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List of Contributors xi Preface xv 1 Overview of the Cell Therapy Field 1 Michael Whitaker, Lucy Foley and Stephen Ward
1.1 The Context of Cell Therapies and Their Manufacturing Challenges 1
1.1.1 Regulation of Cell Therapies 4
1.1.2 Manufacturing Challenges in Cell Therapy 5
1.2 The Cell Therapy Landscape 5
1.2.1 Licensed Cell Therapy Products 7
1.2.2 Companies, Clinicians, Products and Procedures 8
1.2.3 Cell Therapy Clinical Trials 8
1.3 Operations in Cell Therapy Manufacture 11
1.3.1 Cells for Cell Therapy Production 12
1.3.1.1 Cell Source 12
1.4 Upstream Processing of Cellular Therapies 13
1.4.1 Cell Separation 13
1.4.2 Cell Expansion 13
1.4.3 Tissue Expansion 15
1.4.4 Adherent Cell Expansion 15
1.4.4.1 Multi-layer Reactors 15
1.4.4.2 Hollow Fibre Reactors 16
1.4.4.3 Scaffolds 17
1.4.5 Suspension Cell Expansion 18
1.4.5.1 Stirred Tank Bioreactors 18
1.4.5.2 Rocking Platforms 19
1.4.5.3 Perfusion Cell Culture 19
1.4.6 Differentiation 19
1.5 Downstream Processing of Cellular Therapies 20
1.5.1 Harvest, Washing and Concentration 20
1.5.1.1 Centrifugation 21
1.5.1.2 Filtration 21
1.5.2 Separation and Purification 22
1.5.2.1 Centrifugation 22
1.5.2.2 Magnetic Separation 24
1.6 Formulation, Fill and Finish of Cellular Therapies 24
1.6.1 Formulation 25
1.6.2 Fill and Finish 25
1.6.3 Preservation and Shipment 26
1.7 Administration of the Cell Therapy to a Patient 27
1.8 Cell Therapy Manufacturing Facilities of the Future 28
1.8.1 Factory of the Future Requirements 31
1.9 Conclusion 31 References 32 2 Structured Methodology for Process Development in Scalable Stirred Tank Bioreactors Platforms 35 Huaqing Wang, Daniel Kehoe, Julie Murrell and Donghui Jing
2.1 Introduction 35
2.2 Understanding the Engineering of the Stirred Tank Bioreactors 36
2.2.1 Mixing Phenomena in Stirred Tank Bioreactors 37
2.2.2 Understanding Oxygen Transfer Rate (kLa) with Different Sparging Methodologies 40
2.2.3 Heat Transfer in STB (Minimum Volume, Sensor/Sensing Control) 42
2.2.4 How to Choose a Microcarrier for Adherent Cells (hMSCs) 43
2.3 Understanding the Biology of the Cells in Stirred Tank Bioreactors (STB) 45
2.3.1 Cell Types (Adherent and Suspension Cells) 45
2.3.2 Assays for Comparability, Nutrients, Senescence and Doubling Dime 46
2.4 Process Development of Adherent Cells in STB Platforms 47
2.4.1 Standard Comparison to Cell Factory 47
2.4.2 Methodology for Screening of Microcarriers 49
2.4.3 Process Development in Small-scale Bioreactors (3L) 50
2.4.3.1 Cell Seeding Density 51
2.4.3.2 Microcarrier Concentrations (Cells:Bead Ratio) 52
2.4.3.3 Operation Ranges of pH and Dissolved Oxygen 53
2.4.3.4 Feeding Strategy 54
2.4.4 Process Development in Medium-scale Bioreactors (50L) 55
2.4.5 Case Study for Expansion of Bone Marrow Derived MSCs in Stirred Tank Bioreactors 59
2.5 Future Directions 60 References 61 3 The Effect of Scale-up on Cell Phenotype: Comparability Testing to Optimize Bioreactor Usage and Manufacturing Strategies 65 Jason Hamilton and Bart Vaes
3.1 Introduction 65
3.1.1 Cell Characterization in the Development Path 66
3.1.2 The MultiStem® Allogeneic Cell Therapy Product: Mechanisms of Benefit and Target Cells Numbers 69
3.2 Challenges in Cell Product Development 72
3.2.1 Effect of Large-scale Expansion on Stem Cell Properties 72
3.2.2 Serum-free and Xeno-free Media Development 74
3.3 Stem Cell Characterization 75
3.3.1 ISCT Requirements 75
3.3.2 Potency Assays 77
3.3.3 Omics Screens for Therapeutic Stem Cell Characterization 78
3.4 Next-generation Stem Cell Development 80 References 83 4 The Scale-up of Human Mesenchymal Stem Cell Expansion and Recovery 91 Thomas R. J. Heathman, Qasim A. Rafiq, Karen Coopman, Alvin W. Nienow and Christopher J. Hewitt
4.1 Introduction 91
4.2 Scale-up or Scale-out 93
4.3 Understanding the Small Scale 96
4.4 Microcarrier Screening 103
4.5 Spinner Flask Culture 108
4.6 Large-scale Expansion in Conventional Stirred Tank Bioreactors 111
4.7 Cell Recovery from Microcarriers 117
4.8 Conclusions 120 References 121 5 Challenges of Scale-up of Cell Separation and Purification Techniques 127 Marieke A. Hoeve, Paul A. De Sousa and Nicholas A. Willoughby
5.1 Introduction 127
5.1.1 Cell Separation for Cell-based Therapeutics 127
5.1.2 Separation Methodology Design 128
5.1.3 Objective of this
Chapter 128
5.1.3.1 Cell Yield 129
5.1.3.2 Standardisation 129
5.1.3.3 Economical viability 129
5.2 Scalable Cell Separation for Cell Therapy 130
5.2.1 Label Requiring versus Label-free Separation 130
5.2.2 Active versus Passive Method 133
5.2.3 Isolated Purification (Including Off-the-Shelf) versus Embedded Integrated Process 133
5.2.4 Low versus High Resolution 133
5.2.5 Open versus Closed Systems 134
5.2.6 Batch versus Continuous Separation 134
5.3 Currently Developed Cell Separation Techniques 135
5.3.1 Acoustophoresis 135
5.3.2 Aqueous Two-Phase System (ATPS) 137
5.3.3 Centrifugal Techniques 138
5.3.3.1 Centrifugal Counterflow Elutriation (CCE) 138
5.3.3.2 Centrifuge Systems with Integrated Filters 139
5.3.4 Dielectrophoresis (DEP) 140
5.3.5 Deterministic Lateral Displacement (DLD) 141
5.3.6 Genetic Engineering 141
5.3.7 Hydrodynamic Filtration (HDF) 142
5.3.8 Immunoadsorption 142
5.3.9 Immunomagnetic Cell Sorting 145
5.3.10 Inertial Migration 145
5.3.11 Magnetic Cell Sorting - Label-free 147
5.3.11.1 Magnetophoretic Cell Separation 147
5.3.11.2 Magnetic Solution-based Separation 148
5.3.12 Microscale Vortices 150
5.3.13 Normal Flow Filter (NFF) 150
5.3.14 Optical - Label-free 151
5.3.15 Tangential Flow Filters (TFF) 155
5.3.16 Weir and Pillar 156
5.4 Conclusion 157 Acknowledgements 159 References 159 6 Fundamental Points to Consider in the Cryopreservation and Shipment of Cells for Human Application 167 Glyn N. Stacey, Lyn Healy, Jennifer Man, Charles J. Hunt and John Morris
6.1 Introduction 167
6.2 The Role of Cryoprotective Agents (CPA) 168
6.3 Vitrification versus Cryopreservation 169
6.4 Points to Consider in the Development of Cryopreservation Protocols 169
6.4.1 General Considerations 169
6.4.2 Cellular Characteristics and Selection of Appropriate CPAs and Cooling Protocols 170
6.4.3 Key Events in Cryopreservation 172
6.4.3.1 Ice Nucleation 174
6.4.3.2 CPAs: Concentration and Composition 174
6.4.3.3 Cooling Rate 174
6.4.3.4 Storage of Cryopreserved Cells 176
6.4.3.5 Thawing and Recovery of Frozen Cells 177
6.5 Large-volume Freezing 178
6.6 Cryopreservation as
Part of Manufacturing Processes 179
6.6.1 Containers for Cryopreserved Cells 179
6.6.2 Controlled Rate Freezers and Storage Systems 179
6.6.3 The Cold-chain: Challenges and Solutions 180
6.6.4 Biobanking and Regulatory Requirements 181
6.7 Conclusions 181 Acknowledgements 182 References 182 7 Short-term Storage of Cells for Application in Cell-based Therapies 187 Stephen Swioklo and Che J. Connon
7.1 Introduction 187
7.1.1 Advances in Cell-based Therapies 188
7.1.2 The Logistical Landscape for CTPs and the Requirement for Short-term Storage of Cells 188
7.2 Hypothermia and Mammalian Cell Storage 193
7.2.1 Hypothermic Storage of Mesenchymal Stem Cells (MSCs) 194
7.2.2 Optimal Temperature for Cell Storage 202
7.3 The Application of Hypothermic Storage in Cell-based Therapies 204
7.4 Concluding Remarks 205 References 205 8 Cell Therapy in Practice 211 Gustavo S. Figueiredo, Julian R. De Havilland, Majlinda Lako and Francisco C. Figueiredo
8.1 Introduction 211
8.2 The Classification of ATMPs 212
8.3 European Regulations 216
8.3.1 Hospital Exemption (HE) and "Specials" Manufacturing 219
8.3.2 Orphan Medicinal Product Designation 220
8.3.3 Committee for Advanced Therapies 221
8.3.4 Good Manufacturing Practice (GMP) 221
8.3.5 European Union Tissue and Cells Directives (EUTCD) 223
8.4 ATMP Case Study: Autologous Limbal Stem Cell Therapy: the Newcastle Experience 224
8.5 Conclusion 233 References 234 Index 237