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