1.Introduction 1
1.1 Mass Conservation as an Accounting Method 1
1.2 Interpreting Differentials and Integrals:World Population Statistics 3
1.3 Accounting for Diffusion,Convection,and Reaction for Mass Conservation:The Microscopic Scale 5
1.4 Summary 7
1.5 References 7
Part Ⅰ.Commercial Bioseparations and Product Measurement 9
2.Industrial Bioseparation Processes 11
2.1 Bioseparation Process Selection 11
2.1.1 Scale,Concentration,and Price 13
2.1.2 Product Properties 14
2.2 Monoclonal Antibodies 16
2.3 Human Insulin 17
24 Rabies Vaccine 17
2.5 Penicillin 19
2.6 Protease 20
2.7 L-lysine 21
2.8 Citric Acid 22
2.9 Summary 23
2.10 Problems 24
2.11 References 28
3.Concentration Determination and Bioactivity Assays 29
3.1 Amino Acids 29
3.1.1 High-Performance Liquid Chromatography 31
3.1.2 Reverse-Phase High-Performance Liquid Chromatography 32
3.1.3 Capillary Electrophoresis 32
3.1.4 Micellar Electrokinetic Chromatography 33
3.1.5 Electrodialysis 34
3.1.6 Gas Chromatography 35
3.2 Peptides and Proteins 35
3.2.1 Analytical Chromatography 35
3.2.2 Analytical Electrophoresis 37
3.2.3 Immunoassays 40
3.3 Nucleic and Polynucleic Acids 41
3.3.1 Ion-Exchange Chromatography 41
3.3.2 Reverse-Phase High-Performance Liquid Chromatography 42
3.3.3 Ion-Pair Chromatography 43
3.3.4 Slalom Chromatography 43
3.3.5 Gel Electrophoresis 44
3.3.6 Pulsed-Field Gel Electrophoresis 45
3.3.7 Capillary Isotachophoresis 45
3.3.8 Capillary Zone Electrophoresis 45
3.4 Carbohydrates 46
3.4.1 Monosaccharides 46
3.4.2 Oligosaccharides 49
3.4.3 Glycoproteins 51
3.5 Lipids 51
3.5.1 Fatty Acids 51
3.5.2 Fats and Oils 56
3.6 Steroids and Antibiotics 56
3.7 Vitamins 57
3.8 Summary 58
3.9 Problems 59
3.10 References 61
Part Ⅱ.Application of Chemical,Physical,and Biological Properties to Bioseparations 65
4.Thermodynamic and Transport Properties 67
4.1 Chemical Equilibria 67
4.2 Solubility 69
4.2.1 Protein and Amino Acid Solubility 70
4.3 Diffusivity 73
4.3.1 Uncharged Low-Molecular-Weight Biochemicals 73
4.3.2 Proteins 73
4.4 Isoelectric Points and Charge Dependence on pH 74
4.4.1 Carboxylic Acids 74
4.4.2 Amino Acids 76
4.4.3 Proteins 82
4.5 Hydrophobicity-Hydrophilicity Scales 84
4.6 Acid-Base Scales 84
4.6.1 Gutmann Donor-Acceptor Theory 84
4.6.2 Drago E&C Equation 86
4.6.3 Solvatochromatic Comparison Method 86
4.6.4 Hard and Soft Acid and Base Theory 86
4.6.5 Comparison and Correlation of Different Scales 88
4.7 Metal Ion Binding Constants 88
4.7.1 Nucleic Acids 89
4.7.2 Amino Acids 89
4.8 Summary 90
4.9 Problems 91
4.10 References 93
5.Biocolloidal Interactions and Forees 95
5.1 Short-Range Interactions 95
5.2 Long-Range Interactions 96
5.2.1 Van der Waals Forces 96
5.2.2 Electrostatic Interactions and DLVO Theory 100
5.2.3 Hydrophobic Effects 102
5.2.4 Magnetic Interactions 103
5.3 Summary 104
5.4 Problems 105
5.5 References 106
6.Bioaffinity 108
6.1 Molecular Recognition Processes 108
6.2 Receptor-Ligand Interactions 110
6.2.1 Ionic Bonds 110
6.2.2 Hydrogen Bonds 110
6.2.3 Hydrophobic Interactions 111
6.2.4 Van der Waals Forces 111
6.3 Theoretical Aspects of Receptor-Ligand Affinity 111
6.3.1 Thermodynamic Approach 112
6.3.2 Equilibrium Approach 112
6.4 Specific Interactions 115
6.4.1 Antibody-Antigen Interactions 116
6.4.2 DNA-Protein Interactions 117
6.4.3 Cell Receptor-Ligand Interactions 119
6.4.4 Enzyme-Substrate Interactions 120
6.4.5 Biotin-Avidin/Streptavidin Interactions 121
6.4.6 Lectin-Carbohydrate Interactions 122
6.5 Summary 122
6.6 Problems 123
6.7 References 124
Part Ⅲ.Bioseparation Methods 125
7.Crystallization and Precipitation 127
7.1 Saturation and Supersaturation 127
7.2 Nucleation Phenomena 128
7.3 Growth of Crystals 130
7.4 Batch Crystallization 131
7.4.1 Solution Balance 132
7.4.2 Solid-Phase Balance 132
7.4.3 Crystal Size Distribution 134
7.4.4 Organic Solvent and Salt Precipitation 136
7.4.5 Growth Rate Dispersion 137
7.5 Continuous Crystallization 140
7.6 Yield 141
7.6.1 Removal of Solvent and Diluent 141
7.7 Summary 142
7.8 Problems 142
7.9 References 145
8.Membrane Filtration 146
8.1 Membrane Materials 146
8.2 Driving Forces in Membrane Separations 147
8.3 General Theory of Microfiltration 147
8.3.1 Incompressible Cakes 148
8.3.2 Compressible Cakes 149
8.4 Microfiltration 149
8.4.1 Staging in Microfiltration 151
8.5 Ultrafiltration 152
8.5.1 Ultrafiltration Process Application 154
8.5.2 Ultrafiltration Membrane Application and Modification 156
8.6 Reverse Osmosis 156
8.7 Flux Equations 158
8.8 Electrodialysis 158
8.9 Emulsion Liquid Membranes 159
8.10 Summary 160
8.11 Problems 160
8.12 References 163
9.Centrifugation 164
9.1 Governing Principles 164
9.2 Advantages and Disadvantages of Centrifugation 166
9.3 Selection of Centrifuges 166
9.4 Types of Centrifuges 168
9.4.1 Tubular Bowl Centrifuges 169
9.4.2 Disc-Type Centrifuges 170
9.4.3 Batch-Basket Centrifuges 172
9.5 Industrial-Scale Centrifugation 173
9.6 Summary 176
9.7 Problems 176
9.8 References 177
10.Chromatography 178
10.1 Detection Methods 178
10.2 Summary of the Types of Chromatography 181
10.3 Stationary Phases 183
10.4 Six Ways to Analyze Chromatographic Processes 187
10.4.1 Gaussian Solution 187
10.4.2 Staged Models 190
10.4.3 Newtonian Continuum Mechanics and Linear Equilibria 196
10.4.4 Constant Pattern and Saturation Equilibria 200
10.4.5 Van Deemter Equation 205
10.4.6 Gel Partitioning Model 205
10.5 Gel-Permeation Chromatography 206
10.6 Ion-Exchange Chromatography 208
10.7 Affinity Chromatography 213
10.8 Hydrophobic Interaction and Reverse-Phase Chromatography 216
10.9 Perfusion Chromatography 216
10.10 Other Chromatographic Methods 217
10.10.1 Gradient Methods 217
10.10.2 Displacement Chromatography 218
10.10.3 Radial-Flow Chromatography 218
10.10.4 Membrane Chromatography 218
10.11 Scale-Up Strategies and Considerations 219
10.11.1 Scale-Up Method 1:No Change in Stationary-Phase Particle Size 220
10.11.2 Scale-Up Method 2:Increasing Stationary-Phase Particle Size 221
10.11.3 Scale-Up Method 3:Gel Permeation and On-Off Cycling Approach 222
10.12 Summary 223
10.13 Problems 224
10.14 References 228
11.Extraction 230
11.1 Chemical Thermodynamics of Partitioning 230
11.2 Organic-Aqueous Extraction 231
11.2.1 Extractant/Diluent Systems 233
11.2.2 Removing Biochemicals from the Organic Phase 237
11.3 Two-Phase Aqueous Extraction 238
11.3.1 Partitioning Due to Size 239
11.3.2 The Effect of Protein Charge on Partitioning 240
11.3.3 Other Effects 241
11.4 Reverse Micelles 243
11.5 Supercritical Fluids 244
11.6 Large-Scale Vessels for Extraction 246
11.6.1 Mixer-Settlers 246
11.6.2 Extraction Columns 247
11.6.3 Centrifugal Contactors 250
11.6.4 Comparison 251
11.7 Configurations for Stage-Wise Contacting 252
11.7.1 Cocurrent Contacting 252
11.7.2 Crosscurrent Contacting 253
11.7.3 Countercurrent Contacting 254
11.7.4 A Comparison of Contacting Modes 255
11.7.5 Graphical Solution 263
11.7.6 Fractional Extraction 265
11.7.7 Continuous Countercurrent Extraction 269
11.8 Summary 270
11.9 Problems 271
11.10 References 276
12.Electrophoresis 277
12.1 A Brief Introduction to Some Popular Electrophoretic Methods 277
12.1.1 Gel Electrophoresis 278
12.1.2 Capillary Electrophoresis 281
12.1.3 Isoelectric Focusing 282
12.1.4 Isotachophoresis 283
12.1.5 Moving Boundary 283
12.2 Basic Concepts of Electrophoresis 283
12.2.1 Electro-osmosis and the Relaxation Effect as Retardation Forces 286
12.2.2 Situations That Can Hamper Electrophoretic Separation 286
12.3 Zone Electrophoresis 287
12.3.1 Band Dispersion 288
12.4 Isoelectric Focusing 290
12.5 Isotachophoresis 291
12.6 Two-Dimensional Electrophoresis 292
12.7 Summary 294
12.8 Problems 294
12.9 References 298
13.Magnetic Bioseparations 299
13.1 Magnetic Properties of Materials 299
13.2 Magnetic Particle Classification 305
13.3 Theoretical Considerations 306
13.4 Magnetic Particle Separations 308
13.4.1 High-Gradient Magnetic Separations 309
13.4.2 Affinity Chromatography 310
13.4.3 Aqueous Two-Phase Separations 311
13.5 Applications 312
13.5.1 Cell Separation 312
13.5.2 Immunoassays 313
13.6 Summary 313
13.7 Problems 313
13.8 References 314
14.Solvent Removal and Drying 315
14.1 Methods of Solvent Removal 315
14.2 Theory 316
14.2.1 Vapor-Liquid Systems 317
14.2.2 Liquid-Liquid Systems 321
14.2.3 Liquid-Solid Systems 323
14.3 Rayleigh Distillation 325
14.4 Equipment 327
14.4.1 Evaporation 327
14.4.2 Drying 331
14.5 Summary 334
14.6 Problems 334
14.7 References 336
15.Cell Disruption 337
15.1 Cells and Cell Membranes 337
15.2 Cell Disruption Techniques 339
15.2.1 Mechanical Cell Disruption 340
15.2.2 Chemical Cell Disruption 348
15.3 Summary 350
15.4 Problems 351
15.5 References 352
Part Ⅳ.Bioprocess Synthesis 355
16.Integration of Individual Separation Steps 357
16.1 Bioseparation Process Heuristics 357
16.1.1 Reduce Volume Early in the Process Sequence 358
16.1.2 Save the Most Expensive Step for Last 358
16.1.3 Follow the KISS Principle 360
16.1.4 Resolve Components Well as Early as Possible 361
16.1.5 Minimize Inhibition Mechanisms in the Bioreactor 362
16.2 Issues in Concurrent Bioseparation and Bioreactor Process Development 362
16.2.1 Take the Lab-Scale Process and Scale It Directly with No Changes 362
16.2.2 Design a Bioseparation Process Based on the Closest Existing Commercial Product 363
16.2.3 Pilot-Scale Experimentation with"Spiked" Bioreactor Fluid 363
16.3 Expert Systems in Process Synthesis 364
16.4 Integration of Bioreaction and Bioseparation Steps 364
16.5 Making the Bioreactor Step Bioseparation-Friendly 366
16.6 Considerations in Final Product Formulation and Environmental Impact 367
16.7 Summary 368
16.8 Problems 369
16.9 References 371
17.Production Formulation 372
17.1 Formulation Characteristics 372
17.2 Excipients 373
17.2.1 Thickeners and Binders 373
17.2.2 Surface-Active Agents 374
17.2.3 Colors and Flavors 374
17.2.4 Preservatives 374
17.3 Dosage Forms 375
17.4 Encapsulation 375
17.5 Freeze Drying 377
17.5.1 Theory 378
17.5.2 Technique 381
17.6 Summary 383
17.7 Problems 383
17.8 References 383
18.Bioprocess Economics 385
18.1 Resources Available for Cost Estimation 385
18.1.1 Capital Cost Estimation 386
18.1.2 Operating Cost Estimation 388
18.2 Economic Decision-Making Models 389
18.2.1 Internal Rate of Return 391
18.2.2 Payback Period,Including Interest 391
18.2.3 Net Present Value 391
18.2.4 Return on Investment 392
18.2.5 Choosing Among Projects and Alternative Investments 394
18.3 Sensitivity Analyses 394
18.4 Summary 398
18.5 Problems 398
18.6 References 399
Appendix A.The Laplace Transform 400
Appendix B.Numerical Inversion,van der Laan's Theorem,and Huchel and Helmholtz-Smoluchowski Equations 405
Index 409