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Cell Line Development

E-BookPDF1 - PDF WatermarkE-Book
253 Seiten
Englisch
Springer Netherlandserschienen am11.08.20092009
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.mehr
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Produkt

KlappentextMammalian 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.
Details
Weitere ISBN/GTIN9789048122455
ProduktartE-Book
EinbandartE-Book
FormatPDF
Format Hinweis1 - PDF Watermark
FormatE107
Erscheinungsjahr2009
Erscheinungsdatum11.08.2009
Auflage2009
ReiheCell Engineering
Reihen-Nr.6
Seiten253 Seiten
SpracheEnglisch
IllustrationenVIII, 253 p.
Artikel-Nr.1443281
Rubriken
Genre9200

Inhalt/Kritik

Inhaltsverzeichnis
1;175892_1_En_FM1_Chapter_OnlinePDF.pdf;2
2;175892_1_En_1_Chapter_OnlinePDF.pdf;10
2.1;Use of MAR Elements to Increase the Production of Recombinant Proteins;10
2.1.1;1 Introduction to Epigenetic Silencing Issues in the Generation of Production Cell Lines;11
2.1.2;2 Causes of Instability During Mammalian Cell Production;13
2.1.3;3 Use of MAR Elements to Boost and Stabilize Expression;16
2.1.4;4 Identification of MAR DNA Sequences that Mediate Increased Expression;19
2.1.5;5 Identification of the MAR-Binding Proteins as Mediators of Increased Expression;22
2.1.5.1;5.1 Special AT-Rich Binding Protein (SATB1);22
2.1.5.2;5.2 CCCTC-Binding Factor (CTCF);25
2.1.5.3;5.3 High Mobility Group (HMGA);27
2.1.6;6 Effects of MARs on the Copy Number of Integrated Transgenes;28
2.1.7;7 Effects of MARs on Transgene Expression Variegation;31
2.1.8;8 Isolation of Potent MAR Elements via Bioinformatics to Generate Producer Cell Lines;32
2.1.9;9 Conclusions;34
2.1.10;References;35
3;175892_1_En_2_Chapter_OnlinePDF.pdf;42
3.1;Expression Engineering - The IE2 Promoter/Enhancer from Mouse CMV;42
3.1.1;1 Introduction;42
3.1.2;2 Enhancer and Promoter Regions for Gene Expression;43
3.1.3;3 The Human CMV Major Immediate Early Promoter Region;46
3.1.4;4 The Mouse CMV Major Immediate Early Promoter Region;47
3.1.5;5 Dissection of the Bi-directional Mouse CMV MIE Region;48
3.1.5.1;5.1 Putative Binding Sites for Cellular Transcription Factors;48
3.1.5.2;5.2 Enrichment of CpG Content Around the IE2 and IE1 Transcription Start Sites;51
3.1.6;6 Vector Architectures Using IE2 and IE1 Promoter/Enhancers for Expression of Heterologous Genes;52
3.1.7;7 Expression of Recombinant Antibodies Using Mouse CMV MIEP Expression Vectors;55
3.1.8;8 Conclusions;57
3.1.9;References;57
4;175892_1_En_3_Chapter_OnlinePDF.pdf;62
4.1;Defeating Randomness - Targeted Integration as a Boost for Biotechnology;62
4.1.1;1 Transgene Expression in Mammalian Cells - Limitations of the Classical Random Gene Integration Strategy;63
4.1.2;2 Creating a Favorable Platform for Genetic Engineering - Primary Genomic Modification to Integrate a Tag;65
4.1.3;3 Exploiting the Tagged Loci-Reusability via Recombinase Mediated Cassette Exchange (RMCE);67
4.1.3.1;3.1 Site-Specific Recombinases as Tools for Targeting a Previously Tagged Locus;68
4.1.3.2;3.2 Flip-In and RMCE as Versatile Applications of Site-Specific Recombinases;71
4.1.3.3;3.3 Parameters Influencing the Performance of RMCE;73
4.1.4;4 RMCE for Biotechnological Applications;75
4.1.4.1;4.1 Protein Production - Developing RMCE for Protein Production;75
4.1.4.2;4.2 Virus Production - Applicability of RMCE Towards Safer and Efficient Production of Viral Vectors;77
4.1.4.2.1;4.2.1 Retroviral Vectors;77
4.1.4.2.2;4.2.2 Adenoviral Vectors;79
4.1.4.3;4.3 Optimization of Targeting Vector Design with Respect to the Chromosomal Integration Site;80
4.1.5;5 Perspectives;81
4.1.5.1;5.1 The Potential of RMCE for Re-Engineering a Targeted Genomic Locus;81
4.1.5.2;5.2 The Next Generation - Circumventing the Need of Primary Genomic Modifications Employing Zinc Finger Recombinases;84
4.1.6;6 Conclusions;85
4.1.7;References;85
5.1;Importance of Genetic Environment for Recombinant Gene Expression;1
5.1.1;1 Genes and the Eukaryotic Nucleus: From the Simple Onwards;93
5.1.1.1;1.1 Chromatin Organisation: Nucleosomes and Epigenetic Events;94
5.1.1.2;1.2 Beyond the Nucleosome: Into Expression Factories;96
5.1.1.3;1.3 Beyond the Nucleosome: Into Chromosome Territories;97
5.1.2;2 What Does this Mean for Recombinant Gene Expression?;99
5.1.2.1;2.1 CHO and NS0 Myeloma Cell Lines: Production of Biopharmaceuticals;99
5.1.2.2;2.2 Consequences of Genomic Environment Towards Successful or Enhanced Production of Biopharmaceuticals;99
5.1.3;3 Can We Use Knowledge from this Emerging Area to Develop Better Expression Processes?;101
5.1.4;References;102
6;175892_1_En_5_Chapter_OnlinePDF.pdf;106
6.1;Expression Vector Engineering for Recombinant Protein Production;106
6.1.1;Abstract;106
6.1.2;1 Position Effects of the Genomic Sequence Elements;107
6.1.3;2 Position Effects of the Selection Cassette;108
6.1.4;3 Context Dependent Promoter Activities;111
6.1.5;4 Conclusion;115
6.1.6;References;115
7;175892_1_En_6_Chapter_OnlinePDF.pdf;118
7.1;Cell XpressTM Applications in Development and Characterization of Biopharmaceutical Recombinant Protein Producing Cell Li;118
7.1.1;1 Introduction;118
7.1.2;2 Applications of Cell Xpress in Mammalian Cell Line Generation;119
7.1.2.1;2.1 Transfection Protocol Optimization Using Transfection Efficiency;120
7.1.2.2;2.2 Transient Transfection Levels and Manufacturability of Recombinant Therapeutic IgG;122
7.1.2.3;2.3 Cell Population Enrichment by Cell Xpress Laser Processing;125
7.1.2.4;2.4 Single Cell Cloning Using Cell Xpress;126
7.1.3;3 Evaluation of Clonal Recombinant Cell Line Secretion;127
7.1.4;4 Discussion;129
7.1.4.1;4.1 Early Secretion Analysis;129
7.1.4.2;4.2 Cell Cycle and Secretion Analysis;130
7.1.4.3;4.3 Future Applications of Cell Xpress;131
7.1.5;5 Conclusions and Summary;132
7.1.6;References;132
8;175892_1_En_7_Chapter_OnlinePDF.pdf;136
8.1;Selection Methods for High-Producing Mammalian Cell Lines;136
8.1.1;1 Introduction;136
8.1.2;2 Traditional Cloning Methods;138
8.1.2.1;2.1 Single Cell Isolation;138
8.1.2.2;2.2 Basic Cloning Techniques;139
8.1.2.3;2.3 Drawbacks of Traditional Cloning Methods;140
8.1.3;3 Flow Cytometric Methods;141
8.1.3.1;3.1 Cell Surface Expression;142
8.1.3.2;3.2 Intracellular Reporter Proteins;143
8.1.3.2.1;3.2.1 Green Fluorescent Protein;144
8.1.3.2.2;3.2.2 Fluorescent Methotrexate;147
8.1.3.3;3.3 Methods Based on Cell Secretion Rate;148
8.1.3.3.1;3.3.1 Gel Microdrop Technology;148
8.1.3.3.2;3.3.2 Matrix-Based Secretion Assays;149
8.1.4;4 Fluorescent Methods and Automated Systems;151
8.1.4.1;4.1 HTRF (Homogeneous Time Resolved Fluorescence) Based MAb Assay;151
8.1.4.2;4.2 Laser-Enabled Analysis and Processing;152
8.1.4.3;4.3 Automated Colony Picking;153
8.1.4.4;4.4 Fully Automated Robotic Systems;153
8.1.5;5 Concluding Remarks;155
8.1.6;References;156
9;175892_1_En_8_Chapter_OnlinePDF.pdf;161
9.1;Engineering Mammalian Cells for Recombinant Monoclonal Antibody Production;161
9.1.1;1 Introduction;161
9.1.2;2 General Considerations for Mab Expression Vector Design;163
9.1.3;3 Transcriptional Enhancement;164
9.1.4;4 Translational Control;166
9.1.5;5 Modelling the Cellular Recombinant Monoclonal Antibody Production Process;168
9.1.6;6 Engineering the Mammalian Cell Factory for Improved Mab Production Rate;170
9.1.7;7 The Effect of Cell Line Genetic Background on Cellular Productivity;173
9.1.8;8 Stability of Mab Production During Extended Sub-Culture;174
9.1.9;References;175
10;175892_1_En_9_Chapter_OnlinePDF.pdf;182
10.1;Engineering Cell Function by RNA Interference;182
10.1.1;1 Introduction;182
10.1.2;2 Mechanism of RNAi;183
10.1.3;3 Current Challenges for RNAi;185
10.1.3.1;3.1 siRNA Sequence Design;186
10.1.3.2;3.2 siRNA Structure Design;187
10.1.3.3;3.3 siRNA Delivery Methods;188
10.1.4;4 Applications of RNAi in Biotechnology and Biomedicine;191
10.1.5;References;196
11;175892_1_En_10_Chapter_OnlinePDF.pdf;202
11.1;Apoptosis and Autophagy Cell Engineering;202
11.1.1;1 Introduction;202
11.1.2;2 Cell Death in Bioreactors;203
11.1.2.1;2.1 Necrosis;203
11.1.2.2;2.2 Programmed Cell Death;204
11.1.2.2.1;2.2.1 Apoptosis;204
11.1.2.2.2;2.2.2 Autophagy;206
11.1.2.2.3;2.2.3 Characteristic Features of Apoptosis and Autophagy;207
11.1.3;3 Apoptosis Engineering;210
11.1.3.1;3.1 Manipulation of External Cellular Environment;211
11.1.3.2;3.2 Genetic Strategies to Manipulate the Intracellular Environment;211
11.1.3.2.1;3.2.1 Down Regulation of Caspases;212
11.1.3.2.2;3.2.2 Overexpression of Anti-apoptotic Genes;213
11.1.3.2.3;3.2.3 Down-Regulation of Pro-apoptotic Genes;214
11.1.3.2.4;3.2.4 Others;215
11.1.4;4 Autophagy Engineering;215
11.1.4.1;4.1 Manipulation of External Cellular Environment for Autophagy Cell Engineering;216
11.1.4.2;4.2 Genetic Strategies to Manipulate the Intracellular Environment for Autophagy Cell Engineering;217
11.1.5;5 Conclusions;218
11.1.6;References;218
12;175892_1_En_11_Chapter_OnlinePDF.pdf;224
12.1;Glycoengineering and Modeling of Protein N-Glycosylation;224
12.1.1;1 Introduction;224
12.1.2;2 Processing Pathway for N-Glycosylation in Mammalian Expression Systems;225
12.1.3;3 Glycoengineering of Mammalian Expression Systems;228
12.1.4;4 Computer Aided Glycoengineering;231
12.1.5;5 Conclusions;235
12.1.6;References;236
13;175892_1_En_12_Chapter_OnlinePDF.pdf;239
13.1;Engineering the Secretory Pathway in Mammalian Cells;239
13.1.1;1 Introduction;239
13.1.2;2 Overview of the Secretory Pathway;240
13.1.2.1;2.1 Protein Secretion: Concepts and Mechanisms;240
13.1.2.2;2.2 Key Steps Along the Secretory Pathway;241
13.1.2.2.1;2.2.1 Translocation into the Endoplasmic Reticulum;241
13.1.2.2.2;2.2.2 Vesicle Budding from a Donor Compartment;242
13.1.2.2.3;2.2.3 Tethering and Fusion of Vesicles with Acceptor Membranes;243
13.1.2.2.3.1;Vesicle Tethering;243
13.1.2.2.3.2;Mechanisms of Membrane Fusion;243
13.1.2.2.3.2.1;Membrane Fusion and SNARE Proteins;243
13.1.2.2.3.2.2;Membrane Fusion and SM Proteins;245
13.1.3;3 Secretion Engineering;246
13.1.3.1;3.1 Secretory Pathway and Biopharmaceutical Production;246
13.1.3.2;3.2 The Engineering of the Secretory Pathway;246
13.1.3.2.1;3.2.1 Endoplasmic Reticulum Chaperon-Based Secretion Engineering;246
13.1.3.2.2;3.2.2 Xbp-1-Based Organelle Engineering;247
13.1.3.2.3;3.2.3 Vesicle Trafficking-Based Engineering;249
13.1.4;4 Conclusions and Perspectives;249
13.1.5;References;251
14;175892_1_En_BM2_Chapter_OnlinePDF.pdf;255mehr