Cell Line Development (eBook)

Mohamed Al-Rubeai (Herausgeber)

eBook Download: PDF

2009 | 2009
VIII, 253 Seiten
Springer Netherland (Verlag)
978-90-481-2245-5 (ISBN)

Infos

Mammalian cell lines command an effective monopoly for the production of therapeutic proteins that require post-translational modifications. This unique advantage outweighs the costs associated with mammalian cell culture, which are far grater in terms of development time and manufacturing when compared to microbial culture. The development of cell lines has undergone several advances over the years, essentially to meet the requirement to cut the time and costs associated with using such a complex hosts as production platforms.

This book provides a comprehensive guide to the methodology involved in the development of cell lines and the cell engineering approach that can be employed to enhance productivity, improve cell function, glycosylation and secretion and control apoptosis. It presents an overall picture of the current topics central to expression engineering including such topics as epigenetics and the use of technologies to overcome positional dependent inactivation, the use of promoter and enhancer sequences for expression of various transgenes, site directed engineering of defined chromosomal sites, and examination of the role of eukaryotic nucleus as the controller of expression of genes that are introduced for production of a desired product. It includes a review of selection methods for high producers and an application developed by a major biopharmaceutical industry to expedite the cell line development process. The potential of cell engineering approch to enhance cell lines through the manipulation of single genes that play important roles in key metabolic and regulatory pathways is also explored throughout.

Inhalt

175892_1_En_FM1_Chapter_OnlinePDF.pdf 2
175892_1_En_1_Chapter_OnlinePDF.pdf 10
Use of MAR Elements to Increase the Production of Recombinant Proteins 10
1 Introduction to Epigenetic Silencing Issues in the Generation of Production Cell Lines 11
2 Causes of Instability During Mammalian Cell Production 13
3 Use of MAR Elements to Boost and Stabilize Expression 16
4 Identification of MAR DNA Sequences that Mediate Increased Expression 19
5 Identification of the MAR–Binding Proteins as Mediators of Increased Expression 22
5.1 Special AT-Rich Binding Protein (SATB1) 22
5.2 CCCTC-Binding Factor (CTCF) 25
5.3 High Mobility Group (HMGA) 27
6 Effects of MARs on the Copy Number of Integrated Transgenes 28
7 Effects of MARs on Transgene Expression Variegation 31
8 Isolation of Potent MAR Elements via Bioinformatics to Generate Producer Cell Lines 32
9 Conclusions 34
References 35
175892_1_En_2_Chapter_OnlinePDF.pdf 42
Expression Engineering – The IE2 Promoter/Enhancer from Mouse CMV 42
1 Introduction 42
2 Enhancer and Promoter Regions for Gene Expression 43
3 The Human CMV Major Immediate Early Promoter Region 46
4 The Mouse CMV Major Immediate Early Promoter Region 47
5 Dissection of the Bi-directional Mouse CMV MIE Region 48
5.1 Putative Binding Sites for Cellular Transcription Factors 48
5.2 Enrichment of CpG Content Around the IE2 and IE1 Transcription Start Sites 51
6 Vector Architectures Using IE2 and IE1 Promoter/Enhancers for Expression of Heterologous Genes 52
7 Expression of Recombinant Antibodies Using Mouse CMV MIEP Expression Vectors 55
8 Conclusions 57
References 57
175892_1_En_3_Chapter_OnlinePDF.pdf 62
Defeating Randomness – Targeted Integration as a Boost for Biotechnology 62
1 Transgene Expression in Mammalian Cells – Limitations of the Classical Random Gene Integration Strategy 63
2 Creating a Favorable Platform for Genetic Engineering – Primary Genomic Modification to Integrate a Tag 65
3 Exploiting the Tagged Loci–Reusability via Recombinase Mediated Cassette Exchange (RMCE) 67
3.1 Site-Specific Recombinases as Tools for Targeting a Previously Tagged Locus 68
3.2 Flip-In and RMCE as Versatile Applications of Site-Specific Recombinases 71
3.3 Parameters Influencing the Performance of RMCE 73
4 RMCE for Biotechnological Applications 75
4.1 Protein Production – Developing RMCE for Protein Production 75
4.2 Virus Production – Applicability of RMCE Towards Safer and Efficient Production of Viral Vectors 77
4.2.1 Retroviral Vectors 77
4.2.2 Adenoviral Vectors 79
4.3 Optimization of Targeting Vector Design with Respect to the Chromosomal Integration Site 80
5 Perspectives 81
5.1 The Potential of RMCE for Re-Engineering a Targeted Genomic Locus 81
5.2 The Next Generation – Circumventing the Need of Primary Genomic Modifications Employing Zinc Finger Recombinases 84
6 Conclusions 85
References 85
Importance of Genetic Environment for Recombinant Gene Expression 1
1 Genes and the Eukaryotic Nucleus: From the Simple Onwards 93
1.1 Chromatin Organisation: Nucleosomes and Epigenetic Events 94
1.2 Beyond the Nucleosome: Into Expression Factories 96
1.3 Beyond the Nucleosome: Into Chromosome Territories 97
2 What Does this Mean for Recombinant Gene Expression? 99
2.1 CHO and NS0 Myeloma Cell Lines: Production of Biopharmaceuticals 99
2.2 Consequences of Genomic Environment Towards Successful or Enhanced Production of Biopharmaceuticals 99
3 Can We Use Knowledge from this Emerging Area to Develop Better Expression Processes? 101
References 102
175892_1_En_5_Chapter_OnlinePDF.pdf 106
Expression Vector Engineering for Recombinant Protein Production 106
Abstract 106
1 Position Effects of the Genomic Sequence Elements 107
2 Position Effects of the Selection Cassette 108
3 Context Dependent Promoter Activities 111
4 Conclusion 115
References 115
175892_1_En_6_Chapter_OnlinePDF.pdf 118
Cell XpressTM Applications in Development and Characterization of Biopharmaceutical Recombinant Protein Producing Cell Li 118
1 Introduction 118
2 Applications of Cell Xpress in Mammalian Cell Line Generation 119
2.1 Transfection Protocol Optimization Using Transfection Efficiency 120
2.2 Transient Transfection Levels and Manufacturability of Recombinant Therapeutic IgG 122
2.3 Cell Population Enrichment by Cell Xpress Laser Processing 125
2.4 Single Cell Cloning Using Cell Xpress 126
3 Evaluation of Clonal Recombinant Cell Line Secretion 127
4 Discussion 129
4.1 Early Secretion Analysis 129
4.2 Cell Cycle and Secretion Analysis 130
4.3 Future Applications of Cell Xpress 131
5 Conclusions and Summary 132
References 132
175892_1_En_7_Chapter_OnlinePDF.pdf 136
Selection Methods for High-Producing Mammalian Cell Lines 136
1 Introduction 136
2 Traditional Cloning Methods 138
2.1 Single Cell Isolation 138
2.2 Basic Cloning Techniques 139
2.3 Drawbacks of Traditional Cloning Methods 140
3 Flow Cytometric Methods 141
3.1 Cell Surface Expression 142
3.2 Intracellular Reporter Proteins 143
3.2.1 Green Fluorescent Protein 144
3.2.2 Fluorescent Methotrexate 147
3.3 Methods Based on Cell Secretion Rate 148
3.3.1 Gel Microdrop Technology 148
3.3.2 Matrix-Based Secretion Assays 149
4 Fluorescent Methods and Automated Systems 151
4.1 HTRF (Homogeneous Time Resolved Fluorescence) Based MAb Assay 151
4.2 Laser-Enabled Analysis and Processing 152
4.3 Automated Colony Picking 153
4.4 Fully Automated Robotic Systems 153
5 Concluding Remarks 155
References 156
175892_1_En_8_Chapter_OnlinePDF.pdf 161
Engineering Mammalian Cells for Recombinant Monoclonal Antibody Production 161
1 Introduction 161
2 General Considerations for Mab Expression Vector Design 163
3 Transcriptional Enhancement 164
4 Translational Control 166
5 Modelling the Cellular Recombinant Monoclonal Antibody Production Process 168
6 Engineering the Mammalian Cell Factory for Improved Mab Production Rate 170
7 The Effect of Cell Line Genetic Background on Cellular Productivity 173
8 Stability of Mab Production During Extended Sub-Culture 174
References 175
175892_1_En_9_Chapter_OnlinePDF.pdf 182
Engineering Cell Function by RNA Interference 182
1 Introduction 182
2 Mechanism of RNAi 183
3 Current Challenges for RNAi 185
3.1 siRNA Sequence Design 186
3.2 siRNA Structure Design 187
3.3 siRNA Delivery Methods 188
4 Applications of RNAi in Biotechnology and Biomedicine 191
References 196
175892_1_En_10_Chapter_OnlinePDF.pdf 202
Apoptosis and Autophagy Cell Engineering 202
1 Introduction 202
2 Cell Death in Bioreactors 203
2.1 Necrosis 203
2.2 Programmed Cell Death 204
2.2.1 Apoptosis 204
2.2.2 Autophagy 206
2.2.3 Characteristic Features of Apoptosis and Autophagy 207
3 Apoptosis Engineering 210
3.1 Manipulation of External Cellular Environment 211
3.2 Genetic Strategies to Manipulate the Intracellular Environment 211
3.2.1 Down Regulation of Caspases 212
3.2.2 Overexpression of Anti-apoptotic Genes 213
3.2.3 Down-Regulation of Pro-apoptotic Genes 214
3.2.4 Others 215
4 Autophagy Engineering 215
4.1 Manipulation of External Cellular Environment for Autophagy Cell Engineering 216
4.2 Genetic Strategies to Manipulate the Intracellular Environment for Autophagy Cell Engineering 217
5 Conclusions 218
References 218
175892_1_En_11_Chapter_OnlinePDF.pdf 224
Glycoengineering and Modeling of Protein N-Glycosylation 224
1 Introduction 224
2 Processing Pathway for N-Glycosylation in Mammalian Expression Systems 225
3 Glycoengineering of Mammalian Expression Systems 228
4 Computer Aided Glycoengineering 231
5 Conclusions 235
References 236
175892_1_En_12_Chapter_OnlinePDF.pdf 239
Engineering the Secretory Pathway in Mammalian Cells 239
1 Introduction 239
2 Overview of the Secretory Pathway 240
2.1 Protein Secretion: Concepts and Mechanisms 240
2.2 Key Steps Along the Secretory Pathway 241
2.2.1 Translocation into the Endoplasmic Reticulum 241
2.2.2 Vesicle Budding from a Donor Compartment 242
2.2.3 Tethering and Fusion of Vesicles with Acceptor Membranes 243
Vesicle Tethering 243
Mechanisms of Membrane Fusion 243
Membrane Fusion and SNARE Proteins 243
Membrane Fusion and SM Proteins 245
3 Secretion Engineering 246
3.1 Secretory Pathway and Biopharmaceutical Production 246
3.2 The Engineering of the Secretory Pathway 246
3.2.1 Endoplasmic Reticulum Chaperon-Based Secretion Engineering 246
3.2.2 Xbp-1-Based Organelle Engineering 247
3.2.3 Vesicle Trafficking-Based Engineering 249
4 Conclusions and Perspectives 249
References 251
175892_1_En_BM2_Chapter_OnlinePDF.pdf 255

Produkt-Details

• Erscheint lt. Verlag: 11.8.2009
• Reihe/Serie: Cell Engineering
• Reihe/Serie: Cell Engineering
• Zusatzinfo: VIII, 253 p.
• Verlagsort: Dordrecht
• Sprache: englisch
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Biochemie / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie
• Themenwelt: Technik ► Umwelttechnik / Biotechnologie
• Schlagworte: Activation • Biotechnology • CMV • Expression • gene expression • Genetic Engineering • Methodology • Protein • proteins • RNA • transgen • Translation
• ISBN-10: 90-481-2245-7 / 9048122457
• ISBN-13: 978-90-481-2245-5 / 9789048122455