Preface ....................................................... xix
1 Introduction to Bioproducts and Bioseparations ............. 1
1.1 Instructional Objectives ................................... 3
1.2 Broad Classification of Bioproducts ........................ 3
1.3 Small Biomolecules ......................................... 4
1.3.1 Primary Metabolites ................................. 4
1.3.2 Secondary Metabolites ............................... 9
1.3.3 Summary of Small Biomolecules ...................... 12
1.4 Macromolecules: Proteins .................................. 13
1.4.1 Primary Structure .................................. 13
1.4.2 Secondary Structure ................................ 14
1.4.3 Tertiary Structure ................................. 14
Example 1.1. Effect of a Reducing Agent on Protein
Structure and Mobility .................................... 17
1.4.4 Quaternary Structure ............................... 17
1.4.5 Prosthetic Groups and Hybrid Molecules ............. 17
1.4.6 Functions and Commercial Uses of Proteins .......... 19
1.4.7 Stability of Proteins .............................. 21
1.4.8 Recombinant Protein Expression ..................... 25
1.5 Macromolecules: Nucleic Acids and Oligonucleotides ........ 31
1.6 Macromolecules: Polysaccharides ........................... 33
1.7 Particulate Products ...................................... 34
1.8 Introduction to Bioseparations: Engineering Analysis ...... 35
1.8.1 Stages of Downstream Processing .................... 35
Example 1.2. Initial Selection of Purification Steps ...... 36
1.8.2 Basic Principles of Engineering Analysis ........... 37
1.8.3 Process and Product Quality ........................ 39
1.8.4 Criteria for Process Development ................... 39
1.9 The Route to Market ....................................... 40
1.9.1 The Chemical and Applications Range of the
Bioproduct ......................................... 40
1.9.2 Documentation of Pharmaceutical Bioproducts ........ 41
1.9.3 GLP and cGMP ....................................... 42
1.9.4 Formulation ........................................ 42
1.10 Summary ................................................... 42
Nomenclature .............................................. 43
Problems .................................................. 44
References ................................................ 46
2 Analytical Methods and Bench Scale Preparative
Bioseparations ............................................ 48
2.1 Instructional Objectives .................................. 49
2.2 Specifications ............................................ 49
2.3 Assay Attributes .......................................... 51
2.3.1 Precision .......................................... 51
2.3.2 Accuracy ........................................... 52
2.3.3 Specificity ........................................ 52
2.3.4 Linearity, Limit of Detection, and Limit of
Quantitation ....................................... 53
2.3.5 Range .............................................. 54
2.3.6 Robustness ......................................... 55
2.4 Analysis of Biological Activity ........................... 55
2.4.1 Animal Model Assays ................................ 55
2.4.2 Cell-Line-Derived Bioassays ........................ 56
2.4.3 In vitro Biochemical Assays ........................ 57
Example 2.1. Coupled Enzyme Assay for Alcohol Oxidase ..... 58
2.5 Analysis of Purity ........................................ 59
2.5.1 Electrophoretic Analysis ........................... 60
Example 2.2. Estimation of the Maximum Temperature
in an Electrophoresis Gel .......................... 63
2.5.2 High-Performance Liquid Chromatography (HPLC) ...... 76
2.5.3 Mass Spectrometry .................................. 79
2.5.4 Coupling of HPLC with Mass Spectrometry ............ 80
2.5.5 Ultraviolet Absorbance ............................. 80
Example 2.3. Determination of Molar Absorptivity ... 81
2.5.6 CHNO/Amino Acid Analysis (AAA) ..................... 82
Example 2.4. Calculations Based on CHNO Analysis ... 82
2.5.7 Protein Assays ..................................... 83
2.5.8 Enzyme-Linked Immunosorbent Assay .................. 84
2.5.9 Gas Chromatography ................................. 86
2.5.10 DNA Hybridization .................................. 86
2.5.11 ICP/MS(AES) ........................................ 87
2.5.12 Dry Weight ......................................... 87
2.6 Microbiology Assays ....................................... 88
2.6.1 Sterility .......................................... 88
2.6.2 Bioburden .......................................... 88
2.6.3 Endotoxin .......................................... 89
2.6.4 Virus, Mycoplasma, and Phage ....................... 89
2.7 Bench Scale Preparative Separations ....................... 90
2.7.1 Preparative Electrophoresis ........................ 90
2.7.2 Magnetic Bioseparations ............................ 96
2.8 Summary .................................................. 101
Nomenclature ............................................. 103
Problems ................................................. 104
References ............................................... 108
3 Cell Lysis and Flocculation .............................. 111
3.1 Instructional Objectives ................................. 111
3.2 Some Elements of Cell Structure .......................... 112
3.2.1 Prokaryotic Cells ................................. 112
3.2.2 Eukaryotic Cells .................................. 113
3.3 Cell Lysis ............................................... 114
3.3.1 Osmotic and Chemical Cell Lysis ................... 116
3.3.2 Mechanical Methods of Lysis ....................... 117
3.4 Flocculation ............................................. 120
3.4.1 The Electric Double Layer ......................... 121
Example 3.1. Dependence of the Debye Radius on
the Type of Electrolyte ........................... 124
3.4.2 Forces Between Particles and Flocculation by
Electrolytes ...................................... 125
Example 3.2. Sensitivity of Critical Flocculation
Concentration to Temperature and Counterion
Charge Number ..................................... 127
3.4.3 The Schulze-Hardy Rule ............................ 128
3.4.4 Flocculation Rate ................................. 129
3.4.5 Polymeric Flocculants ............................. 129
3.5 Summary .................................................. 131
Nomenclature ............................................. 131
Problems ................................................. 133
References ............................................... 133
4 Filtration ............................................... 135
4.1 Instructional Objectives ................................. 136
4.2 Filtration Principles .................................... 137
4.2.1 Conventional Filtration ........................... 137
Example 4.1. Batch Filtration ..................... 138
4.2.2 Crossflow Filtration .............................. 143
Example 4.2. Concentration Polarization in
Ultrafiltration ................................... 146
Example 4.3. Comparison of Mass Transfer
Coefficient Calculated by Boundary Layer Theory
Versus by Shear-Induced Diffusion Theory .......... 150
4.3 Filter Media and Equipment ............................... 152
4.3.1 Conventional Filtration ........................... 152
4.3.2 Crossflow Filtration .............................. 156
4.4 Membrane Fouling ......................................... 160
4.5 Scale-up and Design of Filtration Systems ................ 162
4.5.1 Conventional Filtration ........................... 163
Example 4.4. Rotary Vacuum Filtration ............. 164
Example 4.5. Washing of a Rotary Vacuum Filter
Cake .............................................. 166
4.5.2 Crossflow Filtration .............................. 171
Example 4.6. Diafiltration Mode in Crossflow
Filtration ........................................ 173
4.6 Summary .................................................. 176
Nomenclature ............................................. 178
Problems ................................................. 179
References ............................................... 184
5 Sedimentation ............................................ 185
5.1 Instructional Objectives ................................. 185
5.2 Sedimentation Principles ................................. 186
5.2.1 Equation of Motion ................................ 186
5.2.2 Sensitivities ..................................... 187
5.3 Methods for Analysis of Sedimentation .................... 189
5.3.1 Equilibrium Sedimentation ......................... 190
5.3.2 Sedimentation Coefficient ......................... 191
Example 5.1. Application of the Sedimentation
Coefficient ....................................... 191
5.3.3 Equivalent Time ................................... 192
Example 5.2. Scale-up Based on Equivalent Time .... 193
5.3.4 Sigma Analysis .................................... 193
5.4 Production Centrifuges: Comparison and Engineering
Analysis ................................................. 194
5.4.1 Tubular Bowl Centrifuge ........................... 195
Example 5.3. Complete Recovery of Bacterial Cells
in a Tubular Bowl Centrifuge ...................... 199
5.4.2 Disk Centrifuge ................................... 200
5.5 Ultracentrifugation ...................................... 203
5.5.1 Determination of Molecular Weight .................. 204
5.6 Flocculation and Sedimentation ........................... 205
5.7 Sedimentation at Low Accelerations ....................... 206
5.7.1 Diffusion, Brownian Motion ........................ 206
5.7.2 Isothermal Settling ............................... 207
5.7.3 Convective Motion and Pйclet Analysis ............. 207
5.7.4 Inclined Sedimentation ............................ 207
5.7.5 Field-Flow Fractionation .......................... 209
5.8 Centrifugal Elutriation .................................. 210
5.9 Summary .................................................. 210
Nomenclature ............................................. 212
Problems ................................................. 214
References ............................................... 217
6 Extraction ............................................... 219
6.1 Instructional Objectives ................................. 219
6.2 Extraction Principles .................................... 220
6.2.1 Phase Separation and Partitioning Equilibria ...... 220
6.2.2 Countercurrent Stage Calculations ................. 226
Example 6.1. Separation of a Bioproduct and an
Impurity by Countercurrent Extraction ............. 230
Example 6.2. Effect of Solvent Rate in
Countercurrent Staged Extraction of an
Antibiotic ........................................ 230
6.3 Scale-up and Design of Extractors ........................ 232
6.3.1 Reciprocating-Plate Extraction Columns ............. 233
Example 6.3. Scale-up of a Reciprocating-Plate
Extraction Column ................................. 235
6.3.2 Centrifugal Extractors ............................. 237
Example 6.4. Increase in Feed Rate to
a Podbielniak Centrifugal Extractor ............... 238
6.4 Summary .................................................. 239
Nomenclature ............................................. 240
Problems ................................................. 241
References ............................................... 243
7 Liquid Chromatography and Adsorption ..................... 245
7.1 Instructional Objectives ................................. 247
7.2 Adsorption Equilibrium ................................... 248
7.3 Adsorption Column Dynamics ............................... 251
7.3.1 Fixed-Bed Adsorption .............................. 251
Example 7.1. Determination of the Mass Transfer
Coefficient from Adsorption Breakthrough Data ..... 256
7.3.2 Agitated-Bed Adsorption ........................... 258
7.4 Chromatography Column Dynamics ........................... 259
7.4.1 Plate Models ...................................... 260
7.4.2 Moment Analysis ................................... 262
7.4.3 Chromatography Column Mass Balance with
Negligible Dispersion ............................. 264
Example 7.2. Chromatographic Separation of Two
Solutes ........................................... 264
Example 7.3. Calculation of the Shock Wave
Velocity for a Nonlinear Isotherm ................. 266
Example 7.4. Calculation of the Elution Profile ... 267
7.4.4 Dispersion Effects in Chromatography .............. 269
7.4.5 Computer Simulation of Chromatography
Considering Axial Dispersion, Fluid-Phase Mass
Transfer, Intraparticle Diffusion, and
Nonlinear Equilibrium ............................. 275
7.4.6 Gradients and Modifiers ........................... 277
Example 7.5. Equilibrium for a Protein Anion in
the Presence of Chloride Ion ...................... 277
7.5 Membrane Chromatography .................................. 279
Example 7.6. Comparison of Time for Diffusion Mass
Transfer in Conventional Chromatography and Membrane
Chromatography ........................................... 282
7.6 Simulated Moving Bed Chromatography ...................... 284
7.7 Adsorbent Types .......................................... 288
7.7.1 Silica-Based Resins ............................... 288
7.7.2 Polymer-Based Resins .............................. 289
7.7.3 Ion Exchange Resins ............................... 290
7.7.4 Reversed-Phase Chromatography ..................... 291
7.7.5 Hydrophobic Interaction Chromatography ............ 292
7.7.6 Affinity Chromatography ........................... 292
7.7.7 Immobilized Metal Affinity Chromatography (IMAC) .. 293
7.7.8 Size Exclusion Chromatography ..................... 293
7.8 Particle Size and Pressure Drop in Fixed Beds ............ 294
7.9 Equipment ................................................ 295
7.9.1 Columns ........................................... 295
7.9.2 Chromatography Column Packing Procedures .......... 296
7.9.3 Detectors ......................................... 297
7.9.4 Chromatography System Fluidics .................... 298
7.10 Scale-up ................................................. 299
7.10.1 Adsorption ........................................ 299
Example 7.7. Scale-up of the Fixed-Bed
Adsorption of a Pharmaceutical Product ............ 302
7.10.2 Chromatography .................................... 306
Example 7.8. Scale-up of a Protein
Chromatography .................................... 308
Example 7.9. Scale-up of Protein Chromatography
Using Standard Column Sizes ....................... 309
Example 7.10. Scale-up of Elution Buffer Volumes
in Protein Chromatography ......................... 310
Example 7.11. Consideration of Pressure Drop in
Column Scaling .................................... 311
7.11 Summary .................................................. 311
Nomenclature ............................................. 314
Problems ................................................. 317
References ............................................... 324
8 Precipitation ............................................ 327
8.1 Instructional Objectives ................................. 327
8.2 Protein Solubility ....................................... 328
8.2.1 Structure and Size ................................ 328
8.2.2 Charge ............................................ 329
8.2.3 Solvent ........................................... 331
Example 8.1. Salting Out of a Protein with
Ammonium Sulfate .................................. 333
8.3 Precipitate Formation Phenomena .......................... 334
8.3.1 Initial Mixing .................................... 335
8.3.2 Nucleation ........................................ 335
8.3.3 Growth Governed by Diffusion ...................... 336
Example 8.2. Calculation of Concentration of
Nuclei in a Protein Precipitation ................. 337
Example 8.3. Diffusion-Limited Growth of
Particles ......................................... 340
8.3.4 Growth Governed by Fluid Motion ................... 341
Example 8.4. Growth of Particles Limited by
Fluid Motion ...................................... 342
8.3.5 Precipitate Breakage .............................. 343
8.3.6 Precipitate Aging ................................. 343
8.4 Particle Size Distribution in a Continuous-Flow Stirred
Tank Reactor ............................................. 344
Example 8.5. Dependence of Population Density
on Particle Size and Residence Time in a CSTR ............ 348
8.5 Methods of Precipitation ................................. 348
8.6 Design of Precipitation Systems .......................... 352
8.7 Summary .................................................. 354
Nomenclature ............................................. 356
Problems ................................................. 358
References ............................................... 360
9 Crystallization .......................................... 362
9.1 Instructional Objectives ................................. 363
9.2 Crystallization Principles ............................... 363
9.2.1 Crystals .......................................... 363
9.2.2 Nucleation ........................................ 364
9.2.3 Crystal Growth .................................... 366
9.2.4 Crystallization Kinetics from Batch Experiments ... 367
9.3 Batch Crystallizers ...................................... 368
9.3.1 Analysis of Dilution Batch Crystallization ........ 369
Example 9.1. Batch Crystallization with Constant
Rate of Change of Diluent Concentration ........... 371
9.4 Process Crystallization of Proteins ...................... 373
9.5 Crystallizer Scale-up and Design ......................... 375
9.5.1 Experimental Crystallization Studies as a Basis
for Scale-up ...................................... 375
9.5.2 Scale-up and Design Calculations .................. 377
Example 9.2. Scale-up of Crystallization Based
on Constant Power per Volume ...................... 378
9.6 Summary .................................................. 379
Nomenclature ............................................. 379
Problems ................................................. 381
References ............................................... 383
10 Evaporation .............................................. 384
10.1 Instructional Objectives ................................. 384
10.2 Evaporation Principles ................................... 385
10.2.1 Heat Transfer ..................................... 385
Example 10.1. Evaporation of a Butyl Acetate
Stream Containing a Heat-Sensitive Antibiotic
in a Falling-Film Evaporator ...................... 388
10.2.2 Vapor-Liquid Separation ........................... 394
10.3 Evaporation Equipment .................................... 396
10.3.1 Climbing-Film Evaporators ......................... 397
10.3.2 Falling-Film Evaporators .......................... 398
10.3.3 Forced-Circulation Evaporators .................... 398
10.3.4 Agitated-Film Evaporators ......................... 399
10.4 Scale-up and Design of Evaporators ....................... 399
10.5 Summary .................................................. 402
Nomenclature ............................................. 403
Problems ................................................. 404
References ............................................... 405
11 Drying ................................................... 407
11.1 Instructional Objectives ................................. 407
11.2 Drying Principles ........................................ 408
11.2.1 Water in Biological Solids and in Gases ........... 408
Example 11.1. Drying of Antibiotic Crystals ....... 411
11.2.2 Heat and Mass Transfer ............................ 412
Example 11.2. Conductive Drying of Wet Solids in
a Tray ............................................ 414
Example 11.3. Mass Flux During the Constant Rate
Drying Period in Convective Drying ................ 420
Example 11.4. Time to Dry Nonporous Biological
Solids by Convective Drying ....................... 421
11.3 Dryer Description and Operation .......................... 422
11.3.1 Vacuum-Shelf Dryers ............................... 422
11.3.2 Batch Vacuum Rotary Dryers ........................ 423
11.3.3 Freeze Dryers ..................................... 424
11.3.4 Spray Dryers ...................................... 426
11.4 Scale-up and Design of Drying Systems .................... 427
11.4.1 Vacuum-Shelf Dryers ............................... 427
11.4.2 Batch Vacuum Rotary Dryers ........................ 428
11.4.3 Freeze Dryers ..................................... 428
11.4.4 Spray Dryers ...................................... 431
Example 11.5. Sizing of a Spray Dryer ............. 432
11.5 Summary .................................................. 435
Nomenclature ............................................. 436
Problems ................................................. 437
References ............................................... 440
12 Bioprocess Design and Economics .......................... 441
12.1 Instructional Objectives ................................. 441
12.2 Definitions and Background ............................... 442
12.3 Synthesis of Bioseparation Processes ..................... 445
12.3.1 Primary Recovery Stages ........................... 445
12.3.2 Intermediate Recovery Stages ...................... 450
12.3.3 Final Purification Stages ......................... 451
12.3.4 Pairing of Unit Operations in Process Synthesis ... 453
12.4 Process Analysis ......................................... 454
12.4.1 Spreadsheets ...................................... 454
12.4.2 Process Simulators and Their Benefits ............. 454
12.4.3 Using a Biochemical Process Simulator ............. 457
12.5 Process Economics ........................................ 460
12.5.1 Capital Cost Estimation ........................... 461
12.5.2 Operating Cost Estimation ......................... 466
12.5.3 Profitability Analysis ............................ 471
12.6 Illustrative Examples .................................... 472
12.6.1 Citric Acid Production ............................ 472
12.6.2 Human Insulin Production .......................... 479
12.6.3 Therapeutic Monoclonal Antibody Production ........ 495
12.7 Summary .................................................. 502
Problems ................................................. 503
References ............................................... 507
13 Laboratory Exercises in Bioseparations ................... 511
13.1 Flocculant Screening ..................................... 511
13.1.1 Background ........................................ 512
13.1.2 Objectives ........................................ 512
13.1.3 Procedure ......................................... 512
13.1.4 Report ............................................ 513
13.1.5 Some Notes and Precautions ........................ 514
13.2 Crossflow Filtration ..................................... 514
13.2.1 Background ........................................ 514
13.2.2 Objectives ........................................ 515
13.2.3 Procedure ......................................... 515
13.2.4 Report ............................................ 515
13.3 Centrifugation of Flocculated and Unflocculated
Particulates ............................................. 516
13.3.1 Background ........................................ 516
13.3.2 Objectives ........................................ 517
13.3.3 Procedure ......................................... 517
13.3.4 Report ............................................ 519
13.4 Aqueous Two-Phase Extraction ............................. 520
13.4.1 Physical Measurements ............................. 520
13.4.2 Procedure ......................................... 521
13.4.3 Calculations and Report ........................... 522
13.4.4 Inverse Lever Rule ................................ 524
13.5 Chromatography Scale-up .................................. 525
13.5.1 Background ........................................ 525
13.5.2 Objectives ........................................ 525
13.5.3 Procedure ......................................... 526
13.5.4 Report ............................................ 528
References ............................................... 530
APPENDIX: Table of Units and Constants ........................ 531
Index ......................................................... 535
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