Cell Culture and Upstream Processing

Contributors     ixAbbreviations     xiPreface     xiiiOverview on mammalian cell cultureCell line development and culture strategies: future prospects to improve yields   Michael Butler     3Introduction     3Cell line transfection and selection     5Increase in efficiency in selecting a producer cell line     6Stability of gene expression     8Optimization of the fermentation process     9Apoptosis     11Bioreactors     11The capacity crunch     12Acknowledgment     13References     13The producer cell lineUse of DNA insulator elements and scaffold/matrix-attached regions for enhanced recombinant protein expression   Helen Kim     19Introduction     19The position effect     20Use of insulators and S/MARs can reduce the effects of heterochromatin on transgene expression     20DNA insulator elements     22The scaffold/matrix-attachment regions     23Binding proteins for DNA insulators and S/MARs     25DNA insulators or S/MARs can be incorporated into expression vectors     26DNA insulators and S/MARs act in acontext-dependent manner     30Conclusion     31Acknowledgements     32References     32Targeted gene insertion to enhance protein production from cell lines   Trevor N. Collingwood   Fyodor D. Urnov     37Introduction     37Identification of genomic 'hot spot' loci     39Recombinase-mediated site-specific gene insertion     39Cre, Flp, and [phiv]C31 recombinase systems     40Recombinase-mediated cassette exchange     40Gene insertion at native 'pseudo' recombinase sites     43Modification of recombinases and their target sites     43Emerging technologies for targeted gene insertion     44Homing endonucleases in HDR-mediated targeted gene insertion     46Targeted gene insertion into native loci by zinc finger, nuclease-mediated, high-frequency, homologous recombination     47Perspective     50References     52Recombinant human IgG production from myeloma and Chinese hamster ovary cells   Ray Field     57Introduction     57The need for recombinant human antibodies     57Recombinant antibodies     58Decoupling antibody isolation and production      58Choice of host cells     59Chinese hamster ovary cells     60Rodent myeloma cells     60The glutamine synthetase system     60Cell line stability     61Bioreactor process strategies     62IgG supply during antibody development     62Strategies for cell line engineering during clinical development     63Cost of goods and intellectual property     64Recombinant human IgG production from myeloma and CHO cells     64Creation of CHO and NS0 cell lines expressing IgG     64Cell expansion, subculture and production reactor experiments     65Northern and western blotting     65Comparison of results of transfections from GS-NS0 and GS-CHO     65Dilution cloning and analysis of clonal heterogeneity     66Analysis of instability of a GS-NS0 cell line     67Output of transfections of GS-NS0 and GS-CHO     68IgG production stability of candidate GS-NS0 clones     69IgG production stability of GS-CHO transfectants     70Fed-batch bioreactor process for GS-NS0 and GS-CHO     71Analysis of IgG quality produced from GS-CHO and GS-NS0 bioreactor processes     71Comparative yield of different human IgGs produced from CHO or NS0 cells     74Summary     74Acknowledgments     76References     76Media developmentCell culture media development: customization of animal origin-free components and supplements   Stephen Gorfien     81Introduction     81Types of cell culture media     82Components of animal origin     83Segregate     85Mitigate     87Replace     88Summary and considerations for the future     95Acknowledgments     98References     98Glycosylated proteinsPost-translational modification of recombinant antibody proteins   Roy Jeffries     103Introduction     103Common post-translational modifications     104Recombinant antibody therapeutics     105Structural and functional characteristics of human antibodies     106The human IgG subclasses: Options for antibody therapeutics     106The structure of human IgG antibodies     108IgG-Fc glycosylation     110IgG-Fab glycosylation     112Cell engineering to influence glycoform profiles     115IgG glycoforms and Fc effector functions     116Glycosylation engineering     118Pharmacokinetics and placental transport     118Antibody therapeutics of the IgA class     119Non-antibody recombinant (glyco)protein therapeutics, 'biosimilar' and 'follow-on' biologics     120Erythropoietin     121Tissue-type plasminogen activator     122Granulocyte-macrophage colony stimulating factor (GM-CSF)     122Granulocyte-colony stimulating factor     122Activated protein C     122Conclusions     123References     123Metabolic engineering to control glycosylation   Amy Shen   Domingos Ng   John Joly   Brad Snedecor   Yanmei Lu   Gloria Meng   Gerald Nakamura   Lynne Krummen     131Introduction     131Manipulation of fucose content using RNAi technology in CHO cells     132Metabolic engineering of fucose content with an existing antibody production line     132Metabolic engineering of fucose content with simultaneous new stable cell line generation     136Effect of fucosylation levels on Fc[Gamma]R binding     140Effects of fucose content on antibody-dependent cellular cytotoxicity      143Discussion     143Acknowledgments     146References     146An alternative approach: Humanization of N-glycosylation pathways in yeast   Stefan Wildt   Thomas Potgieter     149Introduction     149Yeast as host for recombinant protein expression     152N-linked glycosylation overview: Fungal versus mammalian     152A brief history of efforts to humanize N-linked glycosylation in fungal systems     154Sequential targeting of glycosylation enzymes is a key factor     155Replication of human-like glycosylation in the methylotrophic yeast   Pichia Pastoris     157A library of [Alpha]-1,2 mannosidases     157Transfer of N-acetylglucosamine     158Two independent approaches towards complex N-glycans: How to eliminate more mannoses     159Some metabolic engineering: Transfer of galactose     161More metabolic engineering: Sialic acid transfer. The final step     162Glyco-engineered yeast as a host for production of therapeutic glycoproteins     162N-linked glycans and pharmacokinetics of therapeutic glycoproteins     164N-glycans and their role in tissue targeting of glycoproteins     164N-glycans can modulate the biological activity of therapeutic glycoproteins     165Control of N-glycosylation offers advantages     165Conclusions     166References     166The BioprocessPerfusion or fed-batch? A matter of perspective   Marco Cacciuttolo     173Introduction     173Factors affecting the decision on choosing the manufacturing technology     175Technology expertise     175Facility design and scope (product dedicated versus multi-product)     179Impact of switching from perfusion to fed-batch     180Personnel requirements     180Liquid handling     181Equipment     182Manufacturing space     182Decrease in cycle time     182Direct costs of manufacturing     182Productivity and morale     183Conclusions     183Acknowledgments     184References     184

\ From The CriticsReviewer: Bruce A. Fenderson, PhD(Thomas Jefferson University)\ Description: Today, over 40 licensed biopharmaceuticals are produced in bioreactors. These molecules include recombinant proteins, nucleic acid-based products, and monoclonal antibodies. Advances in our understanding of cellular metabolism and molecular biology have allowed improvements in bioreactor product-yield that now approach 5 grams/Liter. This book summarizes state-of-the art strategies for producing pharmaceuticals from cultured cells in nine chapters on topics ranging from use of DNA insulator elements and scaffold/matrix-attached regions for enhanced recombinant protein expression to humanization of N-glycosylation pathways in yeast. The experimental findings presented in this book were developed to maximize the yield of proteins in bioreactors and optimize protein function by manipulating glycosylation pathways. The primary focus of this book is on cell biology and biochemistry.\ Purpose: According to the editor, the aim of this book is to share key developments with a wide audience. He writes, "It is hoped that this [book] will provide useful information for both scientific practitioners of animal cell technology as well as students of biochemical engineering." The authors are all prominent industrial and academic leaders in this field.\ Audience: The book is written for basic science and clinical researchers, particularly those interested in biochemical engineering and biotechnology. Graduate students in these disciplines will appreciate this overview of current approaches to producing high-yield biopharmaceuticals. Readers will have to have good knowledge of cell biology and biochemistry\ Features: This is not a protocol or methods book. Rather, the authors discuss innovative approaches to industrial-scale production of biological products. Major problems encountered using bioreactor systems are identified, and novel solutions are offered. For example, protein glycosylation affects protein stability and function, and so methods for manipulating O-linked and N-linked saccharide additions must be defined. Similarly, the transfection of cells is often complicated by the selective survival of low-expressing clones. To overcome the presumed negative position effects of randomly inserted DNAs, researchers are now using "chromatin borders" that protect transfected DNA against gene silencing. Each chapter provides an introduction, discussion of strategies that work, and interesting examples of results using black-and-white charts, tables, and gels. Each chapter includes an extensive list of references. The book is carefully edited and includes a helpful list of common abbreviations.\ Assessment: Future applications of these technologies may revolutionize the pharmaceutical industry, allowing scientists to design "smart therapies" for devastating human diseases. This book highlights key issues in this field and provides a snapshot of successful approaches. I recommend it for all those interested in the use of bioreactors for product development.\ \