Based on a training course that teaches the theoretical as well as practical aspects of protein bioseparation to bioprocess professionals, this fully updated and revised new edition offers comprehensive coverage of continuous chromatography and provides readers with many relevant examples from the biopharmaceutical industry.
Divided into two large parts, Protein Chromatography: Process Development and Scale-Up, Second Edition presents all the necessary knowledge for effective process development in chromatographic bioseparation, both on small and large scale. The first part introduces chromatographic theory, including process design principles, to enable the reader to rationalize the set-up of a bioseparation process. The second part illustrates by way of case studies and sample protocols how the theory learned in the first part may be applied to real-life problems. Chapters look at: Downstream Processing of Biotechnology Products; Chromatography Media; Laboratory and Process Columns and Equipment; Adsorption Equilibrium; Rate Processes; and Dynamics of Chromatography Columns. The book closes with chapters on: Effects of Dispersion and Rate Processes on Column Performance; Gradient Elution Chromatography; and Chromatographic Column Design and Optimization.
-Presents the most pertinent examples from the biopharmaceutical industry, including monoclonal antibodies
-Provides an overview of the field along with design tools and examples illustrating the advantages of continuous processing in biopharmaceutical productions
-Focuses on process development and large-scale bioseparation tasks, making it an ideal guide for the professional bioengineer in the biotech and pharma industries
-Offers field-tested information based on decades of training courses for biotech and chemical engineers in Europe and the U.S.
Protein Chromatography: Process Development and Scale-Up, Second Edition will appeal to biotechnologists, analytical chemists, chromatographers, chemical engineers, pharmaceutical industry, biotechnological industry, and biochemists.
Table of Contents
Preface ix
Nomenclature xiii
1 Downstream Processing of Biotechnology Products 1
1.1 Introduction 1
1.2 Bioproducts and Their Contaminants 2
1.2.1 Biomolecular Chemistry and Structure 2
1.2.1.1 Proteins 2
1.2.1.2 Primary Structure 3
1.2.1.3 Secondary Structure 6
1.2.1.4 Tertiary Structure 11
1.2.1.5 Quaternary Structure 11
1.2.1.6 Folding 12
1.2.1.7 Post-translational Modifications 12
1.2.1.8 Oligonucleotides and Polynucleotides 16
1.2.1.9 Endotoxins 18
1.2.2 Biochemical and Biophysical Properties 20
1.2.2.1 UV Absorbance 20
1.2.2.2 Size 22
1.2.2.3 Charge 26
1.2.2.4 Hydrophobicity 28
1.2.2.5 Solubility 31
1.2.2.6 Chemical Stability 33
1.2.2.7 Mechanical Stability 34
1.2.2.8 Viscosity 35
1.2.2.9 Diffusivity 38
1.3 Bioprocesses 40
1.3.1 Expression Systems 40
1.3.2 Host Cell Composition 43
1.3.3 Culture Media 44
1.3.4 Components of the Culture Broth 45
1.3.5 Product Quality Requirements 46
1.3.5.1 Types of Impurities 48
1.3.5.2 Validation 50
1.3.5.3 Purity Requirements 51
1.4 Biosimilars 52
1.5 Role of Chromatography in Downstream Processing 53
1.6 Environmental Impact of Biopharmaceutical Manufacturing 59
References 60
2 Introduction to Protein Chromatography 63
2.1 Introduction 63
2.2 Basic Principles and Definitions 63
2.3 Modes of Operation 67
2.3.1 Elution Chromatography 69
2.3.2 Frontal Analysis 70
2.3.3 Displacement Chromatography 71
2.3.4 Periodic Countercurrent and Simulated Moving Bed Separators (SMB) 72
2.4 Performance Factors 76
2.5 Separation Performance Metrics 81
2.5.1 Column Efficiency 81
2.5.2 Chromatographic Resolution 84
2.5.3 Dynamic Binding Capacity 86
2.5.4 Scaling Relationships 87
References 90
3 Chromatography Media 93
3.1 Introduction 93
3.2 Interaction Types and Chemistry 94
3.2.1 Steric Interaction 94
3.2.2 Hydrophobic Interaction 96
3.2.3 Electrostatic Interaction 103
3.2.4 Metal Ion Interaction 106
3.2.5 Biospecific Interaction 108
3.2.6 Mixed Mode Interaction 113
3.3 Buffers and Mobile Phases 117
3.4 Physical Structure and Properties 118
3.4.1 Base Matrices 119
3.4.1.1 Natural Carbohydrate Polymers 121
3.4.1.2 Synthetic Polymers 122
3.4.1.3 Inorganic Materials 123
3.4.2 Porosity, Pore Size, and Surface Area 125
3.4.3 Particle Size and Particle Size Distribution 131
3.4.4 Mechanical and Flow Properties 132
References 135
4 Laboratory and Process Columns and Equipment 139
4.1 Introduction 139
4.2 Laboratory-Scale Systems 140
4.2.1 Pumps 141
4.2.2 Mixers 145
4.2.3 Monitors 146
4.2.4 System Volumes 149
4.3 Process Columns and Equipment 150
4.3.1 Columns 150
4.3.2 Systems 155
4.3.3 Column Packing 157
References 158
5 Adsorption Equilibrium 159
5.1 Introduction 159
5.2 Single-Component Systems 161
5.3 Multicomponent Systems 174
5.4 Empirical Correlation of Equilibrium Data 178
5.5 Protein Conformational Changes upon Adsorption 180
References 180
6 Rate Processes 183
6.1 Introduction 183
6.2 Rate Mechanisms 183
6.2.1 External Mass Transfer 185
6.2.2 Pore Diffusion 188
6.2.3 Solid Diffusion 192
6.2.4 Intraparticle Convection 196
6.2.5 Kinetic Resistance to Binding 201
6.3 Batch Adsorption Kinetics 202
6.3.1 General Rate Equations 204
6.3.2 Analytical Solutions 206
6.3.2.1 External Mass Transfer Control 207
6.3.2.2 Solid Diffusion Control 207
6.3.2.3 Pore Diffusion Control 208
6.3.2.4 Binding Kinetics Control 210
6.3.2.5 LDF Solution 210
6.3.2.6 Combined Mass Transfer Resistances 211
6.3.3 Experimental Verification of Transport Mechanisms 214
6.3.4 Multicomponent Protein Adsorption Kinetics 218
References 223
7 Dynamics of Chromatography Columns 227
7.1 Introduction 227
7.2 Material Balance Equations 227
7.2.1 Boundary Conditions 229
7.2.2 Dimensionless Equations 230
7.3 Local Equilibrium Dynamics 231
7.4 Multicomponent Systems 244
7.5 Displacement Chromatography 256
7.5.1 Prediction of the Isotachic Train 257
7.5.2 Transient Development 262
References 263
8 Effects of Dispersion and Rate Processes on Column Performance 265
8.1 Introduction 265
8.2 Empirical Characterization of Column Efficiency 265
8.3 Modeling and Prediction of Column Efficiency 275
8.3.1 Plate Model 275
8.3.2 Rate Models with Linear Isotherms 278
8.3.3 Rate Models with Nonlinear Isotherms 287
8.3.4 Rate Models for Competitive Adsorption Systems 303
References 308
9 Gradient Elution Chromatography 311
9.1 Introduction 311
9.2 General Theory for Gradient Elution with Linear Isotherms 313
9.3 LGE Relationships and the Iso-resolution Curve in IEC 320
9.3.1 Iso-resolution Curve 329
9.4 LGE Relationships for RPC and HIC 332
9.5 Gradient Elution at High Protein Loads 337
9.6 Separations with pH Gradients 339
References 351
10 Chromatographic Column Design and Optimization 355
10.1 Introduction 355
10.2 Chromatographic Process Steps and Constraints 357
10.3 Design for Capture 361
10.3.1 Load Step 362
10.3.2 Wash Step 363
10.3.3 Elution Step 364
10.3.4 CIP Step 364
10.3.5 Re-equilibration Step 365
10.3.6 Productivity and Capacity Utilization 365
10.3.7 Continuous Capture 370
10.4 Design for Chromatographic Resolution 375
10.5 SMB Design 382
References 391
Index 395
