Process Scale Purification of Antibodies E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 745

Veröffentlichungsjahr: 2011

Beschreibung

Traditional column chromatography dominates current purification technology, and many of the productivity gains that have been achieved have relied on upscaling such devices. However, this comes with a cost penalty and the pharmaceutical industry has reached the point at which further upscaling becomes economically unsupportable. This book offers a broad-based reassessment of old and new purification methods, incorporating an analysis of innovative new trends in purification. The book has wide coverage of different antibody purification strategies and brings together top-tier experts to address problems in process-scale antibody purification.

Leseprobe

CONTENTS

PREFACE

REFERENCES

About the Editor

Contributors

1 Downstream Processing of Monoclonal Antibodies: Current Practices and Future Opportunities

1.1 INTRODUCTION

1.2 A Brief History of cGMP mAb and Intravenous Immunoglobulin (IgIV) Purification

1.3 Current Approaches in Purification Process Development: Impact of Platform Processes

1.4 Typical Unit Operations and Processing Alternatives

1.5 VLS Processes: Ton-Scale Production and Beyond

1.6 Process Validation

1.7 Product Life Cycle Management

1.8 Future Opportunities

1.9 Conclusions

1.10 Acknowledgments

1.11 References

2 The Development of Antibody Purification Technologies

2.1 Introduction

2.2 Chromatographic Purification of Antibodies before Protein A

2.3 Antibody Purification after 1975

2.4 Additional Technologies for Antibody Purification

2.5 Purification of mAbS Approved in North America and in Europe

2.6 Acknowledgments

2.7 References

3 Harvest and Recovery of Monoclonal Antibodies: Cell Removal and Clarification

3.1 Introduction

3.2 Centrifugation

3.3 Microfiltration

3.4 Depth Filtration

3.5 Flocculation

3.6 Absolute Filtration

3.7 Expanded-Bed Chromatography

3.8 Comparison of Harvest and Clarification Unit Operations

3.9 Acknowledgments

3.10 References

4 Protein A-Based Affinity Chromatography

4.1 Introduction

4.2 Properties of Protein A and Commercially Available Protein A Resins

4.3 Protein A Chromatography Step Development

4.4 Additional Considerations During Development and Scale-Up

4.5 Virus Removal/Inactivation

4.6 Validation and Robustness

4.7 Conclusions

4.8 Acknowledgments

4.9 References

5 Purification of Human Monoclonal Antibodies: Non-Protein A Strategies

5.1 Introduction

5.2 Integrated Process Designs for Human Monoclonal Antibody (HuMab) Production

5.3 Purification Process Designs for HuMabs

5.4 Conclusions

5.5 Acknowledgments

5.6 References

6 Purification of Monoclonal Antibodies by Mixed-Mode Chromatography

6.1 Introduction

6.2 A Brief History

6.3 Prerequisites for Industrial Implementation

6.4 Mechanisms, Screening, and Method Development

6.5 Capture Applications

6.6 Polishing Applications

6.7 Sequential Capture/Polishing Applications

6.8 The Future

6.9 Acknowledgments

6.10 References

7 Integrated Polishing Steps for Monoclonal Antibody Purification

7.1 Introduction

7.2 Polishing Steps in Antibody Purification

7.3 Integration of Polishing Steps

7.4 Conclusions

7.5 Acknowledgments

7.6 References

8 Orthogonal Virus Clearance Applications in Monoclonal Antibody Production

8.1 Introduction

8.2 Model Viruses and Virus Assays

8.3 Virus Clearance Strategies from First in Human (FIH) to Biological License Application (BLA) Filing

8.4 Orthogonal Viral Clearance in mAb Production

8.5 Conclusions and Future Perspectives

8.6 Acknowledgments

8.7 References

9 Development of A Platform Process for the Purification of Therapeutic Monoclonal Antibodies

9.1 Introduction

9.2 Chromatography Steps in the Platform Process

9.3 Virus Inactivation

9.4 UF/DF Platform Development

9.5 Platform Development: Virus Filtration and Bulk Fill

9.6 Examples of Platform Processes

9.7 Developing a Viral Clearance Database Using a Platform Process

9.8 Summary

9.9 References

10 Advances in Technology and Process Development for Industrial-Scale Monoclonal Antibody Purification

10.1 Introduction

10.2 Affinity Purification Platform

10.3 Advances in CEX Purification of mAbs

10.4 HPTFF

10.5 A New Nonaffinity Platform

10.6 References

11 Continuous Chromatography for the Purification of Monoclonal Antibodies

11.1 Introduction

11.2 Product Variants and the Separation Problem

11.3 Definition of Performance Parameters

11.4 Gradient Chromatography for Biomolecules

11.5 Continuous Chromatography to Increase Productivity

11.6 The MCSGP Process

11.7 Upgrades for Continuous Processes to Improve Stability

11.8 Impact of Increasing Fermentation Titers

11.9 Outlook

11.10 Acknowledgments

11.11 References

12 Process Economic Drivers in Industrial Monoclonal Antibody Manufacture

12.1 Introduction

12.2 Challenges When Striving for the Cost-Effective Manufacture of mAbs

12.3 Cost Definitions and Benchmark Values

12.4 Economies of Scale

12.5 Overall Process Economic Drivers

12.6 DSP Drivers at High Titers

12.7 Process Economic Trade-Offs for DSP Bottlenecks

12.8 Summary and Outlook

12.9 References

13 Design and Optimization of Manufacturing

13.1 Introduction

13.2 Process Design and Optimization

13.3 Modeling Approaches

13.4 Cost Models in Practice

13.5 Simulation in Practice

13.6 Acknowledgments

13.7 References

14 Alternatives to Packed-Bed Chromatography for Antibody Extraction and Purification

14.1 Introduction

14.2 Increasing the Selectivity of Harvest Procedures: Flocculation of Particulate and Nonparticulate Impurities

14.3 Solutions for Antibody Extraction, Concentration, and Purification

14.4 Nonchromatographic Solutions for Antibody Purification and Formulation

14.5 Membrane Adsorbers

14.6 Conclusions

14.7 Acknowledgments

14.8 References

15 Process-Scale Precipitation of Impurities in Mammalian Cell Culture Broth

15.1 Introduction

15.2 Precipitation of DNA and Protein–Other Applications

15.3 A Comprehensive Evaluation of Potential Precipitants for Impurity Removal

15.4 Industrial-Scale Precipitation

15.5 Cost of Goods Comparison

15.6 Summary

15.7 Acknowledgments

15.8 References

16 Charged Ultrafiltration and Microfiltration Membranes in Antibody Purification

16.1 Introduction

16.2 Charged Ultrafiltration Membranes

16.3 Concentration Polarization and Permeate Flux

16.4 Stagnant Film Model (SFM)

16.5 Osmotic Pressure Model

16.6 Mass Transfer Coefficient

16.7 Sieving Coefficient

16.8 Diffusion–Convection Model

16.9 Scale-up Strategies and the Constant Wall Concentration (Cw) Approach

16.10 Protein Fractionation Using Charged Ultrafiltration Membranes

16.11 Case Study

16.12 Membrane Cascades

16.13 Charged Microfiltration Membranes

16.14 Virus Clearance

16.15 Salt Tolerance

16.16 Conclusions

16.17 Acknowledgments

16.18 References

17 Downstream Processing of Monoclonal Antibody Fragments

17.1 Introduction

17.2 Production of Antibody Fragments for Therapeutic Use

17.3 Downstream Processing

17.4 Improving the Pharmacological Characteristics of Antibody Fragments

17.5 Conclusions

17.6 Acknowledgments

17.7 References

18 Purification of Antibodies Other Than IgG: The Case of IgM and IgA

18.1 Introduction

18.2 Purification of Immunoglobulin M (IgM)

18.3 Purification of IgA

18.4 Conclusion

18.5 Acknowledgments

18.6 References

19 Purification of Antibodies From Transgenic Plants

19.1 Introduction

19.2 Antibody Production in Transgenic Plants

19.3 Downstream Processing of Antibodies Produced in Transgenic Plants

19.4 Purification of Plant-Derived mAbs Using Protein A

19.5 Purification of Plant-Derived mAbs Using Nonprotein A Media

19.6 Polishing Steps

19.7 Conclusions

19.8 Acknowledgments

19.9 References

20 Antibody Purification: Drivers of Change

20.1 Introduction

20.2 The Changing Regulatory Environment–Pharmaceutical Manufacturing for the Twenty-First Century

20.3 Technology Drivers–Advances and Innovations

20.4 Economic Drivers

20.5 Conclusions

20.6 Acknowledgments

20.7 References

Index

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 mammalian cells, and an entire subindustry has grown up around downstream processing 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 antibodies 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 targeting, 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 100 mg/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 1-5 g/L titers now routine and the real prospect of 10-20 g/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 processing, 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 recovery 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 chromatography 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 overview 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 colleagues), Protein A chromatography (Suresh Vunnum and colleagues), nonProtein 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 framework, 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 clearance with capture and purification (Yuling Li and colleagues). Nuno Fontes and Robert van Reis then consider the important aspects of scaling up antibody 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 Muller-Spath 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 manufacture, 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: flocculation, 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 monoclonals (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 considering 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 representing 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 antibodies. 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 antibody processing and can lead to affordable, high-quality pharmaceutical products in the future.

UWE 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 innovation. BioPharm International 20,70-94.

3. Langer, E.S. (2008). Fifth Annual Report and Survey ofBiopharmaceutical Manufacturing 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 GottschalkGroup Vice PresidentPurification TechnologiesSartorius Stedim Biotech

D - 37079 GöttingenTel: (0551) 308 - 2016Telefax: (0551) 308 - 3705Email: [email protected]

Dr. Gottschalk is Group Vice President, Purifi cation Technologies, at Sartorius Stedim Biotech. He worked in different capacities for Bayer Health Care from 1991 to 2004, overseeing the purifi cation of monoclonal antibodies and recombinant 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.

CONTRIBUTORS

Alahari Arunakumari, PhD, Senior Director, Process Development, Medarex, Inc., 519 Route 173 West, Bloomsbury, NJ 08804; Email: [email protected]

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, Universite Victor Segalen Bordeaux 2

John Curling, CEO, John Curling Consulting AB, Swedenborgsgatan 3-5, SE- 753 34 Uppsala, Sweden; Email: [email protected]

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: [email protected]

Suzanne S. Farid, PhD, Senior Lecturer/Associate Professor, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; Email: [email protected]

Nuno Fontes, PhD, Late Stage Purification–Process Research & Development, Genentech, Inc., 1 DNA Way South, San Francisco, CA 94080; Email: [email protected]

Pete Gagnon, Chief Scientific Officer, Validated Biosystems, 240 Av Vista Montana, Ste 7F, San Clemente, CA 92672; Email: [email protected]

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: [email protected]

Judy Glynn, PhD, Senior Principal Scientist, Pfizer, Inc., 700 Chesterfield Parkway West, Chesterfield, MO, 63017; Email: [email protected]

Uwe Gottschalk, PhD, Group Vice President Purification Technologies, Sartorius Stedim Biotech, Gottingen, Germany; Email: [email protected]

John Hickey, Associate Director, Process Sciences–Downstream & Project Management, Bristol - Myers Squibb, Co.

Brian Hubbard, PhD, Executive Scientific Director, Process & Product Development, 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: [email protected]

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: [email protected]

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.

Massimo Morbidelli, PhD, Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland; Email: [email protected]

Thomas Muller-Spath, Dipl.-Ing., Institute for Chemical and Bioengineering, ETH Z urich

Zivko L. Nikolov, PhD, PE, Dow Professor, Biological and Agricultural Engineering, Texas A&M University, College Station, Texas 77843-2117; Email: [email protected]

Rhona O'Leary, PhD, Early Stage Purification, Genentech

Narahari Pujar, PhD, Bioprocess R&D, Merck & Co., Inc., West Point, PA; Email: [email protected]

Jeffrey T. Regan, Biolex Therapeutics

Xavier Santarelli, PhD, Professor, EA 4135, ESTBB, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, Bordeaux cedex, France; Email: [email protected]

Abhinav A. Shukla, Manufacturing Sciences, Bristol-Myers Squibb, Co., East Syracuse, NY 13057; Email: [email protected]

Andrew Sinclair, CEO and Founder, BioPharm Services, Lancer House, Chesham, HP5 1RD, UK; Email: [email protected]

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: [email protected]

Jorg Thommes, 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

Jue Wang, PhD, Senior Manager, Purification Process Development, Medarex, Inc.

Susan L. Woodard, PhD, Department of Biological and Agricultural Engineering, Borlaug Center/TAMU 2123, Texas A&M University

Yaling Wu, PhD, Senior Scientist, Purification Sciences-BioPharmaceutical Development, Human Genome Sciences, Inc.

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: [email protected] or [email protected]

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 processing. 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 manufacturing 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 production at either in–house or contract production facilities. Although mAbs are not commodity products that are substitutable in the clinical setting, they have distinct advantages in production scale and cost, as well as in product development 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 operations 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 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 technology, other than ultrafiltration to formulate and to concentrate the drug substance. 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.

Figure 1.1 Cohn–based IgIV consensus process.

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 knowledge 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; incorporation 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 (). In addition, downstream processing was sometimes performed in the cold. Chromatographic media often provided relatively low loading capacities, 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 selection was based on maximizing volumetric productivity and on the ability to process large volumes of feed rapidly, rather than on the handling of large batches (greater than 20 kg 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.