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Animal Cell Culture - Mohamed Al-Rubeai


Year 2015

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Chapter 1 Cell Line Development1.1 Introduction1.1.1 Product Quantity1.1.2 Product Quality1.2 Two Components of Cell Line Development: The Gene of Interest (GOI) and the Cell1.3 The Classical Approach: Random Integration of GOI1.4 Stable Expression by Episomal Vectors1.5 Targeted Integration and Locus Specific Gene Expression1.5.1 Technologies of Targeted Integration1.5.2 Double Strand Break Induced Homologous Recombination1.5.3 Random Integration Competes with HR1.5.4 Applications of DSB Induced Modifications1.5.5 Cassette Exchange Approaches Based on Site-Specific Recombinases1.5.6 Tagging and Targeting1.5.7 Application of Targeted Integration and RMCE1.5.8 Precautions and Challenges for Targeting into Defined Chromosomal SitesReferencesChapter 2 Transient Recombinant Protein Expression in Mammalian Cells2.1 Introduction2.2 Viral Transient Transfection2.3 Non-viral Transient Transfection2.4 Plasmid Vector Design2.4.1 Plasmid Backbone and Size2.4.2 Vector Elements2.4.3 Episomal Replication2.5 Transfection Principles2.5.1 Calcium Phosphate Coprecipitation and Calfection2.5.2 DNA Lipoplex Formation and Lipid-Based Transfection2.5.3 Cationic Polymers and Transient Transfection with DNA Polyplexes2.5.3.1 Polyplex Uptake, Plasmid Release and Translocation into the Nucleus2.5.3.2 PEI Derivatives and Other Cationic Polymers as Alternatives to PEI2.5.4 Transient Transfection by Electroporation2.6 Cell Lines for Non-viral Transient Transfection2.7 Cell Culture Media Conditions2.7.1 Use of Productivity Enhancers2.8 Process Strategies for Improved Recombinant Protein Production2.8.1 Mild Hypothermia2.8.2 High-Density Transfection and 'Direct' Transfection2.8.3 Co-transfection Strategies2.8.4 Large-Scale and Automated High-Throughput ApplicationsReferencesChapter 3 Production of Antibodies in Hybridoma and Non-hybridoma Cell Lines3.1 Introduction3.2 Antibody Structure and Function3.2.1 Antibody Genes3.3 Therapeutic Antibodies3.3.1 Polyclonal Antibodies3.3.2 Monoclonal Antibodies3.4 Recombinant Antibodies3.4.1 Chimeric Antibodies3.4.2 Humanised Antibodies3.4.3 Human Antibodies3.4.4 Next Generation of Therapeutic Antibodies: AntibodyDrug Conjugates, Bi-specific Antibodies and Antibody Fragments3.4.4.1 Antibody Drug Conjugates (ADCs)3.4.4.2 Bispecific Antibodies3.4.4.3 Antibody Fragments3.5 Role of Therapeutic Antibodies in Cancer and Rheumatoid Arthritis3.5.1 Therapeutic Antibodies and Cancer3.5.1.1 Rituxan®3.5.1.2 Herceptin3.5.1.3 Avastin3.5.2 Therapeutic Antibodies and Rheumatoid Arthritis3.5.2.1 Remicade3.5.2.2 Humira3.5.2.3 EnbrelConclusionReferencesChapter 4 Bioreactors for Mammalian Cells4.1 Introduction to Cultivation Systems for Mammalian Cells4.1.1 Requirements and Categorization of Cultivation Systems4.1.2 Immobilization of Mammalian Cells4.1.3 “Single Use”-Bioreactors4.1.4 Process Strategies4.1.5 Monitoring and Control4.1.6 Parameters for Characterization of Bioreactors4.2 Small-Scale Culture Systems4.2.1 Static Culture Systems4.2.2 Dynamic Culture Systems4.3 Bioreactors for Suspension Culture4.3.1 Cell Damage in Stirred and Bubble Aerated Bioreactors4.3.2 Design of Suspension Bioreactors4.3.2.1 Stirred Tank Bioreactors4.3.2.2 Bubble Columns and Air-Lift Bioreactors4.3.2.3 Wave Mixed Bioreactors4.3.2.4 Devices for Cell and Product Retention4.3.3 Scale-Up Considerations4.4 Bioreactors for Immobilized Cells4.4.1 Fixed Bed and Fluidized Bed Bioreactors4.4.1.1 Fixed Bed Bioreactors4.4.1.2 Fluidized Bed Bioreactors4.4.2 “Hollow-Fiber”-Bioreactors4.5 Bioreactor Concepts for Tissue Engineering4.6 Considerations on Bioreactor Selection and Process DesignReferencesChapter 5 Mass Transfer and Mixing Across the Scales in Animal Cell Culture5.1 Introduction5.2 Oxygen Mass Transfer5.2.1 Basic Oxygen Mass Transfer Concepts and Equations5.2.2 The Volumetric Mass-Transfer Coefficient, kLa5.2.3 The Measurement of kLa5.2.3.1 The Unsteady-State (Dynamic) Method5.2.3.2 The Steady State Technique5.2.4 Correlations for Calculating kLa5.2.4.1 Stirred Bioreactors5.2.4.2 Headspace Aeration and Shaken Bioreactors5.2.4.3 Bubble Columns5.3 Carbon Dioxide Stripping5.3.1 The 'Apparent' Mass Transfer Coefficient Issue5.3.2 CO2 Evolution Rate, CER, and Control of pCO25.3.3 pH and Osmolality5.4 Heat Transfer5.5 Homogeneity Issues5.6 Choice of Agitation Conditions and Agitator5.6.1 Mean Specific Energy Dissipation Rate, εT5.6.2 Hydromechanical Stress Issues Due to Agitation5.7 Hydromechanical Stress from Sparging5.8 Agitator and Sparger Choice5.9 Scale-up and Ultra Scale-Down IssuesConclusionsNomenclatureReferencesChapter 6 Hydrodynamic Damage to Animal Cells6.1 Introduction6.2 Hydrodynamic Forces Acting on Cells6.3 Experimental Studies Attempting to Quantify Cell Damage6.4 Cell Damage from Sparging6.5 Experimental Sublethal Effect of Hydrodynamic StressConclusion and Future DirectionsReferencesChapter 7 Monitoring of Cell Culture7.1 Introduction7.2 Monitoring Principles7.3 Historical Perspective; Scope of This Article7.4 Parameters and Technologies: Viable Cell Density, Total Cell Density and Cell Viability7.4.1 Dye-Based Methods for Monitoring Cell Density and Viability7.4.1.1 Trypan Blue Dye Exclusion7.4.1.2 Fluorescence Based Cell Density and Viability Determination via Flow Cytometry7.4.2 Non-dye-based Methods for Monitoring Cell Density and Viability7.4.2.1 Impedance (Electrical Resistance) Measurement7.4.2.2 Capacitance Measurement7.4.2.3 Electromagnetic Spectroscopic Measurements (NIR, MIR, Raman)7.4.2.4 In Situ Microscopy7.5 Parameters and Technologies: Metabolic Parameters and Recombinant Products7.5.1 Automated Analyzers (Substrate, Metabolite and Product Monitoring)7.5.2 Spectroscopic Methods (Substrate, Metabolite and Product Monitoring)7.5.2.1 Near Infrared Spectroscopy (NIRS)7.5.2.2 Mid Infrared Spectroscopy (MIRS)7.5.2.3 Raman Spectroscopy7.5.3 Automated Systems for Product-Quantification7.5.3.1 Surface Plasmon Resonance (Biacore Systems)7.5.3.2 Bio-Layer Interferometry (Octet Systems)7.5.3.3 Microfluidic Gel Electrophoresis (Caliper LapChip Systems)7.6 Parameters and Technologies: Monitoring Cell Stress and Apoptosis7.6.1 Flow Cytometry (FC)7.6.2 Microplate/Multiwell Plate-Reader7.6.3 Mass Spectrometry7.6.4 DielectrophoresisReferencesChapter 8 Serum and Protein Free Media8.1 Introduction8.2 The Advantages and Disadvantages of Serum8.3 Serum-Free Media8.4 Basal Media8.5 Approaches for the Development of Serum-Free Media8.5.1 Top-Down Approach for Serum Replacement8.5.2 Bottom-Up Approach for Serum Replacement8.6 A Statistical Approach to Serum-Free Media Development8.7 Mitogenic Components Needed to Replace Serum8.7.1 Peptide Hydrolysates8.7.2 Insulin and Insulin-Like Growth Factor8.7.3 Epithelial Growth Factor (EGF)8.8 Transferrin: A Carrier Protein8.9 Attachment FactorsConclusionReferencesChapter 9 Glycosylation in Cell Culture9.1 Introduction9.2 Glycosylation Structures9.3 Cell Type Specific Glycosylation9.4 Culture Parameters Affecting Glycosylation9.5 Glycosylation Engineering and Modification of Glycan StructureConclusionReferencesChapter 10 Modelling of Mammalian Cell Cultures10.1 Scope of Bio-pharmaceutical Industry and Challenges10.2 Critical Review of Mathematical Models of Biological System10.3 Classifications10.3.1 Models Based on Classification of Bioprocesses10.3.2 Classification of the Different Forms of Mathematical (Nonlinear) Models10.3.3 Black-Box, Grey-Box and White-Box Models10.4 Cells, Cell Characteristics and Cell Lines10.5 FDA PAT Initiative10.6 Tools of Modelling10.6.1 Screening of State Variables for Explanatory Correlation with Growth and Productivity10.6.2 Raman Spectrophotometry: Determination of State Variables10.6.3 Flow Cytometry for Determination of Cell Cycle Phases and Organelles10.6.4 Systems Biology in Computational Modelling10.6.5 Regression Analysis and Estimation of Model Constants (Parameters)10.6.6 Data Processing of State Variables10.6.7 Design of Experiments10.7 Modelling of Bioprocesses10.7.1 Growth and Productivity Models10.7.2 Principles Behind Model Formulation10.7.3 Literature Review of Growth Models10.7.4 Unstructured Unsegregated/Segregated Models10.7.5 Structured Non-segregated Models10.7.5.1 Kinetic Approach10.7.5.2 Stoichiometric Approach10.7.5.3 Single Cell Models (SCMs)10.7.5.4 Combination of Single Cell Models (SCMs) and Population Balance Models (PBMs)10.7.6 Stochastic Models10.7.7 Logistic Models10.8 Neural Network (NN)10.9 Fuzzy Logic10.10 Productivity Models10.11 Critical Synopsis and PerspectivesReferencesChapter 11 Mammalian Cell Line Selection Strategies for High-Producers11.1 Introduction11.2 Manual and Semi-Automated Cloning Methods11.2.1 Manual Single Cell Cloning11.2.2 Semi-Solid Suspension Cell Cloning11.2.3 Semi-Automated Single-Cell Cloning11.2.4 Fluorescence-Assisted Cloning and Selection11.2.4.1 GFP and Other Fluorescent Proteins11.2.4.2 Fluorescent Conjugates11.3 Selecting High-Producers with Flow Cytometry11.3.1 Cell Line Selection Via Cell Sorting11.3.1.1 Intracellular Reporters for Cell Sorting11.3.1.2 Cell Surface Expression11.3.1.3 Gel Microdrop Technique11.3.1.4 Matrix Based Secretion Assays11.3.1.5 Secretion Display Technology11.4 Advances in Productivity Screening and Clone Selection Technologies11.4.1 Beyond ELISA11.4.1.1 Homogeneous Time Resolved Fluorescence11.4.1.2 Bio-layer Interferometry11.4.2 Automation of High-Producer Cell Line Selection11.4.2.1 Laser-Enabled Analysis and Processing11.4.2.2 Cell Xpress Module11.4.2.3 Other Automated Screening and Selection SystemsConcluding RemarksReferencesChapter 12 Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain WorldAbbreviations12.1 Introduction12.2 How Much Stability Is Required? Lessons from Industry12.3 Impact of Long-Term Sub-Culture on Cell Populations12.4 Cell Line Instability and Its Impact on Product Quality12.5 Genetic Instability and Its Impact on Biomanufacturing12.6 Epigenetics and Cell Line Instability12.7 Strategies to Combat Cell Line InstabilityConclusionReferencesChapter 13 Perfusion Processes13.1 Introduction13.2 Basics of the Practical Implementation of Perfusion Processes13.3 Cell Separation Devices13.3.1 Basics About Factors Characteristic of the Separation Devices13.3.2 Cell Separation Devices Based on Filtration13.3.2.1 Spin-Filter13.3.2.2 Tangential Flow Filtration (TFF) and Alternating Tangential Flow Filtration (ATF)13.3.2.3 Floating Filter13.3.3 Devices Based on Acceleration or Gravity13.3.3.1 Inclined Settler13.3.3.2 Acoustic Settler13.3.3.3 Centrifuge13.3.3.4 Hydrocyclone13.3.4 Conclusion About the Cell Separation Devices13.4 Development of a Perfusion Process13.4.1 Systems for Process Development13.4.1.1 Screening Systems13.4.1.2 Bioreactor and Scale-Down Model13.4.2 Optimization of the Process Parameters13.4.3 Medium Selection13.4.4 Perfusion Rate Strategy13.4.4.1 Perfusion Rate Strategy Based on CSPR13.4.4.2 Perfusion Rate Strategy Based on Main Substrate Measurement13.4.4.3 Perfusion Rate Tuning for Removal of Toxic By-Products13.4.5 Cell Density: Target, Monitoring and Control13.4.5.1 Selection/Optimization of the Cell Density13.4.5.2 On-line Cell Measurement or Estimation for Perfusion Monitoring13.4.6 Cell Arrest13.4.7 Protein Quality13.4.8 Effect of a Peristaltic Pump and Wall Shear Stress in the Re-circulation Loop13.5 Future ProspectsReferencesChapter 14 Single-Use Bioreactors for Animal and Human Cells14.1 Introduction14.2 Overview of Current SU Bioreactors on the Market14.2.1 Categories/Classes of SU Bioreactors14.2.2 Instrumentation of SU Bioreactors14.3 Often Used Instrumented Dynamic SU Bioreactors and Their Engineering Characteristics14.3.1 Wave-Mixed Bag Bioreactors14.3.2 Stirred Rigid Systems14.3.3 Stirred Bag Systems14.3.4 Orbitally Shaken Bioreactors14.3.5 Fixed Bed Bioreactors14.4 Established and New Applications for Dynamic SU Bioreactors14.4.1 Modern Seed Train Production with Continuous Suspension Cell Lines14.4.2 CHO Cell-Based Production of Monoclonal Antibodies (mAbs) up to Medium Volume Scale14.4.3 Viral Vaccine and Virus-Like Particle Production14.4.4 Expansion of Human Primary Cells for Production of Cell Therapeutics14.5 Scale-Up of Processes Based on SU BioreactorsConcluding RemarksReferencesChapter 15 An Overview of Cell Culture Engineering for the Insect Cell-Baculovirus Expression Vector System (BEVS)15.1 Introduction15.2 Producing Recombinant Proteins Using the BEVS15.2.1 Insect Cells15.2.2 The Baculovirus15.3 Bioprocess Engineering Considerations for the Production of Recombinant Proteins with the BEVS15.3.1 Downstream Processing and Product Quality15.4 PerspectivesReferencesChapter 16 Metabolic Flux Analysis: A Powerful Tool in Animal Cell Culture16.1 Introduction16.2 Metabolic Flux Analysis in Practice16.2.1 Metabolic Network Setup16.2.2 MFA Methods16.2.3 Experimental Design of 13C Studies16.2.4 Metabolomics Techniques16.2.5 Flux Estimation Algorithms for Isotope Models16.3 Optimising Animal Cell Cultures Processes16.4 Applications in Biomedical Research and ToxicologyConclusions and PerspectivesReferencesChapter 17 Cell Immobilization for the Production of Viral Vaccines17.1 Introduction17.2 Cell Substrates in Viral Vaccine Production17.3 Cell Immobilization and Entrapment17.3.1 Microcarriers Technology17.3.2 Hollow Fibers17.3.3 Cell Microencapsulation17.3.4 Cell Aggregates17.4 Bioreactors for Viral Vaccines ProductionConcluding RemarksReferencesChapter 18 Cell Engineering for Therapeutic Protein Production18.1 Introduction18.2 Cell Engineering Strategies18.3 Improving Viable Cell Concentration18.3.1 Anti-cell Death Engineering18.3.1.1 Apoptosis18.3.1.2 Autophagy18.3.2 Cell Proliferation Engineering18.3.3 Metabolic Engineering18.4 Improving Specific Productivity18.4.1 Cell Cycle Engineering18.4.2 Chaperone Engineering18.4.3 Secretion Engineering18.4.4 Unfolded Protein Response-Based EngineeringConclusionsReferencesChapter 19 Proteomics in Cell Culture: From Genomics to Combined 'Omics for Cell Line Engineering and Bioprocess Development19.1 Introduction19.2 Genomics19.3 Proteomics19.3.1 Optimization of Proteomics Methods19.3.1.1 Sample Preparation Methods and Improvements19.3.1.2 Protein Labeling19.3.1.3 MS19.3.2 Proteomics for Bioprocess Development19.3.2.1 Proteomics Analysis to Increase Cell Growth Rate and Viable Cell Density19.3.2.2 Proteomics Analysis to Increase Recombinant Protein Production19.3.2.3 Proteomics to Optimize Media Formulations19.3.2.4 Systems Biology19.3.3 Database Development19.3.4 Combined Metabolomics and ProteomicsConclusionsReferencesChapter 20 Metabolomics in Animal Cell Culture20.1 Introduction20.2 Biological Questions20.2.1 Phenotype Analysis20.2.2 Testing: Drugs and Toxins20.2.3 Production Systems20.3 Experiment Design20.4 Sample Processing20.5 Measurement20.6 Data Analysis20.6.1 Qualitative and Quantitative Analysis20.6.2 Unsupervised and Supervised Analysis20.6.3 System Biology and Cell Culture Metabolomics20.6.4 Metabolomics in Polyomics of Cell CulturesConclusionReferencesChapter 21 Process Analytical Technology and Qualityby-Design for Animal Cell Culture21.1 What Is Process Analytical Technology and Qualityby-Design and Why Is It of Value?21.2 Measurement: Acquisition of Process Data21.2.1 PAT Instruments21.2.2 Electrical Measurements21.2.2.1 Electrochemical Probes21.2.2.2 Capacitance Probes21.2.2.3 Multifunction Analysers21.2.3 Optical-Based Instruments21.2.3.1 Spectroscopy21.2.3.2 Fluorescence21.2.3.3 Image Analysis21.2.3.4 Focussed Beam Reflectance Measurement21.2.4 Chromatographic Techniques21.2.5 Single Use Technologies21.2.6 Automated Sampling and Sample Preparation21.3 Analysis: Transforming Process Data into Process Knowledge21.3.1 Multivariate Data Analysis21.3.1.1 Multivariate Calibration21.3.1.2 Batch Analysis21.3.2 Modelling21.3.2.1 Data-Driven Models21.3.2.2 Mechanistic Models21.4 Control: Harnessing Process Knowledge21.4.1 Automated Process Control21.4.2 Advanced Process Control21.4.2.1 Model Predictive Control21.4.2.2 Fuzzy Logic Control21.4.2.3 Multivariate Statistical Process Control21.5 Optimization: Managing Process PerformanceReferencesChapter 22 Biosafety Recommendations on the Handling of Animal Cell CulturesAbbreviations22.1 Introduction22.2 Biological Risk Assessment22.2.1 Risk Assessment of Cell Cultures22.2.2 Intrinsic Properties of Cell Cultures22.2.3 Intentional Infection of Cell Cultures22.2.4 Adventitious Contamination of Cell Cultures22.2.5 Genetically Modified Animal Cell Cultures22.2.6 Type of Manipulation22.3 Biological Risk Management22.3.1 General Biosafety Recommendations22.3.2 Novel Approaches for Reducing Hazard and/or Exposure Associated with Handling of Cell CulturesConcluding RemarksGlossaryCulture Type: Primary Cell Cultures, Diploid Cell Lines, Continuous Cell LinesReferencesChapter 23 Biopharmaceutical Products from Animal Cell Culture23.1 Introduction23.2 Early Mammalian Based Biopharmaceuticals23.3 Monoclonal Antibodies as Drugs23.4 Fusion Protein Drugs23.5 Drug Approvals and Regulation23.6 Biosimilars or Follow on Biologics ApprovalsConclusionReferences
 
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