flow sheets.
1.3 CURRENT APPROACHES IN PURIFICATION PROCESS
DEVELOPMENT: IMPACT OF PLATFORM PROCESSES
Despite the high degree of homology among humanized mAbs, variations in
complementarity-determining regions and framework sequences make it dif-
ficult to define a truly generic purification process capable of processing many
different mAbs without any changes to the operating conditions. Despite these
variations,many companies have defined platform purification processes based
on a common sequence of unit operations. A frequently used purification
platform for mAbs is shown in Figure 1.3. The conditioned medium is first
clarified by centrifugation, followed by depth filtration. Protein A chromato-
graphy offers direct product capture from the centrate and provides excellent
purification and significant concentration of the product. The low-pH elution
from the Protein A step also provides virus inactivation.Two chromatographic
polishing steps are used to reduce host cell, medium, purification process-
related impurities and product impurities. Additional virus removal is usually
achieved in these polishing steps. One of the polishing steps is almost invari-
ably anion-exchange (AEX) chromatography, often run in the flow-through
mode. The second polishing step is typically cation-exchange (CEX) chroma-
tography, although occasionally ceramic hydroxyapatite or hydrophobic inter-
Cell culture
Centrifuge
Protein A
Cation-exchange
chromatography
Anion-exchange
chromatography
Virus filter
UF/DF
FIGURE 1.3 Typical mAb platform (current).

CURRENT APPROACHES IN PURIFICATION PROCESS DEVELOPMENT
5
action chromatography (HIC) are used. The remaining process steps include
virus filtration (VF) and ultrafiltration/diafiltration (UF/DF) to formulate and
concentrate the product, which is now the bulk drug substance. The efficiency,
robustness, and scalability of this standardized process have resulted in the
rapid convergence of process development groups in the industry around a
similar process flow sheet (4, 5).
The establishment of platform processes for mAb production has already
had an enormous impact on process development strategy and activities, and
is just beginning to affect the world of commercial manufacturing. At this
point, very few companies have two or more commercial mAbs that are puri-
fied by a common platform process. Many mAbs currently in the clinical
pipeline, however, are manufactured by a process similar to the standard
process shown in Figure 1.3. The gradual progression of these early-stage
processes from clinical to commercial production enables additional efficien-
cies in production that will reduce the costs of goods (COGs) and accelerate
responses to surges in product demand. The benefits of efficient facility man-
agement (e.g., reductions in changeover time and the use of common raw
materials and equipment) and flexible commercial production (e.g., balanced
production schedules among multiproduct facilities) will be realized more
slowly than the gains seen today with product candidates in the early stages
of clinical development.The combination of platform processing, multiproduct
facilities, rapid product changeover, and flexible sourcing between contract
manufacturing organizations (CMOs) and in-house production facilities
achieves an industrialization of mAb production that will be unprecedented
in the field of recombinant protein biologics. Antibodies could become a class
of therapeutic biologics that support the treatment of large patient popula-
tions while remaining cost competitive with small molecules. To achieve this
vision, the biopharmaceutical industry must take advantage of the opportuni-
ties presented by the ease of development, validation, and production afforded
by platform processes.
Given the value and broad adoption of processing platforms, combined with
an installed production facility base designed for them, there is enormous
pressure to conform to such platforms with future products.As a result, options
for unit operations, raw materials, step sequences, control systems and algo-
rithms, and processing equipment are limited. While these restrictions may at
first seem highly constraining, they require other challenges to be addressed,
e.g., the establishment of highly efficient work processes that rapidly define
the appropriate processing conditions for each new mAb that enters the pipe-
line, as well as the definition of a common set of optimization approaches and
process characterization studies that will streamline late-stage development of
clinical products.
Clarification operations such as centrifugation often vary little from product
to product, provided that the cell culture process is not radically different.
Large changes in the cell concentration or viability in a bioreactor will affect
clarification, but provided that the unit operations are designed for the worst-

6
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
case feedstream, few if any modifications will be needed for new mAbs. The
capacity of centrate depth filters can vary significantly, depending on feed-
stream, and should be optimized for robustness while the costs of raw materi-
als are minimized. Similarly, the platform’s ultrafiltration steps (VF and final
ultrafiltration) should be largely unaffected by the change in the mAb. The
unit operations that are most likely to require tuning are the chromatography
steps. Even there, the standardization of many elements in a chromatographic
unit operation will streamline development time lines by focusing on key
factors influenced by product characteristics (5). Process variables that are
often specified for platform processes include resin and membrane selection,
column bed height, wash volumes, loading capacity, membrane flux, and target
bulk concentration. This effort simplifies and accelerates early-stage process
development.
The Protein A capture step is generally a very robust operation that can
tolerate changes in bioreactor harvest conditions and product characteristics
(see Chapter 4). The variables that may be influenced by product or feed-
stream variations are dynamic binding capacity, the optimal composition of
the column wash solution, and the elution conditions. Variations in these
process parameters arise from differences in the affinity of Protein A for the
mAb, the steric hindrance among molecules (6), and variations in impurity
levels and species in the feedstream, probably caused either by the cell line
and bioreactor management or by the properties of the mAb itself.
The most common variables for the ion-exchange polishing steps include
the column-loading and solution compositions (e.g., pH and counterion con-
centration) and the wash and elution compositions. In some cases, there can
be major changes to the platform, as when a highly acidic mAb has strong
affinity for an AEX resin and the typical flow-through operation must be
abandoned in favor of a bind–elute step. Broad ranges in the affinities of ion-
exchange resins have been described (5, 7) and highlight one area where the
diversity of mAb properties has an impact on the purification process.
Given that chromatography variables may be optimized independently for
each mAb, there are choices to be made regarding the investment in early-
stage process development. Two extremes are represented by tailored, as
compared to generic, processing conditions. An example of the tailored
approach is the use of high-throughput screening to define unique operating
conditions for each mAb, e.g., to optimize an AEX polishing step (8). Similarly,
scouting studies using gradient elution for bind–elute steps could be used to
tune the elution conditions for a CEX step. The generic approach to process
development would use a single pH for the AEX step and adjust the load
counterion concentration only by dilution. This minimum counterion concen-
tration would vary from mAb to mAb; an even more flexible step would use
a single counterion concentration, which allows successful processing of the
majority of mAbs without significant product losses of the most acidic family
members. Likewise, in a generic approach for CEX, the step could be designed
with a very low conductivity for the load combined with a broad gradient

CURRENT APPROACHES IN PURIFICATION PROCESS DEVELOPMENT
7
elution, which could potentially encompass successful processing of a large
number of mAbs (see Chapters 5–7, which provide examples of this approach).
The trade-offs of the two approaches would be influenced by the interplay of
process development resources and time (more for the tailored approach, less
for the generic), manufacturing efficiency (higher for tailored, lower for
generic), and other factors, such as mAb characteristics.
After Phase I process development, companies typically engage in at least
one additional cycle of process development (commonly termed late-stage
development), which defines the Phase III process and is subsequently used
for product launch.This second cycle often involves changes to the cell culture
process, including media reformulation, changes to the feeding strategy, opti-
mization of culture duration, and potentially the introduction of a new cell
bank or cell line. Although changes to the purification process during this
second cycle are unlikely to have the same regulatory impact as cell line
changes, the elimination of a step (e.g., the elimination of one of two polishing
steps) could cause a problematic change in an impurity profile. Given that
several years may have passed between the development of the Phase I and
Phase III processes, consideration should be given to upgrading the purifica-
tion process to include the superior separation media that have been recently
introduced to the market. Additional optimization studies will define the final
process control ranges for key and critical process parameters and will inves-
tigate the processing parameters that are unique to each mAb (e.g., column
capacities, resin and membrane lifetimes, in-process hold times, and maximum
bulk concentration).
If the implementation of radically new processing technologies is consid-
ered, this option is typically weighed at the late-stage development cycle rather
than at the early stage. Alternate, off-platform technologies (9) would require
sustained and significant effort to define processing parameters; establish
process robustness; acquire and test novel raw materials; and specify, purchase,
and validate new equipment. These factors strongly support an approach
where implementation of new technology on a Phase I time line would be used
only if the new process technology represented the sole means of enabling
clinical production. The “speed-to-clinic” driver would almost always trump
the benefits in COGs or the productivity benefits that novel technology would
offer at this stage of development. This would force novel technologies to be
considered during the next two cycles of process development, either for the
Phase III definition or as a post-licensure change.
This argument may appear to establish a conundrum for the introduction
of novel, off-platform unit operations. If they will not be introduced in Phase
I processes due to speed-to-market pressures, they must be delayed to the
Phase III process. Yet the introduction of significant process changes
(especially those that could negatively affect the impurity profile of the
product) after Phase I safety studies is a high hurdle that may require addi-
tional clinical trials, increasing the cost of the drug development and poten-
tially delaying product launch. The introduction of new technology after

8
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
licensure may appear to have an even higher bar, even if the risk of an untow-
ard effect of process changes is very low. There have been instances of signifi-
cant post-licensure changes in purification processes for recombinant proteins
(10–12), which in some cases required additional clinical studies.The U.S. Food
and Drug Administration (FDA) allows process changes to be managed under
comparability protocols without clinical trials if the process change has no
impact on product safety, potency, or efficacy, and if the product is well
characterized.
A major benefit of establishing a purification process platform is realized
when a reasonably long period elapses without significant changes that alter
process development or manufacturing operations. This leads to an approach
whereby process improvements are bundled and introduced through a con-
trolled, internal review and decision-making process (a “punctuated equilib-
rium” for the platform). The evolution of a company’s platform is a healthy
process, as superior separation media and accumulated development knowl-
edge combine to offer significant advantages in development speed, reduction
in COGs (see Chapter 9) or improved plant productivity. The change-control
process for the platform should be managed to prevent changes from occur-
ring too frequently, while allowing the introduction of clearly superior technol-
ogy when appropriate.
1.4 TYPICAL UNIT OPERATIONS AND
PROCESSING ALTERNATIVES
Centrifugation is currently the method of choice for harvest operations (13,
14). Continuous-flow disk-stack centrifuges are robust, are easily scaled across
various fermenter volumes, and use a relatively generic set of processing
parameters. It is unlikely that centrifugation can provide a sufficient degree of
solids removal to allow the centrate to be processed directly by the initial
capture chromatography without further filtration, although processing alter-
natives such as flocculation may offer a significant reduction in the filtration
area required (15). Depth filters may remove protein [host cell protein (HCP)
or product] during filtration (16), and changes to filtration media should be
evaluated carefully. Centrifugation parameters can affect the filter area needed
and can influence the colloidal properties of the feedstream. Therefore, con-
sideration of both centrifugation and filtration parameters is required for
optimal performance and integration of these steps. Although centrifugation
is likely to remain the preferred choice for large-scale operations, smaller-scale
cell culture harvests (≤400L) can also be performed using a combination of
depth and size filtration. Newer technologies, such as pod harvest units, make
filtration a more attractive option at small scales.
The majority of processes use Protein A chromatography as the initial
capture chromatography step. Two potential issues associated with Protein A

TYPICAL UNIT OPERATIONS AND PROCESSING ALTERNATIVES
9
chromatography are leaching of the Protein A ligand from the resins into the
product pool and the higher cost of Protein A resins as compared with nonaf-
finity resins. Alternative ligands to Protein A that are either small molecules
or polypeptide mimetics of Protein A have generally not delivered increased
load capacity while maintaining selectivity (17). Most of these resins are not
known to be under active investigation in industrial laboratories. Other alter-
natives to Protein A chromatography as a capture step typically include ion-
exchange chromatography, and because many mAbs have pIs greater than 7,
CEX resins are the primary choice (see Chapter 5). With CEX, the pH and/or
conductivity of the harvest cell culture fluid may need to be adjusted, and in
some cases the product in the feedstream should be concentrated using ultra-
filtration, to obtain optimal capacity and throughput. This conditioning opera-
tion should be evaluated when the overall costs of affinity capture are compared
to those of nonaffinity capture. The lower pH and conductivity may affect
antibody stability in the feedstream (because of acid-activated proteases) or
precipitation of proteins (either product or HCP), thus necessitating increased
filter area. However, precipitation of HCP during conditioning may contribute
to HCP removal during the process (see Chapter 5), although it could cause
complications during ultrafiltration. The potential of impurity precipitation as
an up-front conditioning step is addressed later (see Chapter 15), although the
economics of the technique do not appear to be favorable, at least at the high
concentrations of the precipitant evaluated by those authors.
The number and types of polishing steps will be determined by the nature
of the product and process-related impurities. In general, a Protein A pool will
be more pure than a nonaffinity pool. Cell culture conditions that increase
expression but also result in higher levels of aggregate or charge variants may
influence the choice of affinity or nonaffinity process or may require additional
polishing steps. A nonaffinity process is generally less amenable to a platform
approach because not all antibodies bind well to the capture column without
custom feedstream modifications, as in the case of CEX resins. Furthermore,
a Protein A capture step offers greater freedom to match the subsequent
processing step without feedstream adjustments (see Chapter 4). If the affinity
pool is relatively pure, only one additional chromatography polishing step may
be necessary. There have been platforms that use just two chromatography
steps, where the AEX step is run under conditions of weak partitioning (8), as
shown in Figure 1.4. Two-column processes offer advantages beyond the
obvious cost reduction associated with eliminating a step from the process (see
Chapter 5, which provides examples of processes comprising two columns plus
a membrane adsorber) New, mixed-mode chromatography resins may also aid
in minimizing the number of polishing steps by increasing the separation per-
formance for each resin (see Chapter 6). Product-related impurities with only
slight charge or size differences can be the greatest challenge for the purifica-
tion process because of their similarity to the product. Controlling these impu-
rities to acceptable levels during the cell culture process facilitates a two-column
process.

10
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
Cell culture
Centrifuge
Protein A
Anion-exchange
Mixed-mode
chromatography
chromatography
(weak partitioning)
Virus
filtration
UF/DF
FIGURE 1.4 Emerging two-column platforms.
Cell culture
Centrifuge
Protein A
CEC
AEC
Mixed
(WPC)
Mode
Anion
membrane
Virus filter
Virus filter
UF/DF
Virus filter
Continuous process
(no in-process pool)
FIGURE 1.5 Future high-efficiency platforms. AEC = anion-exchange chromato-
graphy; CEC = cation-exchange chromatography; WPC = weak partitioning
chromatography.
Future platforms could build on continuous processing, two-column pro-
cesses, and alternative unit operations (Figure 1.5). Alternatives to chroma-
tography may simplify processes, reduce costs, and facilitate the processing of
large batches. Currently, binding capacities for adsorptive membranes are
lower than those of resins, making them most suitable for flow-through chro-
matography operations in which relatively low amounts of impurities are
bound. Membrane adsorbers could replace an ion-exchange polishing step and
may offer reduced buffer consumption and small pool volumes when com-
pared to resin column chromatography (18).They may also be more conducive

VLS PROCESSES: TON-SCALE PRODUCTION AND BEYOND
11
to a continuous process in which the feedstream moves through each unit
operation without the collection of a discrete pool, although pH and conduc-
tivity targets must coincidently match subsequent unit operations. Other alter-
native technologies include crystallization and precipitation (9) (see Chapter
14). For individual antibodies, these technologies may provide a performance
benefit in cost or productivity that would merit their implementation in a
process. However, because of mAb variability, they are not likely to be ame-
nable to a platform process, and they require additional resource investment
during process development. Factors to consider in evaluating such novel
process options include annual production requirement, expression level,
COGs target, and production costs. As outlined later (see Chapter 12), eco-
nomic considerations must include capital investment, operating expenses,
economies of scale, the impact of opportunity costs of extended changeover
periods, and the processing success rate.
The concentration of drug substance is linked to the requirements for drug
product. For intravenous delivery, the drug product concentration may only
be 5–20mg/mL. Drivers for concentrations of 100mg/mL or greater include
the benefits of subcutaneous administration and a reduction in the number of
drug substance storage vessels (often large stainless steel tanks) for very large
batches. Achieving concentrations greater than 100mg/mL depends on the
solubility of the antibody as well as the formulation excipients, concentrations,
and pH. Operational challenges to high-concentration formulations include
mAb stability during concentration and diafiltration, product losses due to
system hold-up volume and incomplete recovery, and the extreme viscosity of
some antibodies as the concentration increases. These challenges may be
addressed by careful equipment design and by a formulation that minimizes
viscosity and maximizes product stability.
1.5 VLS PROCESSES: TON-SCALE PRODUCTION AND BEYOND
The recent increases in cell culture titers are likely to continue as cellular
productivity and cell density improve and as production phases are extended.
Titers as high as 10g/L have already been reported (19), and titers of 2−5g/L
or more will be common.The rise in titers combined with predictable scale-up
to larger bioreactors has sparked debate about purification bottlenecks that
limit a production plant’s capacity. However, efforts to intensify the purifica-
tion process should stave off capacity limits for all but the largest-volume
products (20, 21). The benefits of chromatography resins and ultrafiltration
membranes with elevated capacities and improved volumetric productivities
allow purification processes to keep pace with advances in cell culture. The
most common limitations are buffer make-up and storage capabilities,
in-process pool tank volumes, and the scale of the production equipment.
High-capacity resins and membranes reduce buffer consumption, generate

12
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
high-concentration in-process pools that minimize process volumes, and allow
larger batches to be processed with existing equipment.A two-column process
is significantly more productive than three-column or four-column purification
trains, and for almost all products, it should enable VLS production without
constraint.
Increased cell culture titers generated by extended fed-batch operations
and high cell densities give rise to very large batch volumes (e.g., a 10g/L titer
in a 25,000-L bioreactor would generate 250kg of harvest material and a 200-
kg batch of drug substance). An extremely long bioreactor production phase
affects facility utilization and staffing. At this scale, very few batches would be
required to supply the market for most mAbs. A 1-ton demand would be met
with only five batches, requiring very little production time in a plant operating
multiple bioreactors. Efforts to level the staffing load or to manage product
inventory may result in the operation of production bioreactors at less than
maximum volume, if plant capacity is not appropriately matched to demand
(this constraint is relaxed if the mAb is produced by a CMO). Furthermore,
as the bioreactor production stage lengthens,the mismatch between the shorter
cycle of the downstream process and the bioreactor becomes more problem-
atic (22). Harvesting a single reactor for an extended culture (e.g., every 24d
or more) and using a 3-d purification process means that the purification staff
may be unoccupied for a large portion of the interval between harvests. While
this ratio allows a single purification train to service eight bioreactors, the
enormous production capacity of a plant running high-titer processes would
invariably result in inefficiencies unless the plant is also making other
products.
Purification costs are the dominant drivers for VLS processes, but they are
still low when expressed on a per-gram basis. Key COG components are the
Protein A resin and the virus filter. Currently, the reuse of high-capacity
Protein A resins for 200 cycles or more (23) reduces costs associated with this
unit operation to approximately $1 per gram. This analysis presumes that the
resin is used to its full lifetime, which will be the case for large-volume products
in steady production over several years. When demand does not require
enough runs to extend the Protein A resin to its validated lifetime, COGs will
increase, with the cost per gram declining over time as the initial investment
in the resin is diluted over successive batches (e.g., if a 100-kg annual demand
can be produced with two 50-kg batches, each requiring five loading cycles
for the Protein A step per batch, the resin will not reach 200 cycles of use
until 20 years after launch, so limits on chronological resin age almost
certainly dictate a shorter life span). Another significant purification cost is
the VF filter. Provided that the membrane can offer reasonable fluxes with
highly concentrated feedstreams, an extended operation of several hours will
minimize the necessary membrane area and the cost. The reuse of VF mem-
branes is attractive, especially for new membranes that are developed to
withstand harsh sanitization cycles and do not require destructive post-use
integrity testing.

PROCESS VALIDATION
13
1.6 PROCESS VALIDATION
Process validation of purification processes defines process parameter ranges,
critical process parameters, virus removal, membrane and resin lifetimes, and
process pool stability. Control ranges ensure that the goals of the unit opera-
tion, including the appropriate product quality, are met. The variability of the
incoming feedstream, the capability of the downstream process steps, and the
potential interactions of all parameters for each step must be considered. A
chromatography step may have 10−20 operating parameters. A series of
designed experiments in which all parameters are examined would result in an
impossibly large study. By applying an understanding of the process, along with
a risk assessment, parameters can be placed into two categories:those that may
have interactions with other variables and those that can be studied in single-
parameter studies. For an ion-exchange column, examples of the potentially
interactive variables include elution pH, elution conductivity, column loading,
and temperature. Single-parameter studies might include flow rate, bed height,
wash volumes, and pooling conditions. The risk assessment should contain the
rationale for the type of study chosen for each parameter.
The process platform also offers significant benefits during process charac-
terization and validation. Modular validation leverages data from previous
studies to support the control ranges for a new antibody (24).The criteria that
allow a modular approach should be defined, as should the parameters that
qualify for modular or product-specific validation. For example, if a study for
antibody X had shown that Protein A bed heights between 15 and 25cm had
no effect on the process, that study could be used to support the same bed-
height range for antibody Y. However, because different antibodies have dif-
ferent dynamic binding capacities on Protein A resins, a product-specific study
for the load range would be necessary. Unit operations that tend to be less
product specific, such as centrifugation, virus filtration, low pH virus inactiva-
tion, and UF/DF, are the most amenable to the modular approach.
The quality by design (QbD) initiative is a comprehensive approach to
product development and life cycle management in which the product and
process parameters are designed to meet specific objectives. A thorough
understanding of process parameters and their interactions and impact on
product attributes defines the design space (25). Once approved, this design
space offers regulatory flexibility for post-approval process changes.Validation
studies must address the input variables (e.g., impurity levels) to the unit
operation from the preceding step. Modular validation complements QbD and
the definition of design space for platform processes. A company can use data
from validation studies on similar molecules, combined with risk analysis, to
define the design space for a related molecule.As a company accumulates data
on several mAbs, it may be possible to develop a master file that provides the
foundation for the design space for each new mAb manufacturing process.
For mAbs produced in mammalian cells, validation of the purification
process to remove or to inactivate adventitious viruses is a requirement (see

14
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
Chapter 8). In many cases, the platform processes for mAb manufacturing
have an exemplary viral safety profile: they use cells, such as Chinese hamster
ovary cells, which do not express viable retroviruses (26); use a culture medium
with no animal-derived raw materials; include viral safety tests and procedures
for postproduction cells for each lot; and establish the capability of the purifi-
cation process to clear or to inactivate viruses. The nature and scope of these
studies—including the number and types of viruses studied, determination of
the overall clearance of the model viruses, and confirmation of the validity of
the scale-down model and of the ability of resins to remove virus over their
claimed lifetime—are described in the International Conference on Harmoni-
sation Q5A (27). The concept of modular viral validation was first established
by the FDA in 1997 (28). It is critical to establish criteria for the use of modular
validation data. As an example, for a chromatography step, these criteria
should include the exact resin type, its position in the process, the robustness
of performance, and the comparability of process parameters (e.g., flow rate,
protein load, and bed height).
An understanding of the mechanism of virus removal or inactivation is an
important element of the modular approach. The design space for a step is
established by assessing the effect of process parameter ranges on product
purity (e.g., product variants, aggregates, and HCP). If a step contributes to
viral safety, an understanding of how these parameter ranges affect virus
removal is also needed. However, virus removal validation studies are com-
plicated and expensive. Therefore, studies are performed at the set point for
process parameters.To address this issue, modular data from a comprehensive
study on the effect of process parameters on viral clearance performed for one
mAb (to define its design space) are used to support the design space for
subsequent mAbs. With the mechanism of action and process robustness
established, companies may make modular claims for pH inactivation and VF
(29). Typically, the other step that contributes to viral safety is AEX chroma-
tography. If this step is amenable to modular claims, then there may be no
need to conduct product-specific viral clearance studies for clinical studies if
the extent of viral clearance from all modular studies is great enough to
provide a sufficient safety factor.
Even when modular validation can be applied, unique studies will be
required for each mAb. The hold times for conditioned media and in-process
pools will be a unique combination of the feedstream, impurities, and the mAb
sequence and structure. Forced decomposition studies should be performed
to identify degradation pathways. Resin reuse studies are unlikely to be ame-
nable to a modular validation approach because resin lifetime will be dictated
by unpredictable effects of the antibody’s properties as well as the impact of
feedstream variability arising from the unique characteristics of individual cell
lines.

PRODUCT LIFE CYCLE MANAGEMENT
15
1.7 PRODUCT LIFE CYCLE MANAGEMENT
Whereas the preapproval process development phase typically lasts 5−7 years,
the post-approval commercial manufacturing life cycle may extend to 20 years
or more. The manufacturing life cycle includes post-approval process changes,
transfer to new manufacturing sites, and possibly process scale-up.The breadth
and depth of process development, characterization, and validation can greatly
facilitate these changes. The reasons for making post-licensure changes are
varied, but the most common motivation is to increase production capacity to
meet increased demand. The approaches to increased capacity take a variety
of forms and present different technical and regulatory challenges. Transfer-
ring the process to a new manufacturing facility at a similar scale is the most
straightforward approach, but if the second facility is not an exact fit for the
process, adjustments may be necessary. Changes in scale are usually associated
with a facility change. If the scale-up of the cell culture and purification pro-
cesses is not aligned, particularly if the purification equipment is undersized
relative to cell culture, then it is likely that process adjustments will be
necessary.
The interval between the definition of the cell culture and purification pro-
cesses and product approval may be several years.During this time,advances in
cell culture technology may enable increases in titers of 50% or more, which
could be used to increase productivity of current products through post-
licensure process changes.The effects of this increased titer on the purification
process and facility are far-reaching and will probably require both process and
equipment changes. If no changes are made to the operating conditions of the
purification process, direct scale-up requires increases in column diameter and
volume,membrane and filter areas,flow rates,and buffer and pool volumes.For
facilitiesdesignedseveralyearspriortotheprocessimprovement,themaximum
titer used as a basis for the plant design is typically lower than that provided by
the newest product.
Before process changes are made, a thorough assessment of the locations
of purification bottlenecks is necessary. Changes to equipment, although
perhaps more straightforward from the technical and regulatory perspectives,
may be constrained by space availability, plant downtime during retrofitting,
and capital expense. Purification process changes fall into two general catego-
ries. The first category includes modifications to optimize wash and elution
volumes, to narrow pooling conditions, and to increase column capacities. How
the ranges for these parameters are specified in the license will determine the
regulatory pathway to the implementation of these changes. The second cate-
gory of changes involves substantial modification to the process and requires
regulatory approval prior to implementation. High-capacity chromatography
resins can be used to address process bottlenecks. Pool-volume reduction
affects both the step’s pool volume and the downstream pool volumes if that
step is operated in the flow-through mode. Because one main facility limitation
is tank number and volume, changing the order of unit operations to minimize

16
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
the effect of pH and conductivity adjustments for downstream steps can be
beneficial. Of course, adding UF/DF steps is a one way to address tank limita-
tions, but this change involves new capital equipment, downtime, and space
considerations.
Increases in the product titer may not be the only changes to the feedstock.
Modifications to the cell culture process may also change the product and
process impurity profiles. Other changes to the feedstock include increased
debris load with different physical properties, which will challenge the harvest
operation. Evolving regulatory requirements are also drivers for process
changes. These regulatory drivers may not be limited just to requirements of
the national and international regulatory agencies [e.g., the FDA and Euro-
pean Medicines Evaluation Agency (EMEA)]; they may also include state and
local agencies that govern areas such as hazardous waste and discharge into
the local water system.
Risk assessment should assess the scope of process characterization work
needed to support process changes.This assessment should evaluate the impact
of the changes on the overall process and the applicability of validation studies
from the original process. For example, if changes were made only to the cell
culture process, the Protein A step would need to be revalidated. If the Protein
A pool from the new process was comparable (i.e., had the same level of
product and process impurities), then the unit operations downstream of
Protein A would not need to be revalidated.
Commercial manufacturing is critical to the patients who rely on these
drugs, and uninterrupted product supply is dependent on the availability of
raw materials. For many purification raw materials, such as chemicals, supply
is not critical because multiple suppliers can meet the necessary quality and
quantity requirements. However, for some raw materials (e.g., chromatogra-
phy resins), similar raw materials are not interchangeable among vendors.
During process development, a specific resin is chosen for each step and the
process optimized for that resin. Changing to a similar resin may not result in
the same purity and yield under the same operating parameters. Certain mem-
branes and filters may require unique operating conditions. For these process-
specific raw materials, it is critical to have a risk-mitigation plan that will
ensure uninterrupted manufacturing should a vendor be unable to supply a
raw material (30). The risk can be mitigated by carrying an additional inven-
tory of these raw materials or by developing processes with alternative raw
materials. When alternative raw materials are used, the product quality of the
drug substance must still meet the appropriate specifications. Ideally, an alter-
native raw material will provide the same process performance as the original
raw material, although other factors (e.g., cost, ease of use, and process robust-
ness) may make it a second choice. If the alternative raw material does not
result in the same product quality in the intermediate process pool, it will be
necessary to assess whether downstream steps can achieve the same product
quality either with or without modification. Once alternative raw materials
have been identified, an implementation strategy should be developed. The

FUTURE OPPORTUNITIES
17
cost of the work necessary to develop, characterize, and validate an alternative
raw material should be balanced against the cost of carrying an additional raw
material inventory. Other factors to consider include the resin’s lifetime, cost,
and annual demand. When a raw material is used in more than one process,
cumulative use and business risks also need to be considered. A platform
process that relies on a small number of raw materials minimizes the number
of raw materials in inventory and may reduce the overall backup required.
However, this approach increases business risk, because a supply disruption
may affect many products.
Raw materials may be grouped into classes such as chromatography resins,
sterilizing filters, virus filters, and ultrafiltration membranes. The degree to
which such raw materials can be substituted in these classes can be considered
according to their intended use. For example, there may be significant differ-
ences among CEX resins in terms of resolution, with each resin requiring dis-
tinct operating conditions. These differences are based on the unique physical
(e.g., bead and pore size) and chemical (e.g., ligand and resin matrix) proper-
ties of resins.Alternatively, sterilizing filters have a clear mechanism of separa-
tion (a size differential), and the operating parameters or design space can be
defined to cover various filters. Taking this a step further, as the QbD concept
evolves and design space is increasingly defined on the output of a unit opera-
tion, it may be possible to use different raw materials for a step as long as the
output of the processing step (e.g., product purity and isoform distribution) is
maintained.
For post-approval process changes, QbD and design space can greatly facili-
tate such changes. Knowledge gained during process development leads to a
thorough understanding of process input ranges and process outputs, particu-
larly for critical quality attributes, and thus lays the foundation for a robust
design space. Such a design space is the basis for consistent, reproducible
manufacturing operations, but also allows changes within the design space
over the product’s life cycle in response to changing conditions.
1.8 FUTURE OPPORTUNITIES
With standard mAb purification platform processes firmly entrenched in the
industry and representing the current state of the art, one might ask what the
future will hold. Several opportunities addressing this question will be pre-
sented in this section. Some of these suppositions may come to pass, while
others may never be adopted.
Because mAbs are a group of compounds large enough to warrant consid-
eration as a separate class (28),the adoption by regulatory agencies of common
targets for impurity levels could be of great value to the industry.These targets
would define process development design principles that are generally regarded
as safe and would represent impurity levels that are readily achievable with
standard processes. Currently, the only impurity that has a well-accepted limit

18
DOWNSTREAM PROCESSING OF MONOCLONAL ANTIBODIES
is host cell DNA. The FDA initially issued guidance that specified 10pg/dose
(31), and then 100pg/dose (28), and this was followed by World Health Orga-
nization guidelines that specified 10ng/dose (32).
At the 2003 Well Characterized Biologicals conference, FDA representa-
tives cited typical levels of HCP, aggregates, and residual Protein A levels for
mAbs described in Phase I Investigational New Drug Applications (33).
Although levels of process- and product-related impurities have not been
translated into regulatory guidance documents, the presentation suggests these
levels are generally regarded as safe with the caveat that dose, dosing fre-
quency, and route of administration are all important factors have not been
issued in regulatory guidance documents, and while they do not reflect a
common international position, the presentation of these levels hints at the
possibility of establishing limits that are generally regarded as safe for these
host cell and process-related impurities. Clarity regarding these targets could
greatly simplify process development, enable more productive processes that
do not “overdevelop or overengineer” without compromising product safety,
and aid in the regulatory review of both clinical filings and commercial license
applications.
Of course, the appropriate limits on impurity levels should be evaluated on
a case-by-case basis, with risk adjustment for therapeutic dose, disease indica-
tion,dosing frequency,and other clinical factors.Ideally,a set of common target
impurity levels would cover the most conservative case, so that adjustments
would generally be made only to increase the impurity levels if warranted by
process capability, following a thorough review of the clinical context for the
product. In the opposite case, companies would be expected to recognize
the highly unusual combination of factors that would warrant a reduction in the
target impurity level in order to lower the risk of adverse clinical events.
Programs are under development at several companies for identifying, early
in the discovery phase, mAbs that have inherent processing or quality issues,
such that mAbs directed at the same target but with alternate sequences (and
thus improved characteristics) could be chosen instead. Examples of the
product attributes that may be included in a manufacturing feasibility assess-
ment include stability (both drug substance and drug product), maximum
solubility and suitability for subcutaneous administration, viscosity at high
concentrations, tendency toward unwanted microheterogeneity, and compati-
bility with a platform purification process. Recognizing that the value of many
of these attributes does not manifest itself until late in a product’s life cycle,
early intervention based on the feasibility assessment prior to the initiation
of clinical trials is a prudent approach to accommodate manufacturing
objectives.
Several companies are evaluating production hosts other than mammalian
cells, including transgenic animals or plants, recombinant yeast engineered to
express the appropriate glycosyltransferases and thus provide natural oligo-
saccharides, and full-length mAbs from Escherichia coli. It is unlikely that the
purification platform developed for mAbs expressed in mammalian cells will
transfer cleanly to mAbs produced by these alternate hosts. The up-front