Cell-Culture-Bioprocess-Engineering Book.pdf - Cell Culture Bioprocess Engineering Cell Culture Bioprocess Engineering Wei-Shou Hu Department of

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Unformatted text preview: Cell Culture Bioprocess Engineering Cell Culture Bioprocess Engineering Wei-Shou Hu Department of Chemical Engineering and Material Science University of Minnesota Minneapolis, MN With Contributions From: Weichang Zhou Gargi Seth Sadettin Ozturk Chun Zhang Edition 1.1 Copyright © 2013 by Wei-Shou Hu ISBN: 978-0-9856626-0-8 Preface For over two decades, we have assembled innovative guest lecturers to share their research and best-practices at our annual cell culture bioprocessing short course at the University of Minnesota. This course was created for industrial practitioners of the production of biologics. This book is the culmination of two decades of accumulated expertise, practical know-how and insight into future trends. There have been many books and courses on cell culture technology covering topics from a technical or business perspective. The goal of this course and this book is to bring new knowledge from cutting-edge research into the very practical setting of today’s industrial laboratories. A second goal of this course is to prepare industrial practitioners and students from different academic disciplines to collaborate in today’s cross-disciplinary teams. In the course of delivering a molecule from a gene sequence in the laboratory to a product in the manufacturing plant, scientists and engineers must quickly communicate, troubleshoot and innovate. The fundamental knowledge for practicing industrial cell culture spans from cell biology and physiology to process engineering principles in stoichiometry, reactor kinetics and scale up. Thus, we have designed this course for students of diverse backgrounds. The book is used in the classroom of our annual course. The layout of the book is thus designed to facilitate the delivery of information. The left panels are graphs, tables, diagrams, highlights of key points and space for note taking; while the right panels are descriptive text. This course has been given around the world: in Europe, East and South Asia, South America and as an internal course at many corporations. Over three thousand industrial biotechnologists have taken this course. With the technology of biologics production spreading to wider regions of the world, this book will meet a timely need of many who practice the technology but cannot attend the course in Minnesota. The book is published in an electronic form to allow for more frequent future updates, and for easy distribution to the parts of the world where the biologics manufacturing is quickly expanding. Wei-Shou Hu Department of Chemical Engineering and Material Science University of Minnesota Acknowledgements The authoring of this book has been influenced by many who have lectured in the summer course at the University of Minnesota over the years. Foremost, thanks go to Anthony J. Sinskey, Michael C. Flickinger, Donald McClure and Fredrick Srienc who started the course with me originally. Konstantin Konstantinov, James Piret, James N. Thomas, Randall Kaufman, Florian Wurm, John Aunins, Michael Betenbaugh, Sadettin Ozturk, Matthew Croughan, Weichang Zhou, Chun Zhang and Gargi Seth all contributed to enrich the course. Many former and current members of my research laboratory at the University of Minnesota contributed to the preparation of course materials. These include Derek Adams, Marlene Castro, Bhanu Chandra Mulukutla, Anushree Chatterjee, Anna Europa, Patrick Fu, Chetan Gadgil, Mugdha Gadgil, Anshu Gambhir, Patrick Hossler, Claire Hypolite, Nitya M. Jacob, Kathryn Johnson, Anne Kantardjieff, Edmund Kao, Anurag Khetan, Rashmi Korke, Huong Le, Jongchan Lee, Marcela de Leon Gatti, Sarika Mehra, Jason D. Owens, Yonsil Park, Gargi Seth, Shikha Sharma, Kartik Subramanian, Siguang Sui, Katie Wlaschin, and Kathy Wong. Gargi Seth, Sadettin Ozturk, Weichang Zhou and Chun Zhang, whose participation in the course led to the development of new chapters, are noted as contributors. This book, which began as a set of lecture notes, has gone through many years of refinement in organization by many skillful hands. Kimberly Durand first took the notes to digital form in a CD ROM. Ruth Patton, Radha Dalal, Katherine Matthews, Heather Wooten, Kirsten Keefe, Jessica Raines-Jones, Kimberly Coffee and Kaitlyn Pladson continued to shape it. At the long last, Erin Fenton and Jenna Novotny took it to current form. Kimberly Durand also coordinated our final publication efforts. This book is dedicated to the students, fellows and staff formerly and currently in my laboratory at the University of Minnesota. It is through working with them that the materials used in the book were distilled. It was also through their educating me with new knowledge, new concepts, and new tools that this book took its shape. I must also thank my dear friend and close colleague, Miranda Yap of Bioprocess Technology Institute, Singapore, with whom I have had a wonderful and long collaboration. Finally, I wish for my lovely family, Jenny, Kenny and my wife, Sheau-Ping to share the joy of the book’s completion. Wei-Shou Hu Department of Chemical Engineering and Material Science University of Minnesota Contents In Brief Overview of Cell Culture Technology. . . . . . . . . . . . . . . . . . . . . . . 1 Cell Biology for Bioprocessing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Cell Physiology for Process Engineering. . . . . . . . . . . . . . . . . . . . . 57 Medium Design for Cell Culture Processing. . . . . . . . . . . . . . . . . . 97 Cell Line Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Stoichiometry and Kinetics of Cell Cultivation. . . . . . . . . . . . . . . . 147 Cell Culture Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Metabolic Flux Analysis in Cell Culture Systems . . . . . . . . . . . . . . 175 Cell Culture Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Oxygen Transfer in Cell Culture Bioreactors . . . . . . . . . . . . . . . . . 213 Fedbatch Culture and Dynamic Nutrient Feeding. . . . . . . . . . . . . 233 Cell Retention and Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Scaling Up and Scaling Down for Cell Culture Bioreactors. . . . . . 263 Cell Culture Genomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 ACKNOWLEDGEMENTS | VII Overview of Cell Culture Technology Cell Culture Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Cell Culture Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Virus Vaccines and Protein Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Protein Molecule as Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Industrial Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Biosimilars or Follow-on-Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Manufacturing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Alternative Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Product Quality and Process Robustness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Critical Feature of rDNA Proteins from Mammalian Cells . . . . . . . . . . . . . . . . . . 14 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Cell Culture Engineering In the past decade we have seen continuous growth in mammalian cell culture bioprocessing, driven primarily by the expansion of therapeutic antibody production in the pharmaceutical industry. The range and quantity of products have both significantly increased over the past ten years. Also fueling this growth are the increasing numbers of therapeutic protein candidates in the drug development pipeline that can potentially render many more untreatable diseases treatable. Recombinant therapeutic proteins have yielded major advances in healthcare. Their societal impacts may even rival those of antibiotics, whose discovery and clinical applications transformed much of modern medicine. Microbial fermentation technology enabled pharmaceutical industry to make penicillin widely available between 1950 and 1970. Today, we see cell culture processing technology enabling this new class of protein biologics to reach needy patients. As with any product, manufacturers are under continual pressure to produce more with less. In OVERVIEW | 1 the case of cell culture bioprocess technology, we see increasing demand for therapeutic proteins, coupled with the strain of often prohibitively high investment costs for new manufacturing facilities. Thus, we must constantly re-evaluate, streamline and refine, to increase production without the luxury of totally new and improved facilities. Fig. 1.1: Historical trend of penicillin titer and value As we look to the future of cell culture processing, it is useful to look back at the history and development of penicillin. This specific case highlights lessons that are almost universally relevant for the manufacturing of other products. Today’s innovations will all travel through some variation of these phases, from the moment of discovery, expansion and distribution, maturation and even demise of the product. Pencillin is also representative of the many strides made in the broader field of microbial natural products that preceded today’s protein biologics. Sir Alexander Fleming’s discovery of penicillin began a new chapter in biotechnology. In the twentyfive years following the first clinical applications (pioneered by Edward Penley Abraham) both the product titer and the production volume of penicillin increased almost exponentially. This rapid expansion in production quantities and titer was then followed by a period of slower but steady growth over the next fifty years. The roughly three orders of magnitude increase in production volume and product concentration was the result of relentless effort on the part of process scientists and engineers. These engineers looked for hidden opportunities for strain improvement, media development, and much more. As a result, we have seen steady productivity growth due to improvements in oxygen transfer, heat transfer, and mixing characteristics. Additional advances in on-line sensing, sterility control, equipment reliability and process control all contributed to technological success. It should be noted that the success of process technology also eventually drove down the price. Penicillin G is no longer produced in the United States; the cost of production is now OVERVIEW OF CELL CULTURE TECHNOLOGY | 2 dramatically lower in other parts of the world. Now, two decades after the first introduction of therapeutic biologics, we have seen titers in large manufacturing processes increase from tens of milligrams per liter to more than five grams per liter for many immunoglobulin products today. Although little published information is available, the production cost has also decreased by at least an order of magnitude since the beginning of cell culture products. A graph of historical data for cell culture products plotted one or two decades from now will likely resemble that of penicillin. Cell culture production today is likely around the transition from the exponential growth stage to the steady and slower growth period. However, it is important to note that, in terms of both absolute quantity of product produced and economic value, the slower and steadier phase is as critical as the early rapid growth stage for the product life cycle. Even for penicillin, there were tremendous process enhancements after the initial rapid growth phase. Due to these improvements, major medicines became affordable for the world’s population. The next question for bioprocess scientists and engineers is: How can cell culture processing accomplish what the antibiotics industry has achieved for our society? Bioprocess scientists and engineers possess genomics and genome engineering tools that were not previously available to antibiotic researchers or even to the early innovators of cell culture processes. These new genome-wide investigative and engineering tools will greatly facilitate the designing and engineering of cells with desired growth and production characteristics. Process technologists will need to harness the power of genomics and genome engineering to enhance productivity and process robustness. This will also facilitate the expansion of biosimilars (i.e., “Follow-on” biologics) and make many medicines available to needy patients around the world at an affordable cost. Much of the process technology employed in cell culture biologics was developed for antibiotic OVERVIEW OF CELL CULTURE TECHNOLOGY | 3 Cell Culture Products production. In transforming cell culture products from laboratory discovery to clinical reality, many innovations in the design and engineering of gene constructs, cells, products, and processes have been conceived and implemented. These technologies are also likely to help new technologies move forward. The next generation technology that will benefit most from cell culture innovations is stem cell based therapy. This technology is still in its infancy, but its significant potential impact on our society will compel cell culture technologists to push the evelope. Virus Vaccines and Protein Therapeutics Cell culture processes have been used to produce viral vaccines for over half a century. Virus production in animals or in tissues has been in practice for over two centuries. The most notable example is the pox vaccine from cow. Most of the tissue-based production methods have since been replaced by cell culture processes. A tissue system that is still in use is the chick egg. This process is begun by seeding a virus into 10-day-old embryos in chicken eggs. A few days later, the replicated virus is then isolated from infected embryos. Early cell culture processes were an extension of tissue culture, using primary cells explanted from various tissues (such as chick embryos and monkey kidneys) for the virus to infect and replicate. The primary cells used in virus production have mostly been replaced by cell strains or even cell lines, which can be cultivated over many generations to build up stocks (or a cell bank) for routine use to ensure consistent quality. Most viruses used as vaccines have been inactivated by formalin treatment to render the virus incapable of infection. However, the treated virus particles retain a small degree of immunogenicity to elicit the immune response in vaccine applications. There are cases in which live attenuated viruses are used. These attenuated viruses have been adapted, OVERVIEW OF CELL CULTURE TECHNOLOGY | 4 Table 1. Principal Viral Vaccines Used in Prevention of Human Virus Diseases Disease Source of vaccine Condition of virus Poliomyelitis Tissue culture (human diploid cell line, monkey kidney) Live attenuated, inactivated Measles Tissue culture (chicken embryo) Live attenuated Mumps Tissue culture (chicken embryo) Live attenuated Rubella Tissue culture (duck embryo, rabbit, or human Live attenuated diploid) Smallpox (vaccinia) Lymph from calf or sheep Live vaccinia (glycerolated, lyophilized) Smallpox (vaccinia) Chorioallantois, tissue cultures (lyophilized) Vaccinia Yellow fever Tissue cultures and eggs (17D strain) Live attenuated Influenza Highly purified subunit forms of chicken embryo allantoic fluid (formalinized UV irradiated) Inactivated Influenza Cell culture (MDCK, Vero) Attenuated Rabies Duck embryo or human diploid cells Inactivated Adenovirus Human diploid cell cultures Live attenuated Japanese B encephalitis Mouse brain (formalinized), cell culture Inactivated Venezuelan equine cephalomyelitis Guinea pig heart cell culture Live attenuated Eastern equine Chicken embryo cell culture Inactivated Western equine Chicken embryo cell culture Inactivated Russian spring summer encephalitis Mouse brain (formalinized) Inactivated often by the prolonged cultivation in a non-human host species so that the adapted strain is no longer virulent to humans. These viruses are still capable of replication, which significantly reduces the dose required for immunization. However, they also carry a very low, but non-zero, risk of reverting to their wild type form and causing an infection in the patient. Vaccine technology predated modern cell culture for recombinant protein production by over two decades. Although recombinant therapeutic proteins propelled the advances in cell culture technology, proteins derived from tissues, and even cell culture, were used for therapeutic purposes even before the arrival of recombinant DNA technology. These examples include insulin, urokinase, Factor VIII, and interferon. The generation of recombinant DNA therapeutic proteins, such as human growth hormone and insulin, were first produced in microorganisms. The next wave were human proteins, which naturally circulate in human blood and require post-translational modifications, such as complex disulfide-bond formation and glycosylation. These proteins can not be replicated in microbial systems. For the production of those proteins, mammalian cells must be, and were, employed. Initially, hybridoma cells were used. These are fusion products of the non-antibody-secreting, but continuously proliferating, myeloma cell and the antibody-secreting, but non-dividing, lymphocyte. This soon gave way to recombinant DNA technology. After the introduction of tissue plasminogen activation (tPA) by Genentech in 1987, erythropoietin (EPO) and Factor VIII also reached the market in following years. Antibody products and antibody-based fusion proteins have since blossomed. They make up the bulk of the protein drugs in clinical use. OVERVIEW OF CELL CULTURE TECHNOLOGY | 5 Table 2. Therapeutic Protein Biologics Produced in Non-Mammalian Host Activity/Use Granulocyte colonystimulating factor (Neupogen) White blood cell growth for Neutropenia Insulin (Humulin) Diabetes α-Interferon (Intron-A) Anticancer, viral infections Somatropin [human growth hormone] (Humatrope) Growth deficiencies Somatropin [human growth hormone] (Protopin/ Nutropin) Growth deficiencies Interleukin-2 (Proleukin) Kidney Cancer Table 3. Non-Antibody Products Produced in Mammalian Cells Trade name Type Therapeutic Use Manufacturer U.S. approval year Host Aldurazyme Laronidase Mucopolysaccharid-eosis I Genzyme 2006 CHO Cerezyme β-glucocerebrosidase Gaucher’s disease Genzyme 1994 CHO Myozyme Fabrazyme -galactosidase Pompe disease Genzyme 2006 CHO -galactosidase Fabry disease Genzyme 2003 CHO Naglazyme N-acetylgalactosamie Mucopolysaccharideosis VI 4-sulfatase BioMarin Pharmaceutical 2005 CHO Orencia Ig-CTLA4 fusion Rheumatoid arthritis Bristol-Myers Squibb 2005 CHO Luveris Luteinizing hormone Infertility Serono 2004 CHO Activase Tissue plasminogen activator Acute myocardial infraction Genentech 1987 CHO Epogen/ Procrit EPO Anemia Amgen/Ortho Biotech 1989 CHO Aranesp EPO (engineered) Anemia Amgen 2001 CHO Pulmozyme Deoxyribonuclease I Cystic fibrosis Genentech 1993 CHO Avonex Interferon-β Relapsing multiple sclerosis Biogen Idec 1996 CHO Rebif Interferon-β Relapsing multiple sclerosis Serono 2002 CHO Follistim/ Gonal-F Follicle stimulating hormone Infertility Serono/NV Organon 1997 CHO Benefix Factor IX Hemophillia A Wyeth 2000 CHO Enbrel TNF receptor fusion Rheumatoid arthritis Amgen, Wyeth 1998 CHO Tenecteplase Tissue plasminogen activator (engineered) Myocardial infrac...
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