Protein Chromatography Process Development and Scale-Up

Giorgio Carta, PhD, is a professor in the Department of Chemical Engineering at the University of Virginia, Charlottesville (USA), where his research focuses on transport phenomena and bioseparations. He regularly organizes professional courses on various aspects of bioseparations, including a course on protein chromatography development and scale-up together with Alois Jungbauer.

Alois Jungbauer, PhD, is the head of protein technology and downstream processing at the Department of Biotechnology of the University of Natural Resources and Life Sciences (BOKU) in Vienna (Austria). For more than 30 years, Professor Jungbauer has worked in biochemical engineering, with a focus on bioseparation, where he has published widely and holds 15 patents. He has also organized a biannual professional course in protein chromatography focused on mass transfer, dispersion, and scale-up.

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

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