Aggregation of Therapeutic Proteins

AGGREGATION OF THERAPEUTIC PROTEINS......Page 2
CONTENTS......Page 10
PREFACE......Page 20
CONTRIBUTORS......Page 24
1.1 THE PROBLEM OF PROTEIN AGGREGATION......Page 26
1.1.1 Structural Features of Proteins......Page 29
1.1.2 Structural Features of Protein Aggregates......Page 30
1.2 PARALLELS TO PROTEIN FOLDING......Page 36
1.3.1 Physicochemical Properties of Proteins......Page 37
1.3.2 Surface Properties and Packing Arrangements......Page 43
1.3.3 Solvent Interactions......Page 46
1.4 MODELS OF AGGREGATION......Page 47
1.4.1 Monomer Conversion......Page 48
1.4.2 Oligomeric Intermediates......Page 49
1.4.3 Nucleation in Protein Folding......Page 50
1.4.4 Domain Swapping......Page 51
1.5.1 Classical Models Based on Chemical Equilibrium......Page 54
1.5.2 Statistical Mechanical Models......Page 56
1.5.3 Computational Models......Page 58
1.5.4 Application of Coarse-Grained Models......Page 60
1.5.5 Information Theory (IT)......Page 62
1.6 INFLUENCES OF CHEMICAL ALTERATION ON AGGREGATION......Page 65
1.6.2 Methionine Oxidation......Page 66
1.6.3 Covalent Cross-Linking of Strands......Page 67
1.6.4 Deamidation......Page 69
1.6.5 Proline Isomerization......Page 70
1.7 APPROACHES TO PREDICTING AGGREGATION......Page 71
1.8 CONCLUSIONS......Page 74
REFERENCES......Page 75
2.1 INTRODUCTION......Page 88
2.2 NATIVE AND NONNATIVE AGGREGATION PATHWAYS......Page 91
2.3 THERMODYNAMICS OF REVERSIBLE SELF-ASSOCIATION......Page 94
2.3.1 Self-Association of Folded/Native Proteins......Page 95
2.3.2 Nonnative Self-Association......Page 98
2.4.1 Aggregation Kinetics......Page 100
2.4.2 Distinguishing Aggregation Pathways and Rate-Limiting Steps......Page 103
2.5 CHEMICAL MODIFICATIONS......Page 105
2.6.1 Transfer Free Energies and Preferential Interaction Parameters......Page 107
2.6.2 Relating G32 to Molecular Interactions......Page 110
2.6.3 An Additive Approach to Δμ2tr, ∂μ2 ∂ 3ex m , and G32(m3)......Page 114
2.6.4 Predicting Cosolvent/Cosolute Effects on Unfolding, Association, and Aggregation......Page 118
APPENDIX—DERIVATION OF Γ32 FOR VAN DER WAALS (vdW) MIXTURE......Page 119
REFERENCES......Page 122
3.1 INTRODUCTION......Page 128
3.2 ENERGY LANDSCAPES, PROTEIN FOLDING, AND AGGREGATION......Page 130
3.3.1 Computational Tools......Page 131
3.3.2 Experimentally Studied Aggregation-Prone Sequences and Sequence Patterns......Page 134
3.3.3 Prediction of APRs in Therapeutic mAbs......Page 135
3.3.4 Other Useful Studies......Page 138
3.4 CONCLUSIONS AND FUTURE DIRECTIONS......Page 139
REFERENCES......Page 140
4.1 INTRODUCTION......Page 144
4.2.1 Physical Aggregation through Formation of Unfolding Intermediates......Page 145
4.2.2 Direct Aggregation through Self-Association or Chemical Linkages......Page 152
4.2.4 Protein Aggregation from the Unfolded State......Page 154
4.2.5 Aggregation Nucleation......Page 155
4.2.6 Multiplicity in Protein Aggregation......Page 156
4.3.1 Relationship between Temperature and Protein Stability......Page 157
4.3.2 Effect of High Temperature on Protein Aggregation......Page 158
4.3.3 Melting Temperature (Tm) and Protein Aggregation......Page 160
4.4 EFFECTS OF SOLUTION CONDITIONS AND COMPOSITION ON PROTEIN AGGREGATION......Page 161
4.4.1 Solution pH......Page 162
4.4.2 Type and Concentration of Buffering Agents......Page 165
4.4.3 Ionic Strength......Page 166
4.4.4 Excipients/Additives......Page 168
4.4.5 Protein Concentration......Page 179
4.4.6 Metal Ions......Page 182
4.4.7 Denaturant and Reducing Agents......Page 184
4.4.8 Impurities......Page 185
4.4.9 Organic Solvents......Page 186
4.4.10 Containers/Closures......Page 187
4.4.12 Light......Page 188
4.5.1 Fermentation/Expression......Page 189
4.5.2 Unfolding/Refolding......Page 190
4.5.4 Freeze-Thaw......Page 193
4.5.5 Agitation......Page 194
4.5.6 High Hydrostatic Pressure......Page 195
4.5.7 Drying......Page 196
4.5.10 Miscellaneous Processes......Page 198
4.6.2 Excipients and Excipient Levels......Page 199
4.6.3 Physical State of the Solid......Page 201
4.7 SUMMARY......Page 202
REFERENCES......Page 203
5.1 INTRODUCTION......Page 230
5.2 AGGREGATE CLASSIFICATION......Page 231
5.2.1 Aggregate Nomenclature......Page 234
5.3 ANALYTICAL TOOLS FOR THE CHARACTERIZATION OF AGGREGATES......Page 237
5.3.1 SEC......Page 246
5.3.2 Light Scattering......Page 248
5.3.3 Analytical Ultracentrifugation (AUC)......Page 253
5.3.4 FFF......Page 256
5.3.5 Electrophoresis......Page 258
5.3.6 Turbidimetry and Nephelometry......Page 260
5.3.7 Analytical Techniques for Subvisible Particles......Page 262
5.3.8 Visible Particles......Page 265
5.3.9 Miscellaneous Technologies for Characterization of Protein Aggregates......Page 266
5.3.10 Protein Structural Characterization in Aggregates: Spectroscopy-Based Techniques......Page 270
5.4 SUMMARY......Page 271
REFERENCES......Page 272
6.2 CANDIDATE SELECTION......Page 282
6.2.1 Sequence Analysis......Page 283
6.2.2 Stability to Process Conditions......Page 284
6.2.3 Focus on Stability to Formulation and Long-Term Storage Conditions......Page 290
6.2.5 Specific Considerations for Bacteria-Derived Protein......Page 293
6.3.1 Expression Strategies......Page 294
6.3.2 Bioreactor Conditions......Page 295
6.4 PROTEIN AGGREGATION AND PURIFICATION......Page 296
6.4.1 Preventing Aggregation with Appropriate Solvent Conditions......Page 299
6.4.2 Removal of Aggregates during Processing......Page 306
6.4.4 Specific Considerations for Bacterial-Derived Proteins......Page 310
6.4.5 Bulk DS Stability and Storage......Page 317
6.4.6 Scale-Up......Page 318
REFERENCES......Page 320
7.1 INTRODUCTION......Page 326
7.2 ROLES OF CONFORMATIONAL AND COLLOIDAL STABILITY IN REDUCING RATES OF AGGREGATION......Page 327
7.2.1 Conformational Stability......Page 328
7.3.1 Effects of Air–Water Interface......Page 330
7.3.2 Protein Aggregation at Solid–Liquid Interfaces......Page 331
7.3.3 Effects of Excipients on Structural Perturbation and Aggregation at Surfaces......Page 332
7.3.6 Shear Effects on Protein Aggregation......Page 334
7.4.1 Freezing and Thawing Operations......Page 335
7.4.3 Preparation of Final Formulation with Excipients......Page 338
7.4.5 Filling......Page 339
7.5.2 The History of Visible Inspection and Particle Contamination Control in Parenteral Products......Page 341
7.5.4 Subvisible Particle Measurements......Page 342
7.5.5 Subvisible Particle Acceptance Criteria......Page 343
7.5.6 Visual Inspection Requirements......Page 345
7.5.7 Visual Inspection Methods......Page 346
7.5.8 What Does “Visible” Mean?......Page 347
7.5.9 Potential Causes of Particulate Contamination in Parenteral Products......Page 348
7.6 SUMMARY AND OUTLOOK......Page 349
REFERENCES......Page 350
8.1 INTRODUCTION......Page 358
8.2.1 Traditional Formulation Development......Page 359
8.2.3 Computer-Assisted Design of Formulations......Page 369
8.3 PROTECTION OF PROTEINS IN VARIOUS PROCESSING STEPS......Page 370
8.3.2 Freezing/Thawing......Page 371
8.3.3 Manufacturing Processes for Liquid Drug Product......Page 372
8.3.4 Drying......Page 373
8.3.6 Preparation of Controlled Protein Delivery Systems......Page 375
8.4.1 Mutagenesis......Page 376
8.4.2 PEG ylation......Page 377
8.5 SUMMARY......Page 378
REFERENCES......Page 379
9.1 INTRODUCTION......Page 392
9.2.1 Background......Page 393
9.2.3 Results and Interpretation......Page 394
9.2.4 Discussion and Lessons Learned......Page 397
9.3.1 Background......Page 401
9.3.3 Results and Interpretation......Page 403
9.3.4 Discussion and Lessons Learned......Page 405
9.4.1 Background......Page 406
9.4.3 Results and Interpretation......Page 407
9.4.4 Discussion and Lessons Learned......Page 408
9.5.1 Background......Page 410
9.5.2 Model Protein and Conditions (Case Study 4)......Page 411
9.5.3 Results and Interpretation (Case Study 4)......Page 412
9.5.4 Model Protein and Conditions (Case Study 5)......Page 413
9.5.5 Results and Interpretation (Case Study 5)......Page 414
9.5.6 Discussion and Lessons Learned......Page 415
9.6.2 Model Protein and Conditions......Page 416
9.6.3 Results and Interpretation......Page 417
9.6.4 Discussion and Lessons Learned......Page 418
9.7.1 Are Aggregates Created Equal?......Page 419
REFERENCES......Page 421
10.1 INTRODUCTION......Page 428
10.2.1 Incidence of Immunogenicity during Therapy......Page 429
10.2.2 Consequences of Protein Immunogenicity for Patients......Page 430
10.2.3 Factors Playing a Role in Protein Immunogenicity......Page 431
10.3 IMMUNE MECHANISMS RELATED TO PROTEIN IMMUNOGENICITY......Page 434
10.3.1 Immune Responses to Therapeutic Proteins......Page 435
10.3.3 B-Cell Activation Mechanisms......Page 436
10.4 AGGREGATES AND IMMUNOGENICITY......Page 440
10.4.1 Evidence of Aggregates as a Risk Factor for Immunogenicity......Page 442
10.4.2 Proposed Mechanisms of Antibody Induction by Protein Aggregates......Page 445
10.4.3 Possible Factors Responsible for the Immunogenicity of Protein Aggregates......Page 448
10.4.5 Predictive Models to Access the Immunogenicity of Aggregates......Page 451
10.5 CONCLUSIONS......Page 452
REFERENCES......Page 453
11.1 INTRODUCTION......Page 460
11.2.1 Drug Development to Licensure......Page 461
11.3.1 Aggregates and Immunogenicity......Page 463
11.4.2 Selection and Validation of Detection Methods......Page 465
11.4.3 Setting Specifi cations for Aggregates as Part of Lot Release and Stability Testing......Page 469
11.4.4 Commonly Used Guidance Documents Relating to Control of Aggregates......Page 470
11.5.2 Assessment of Biosimilars......Page 471
DISCLAIMER......Page 472
REFERENCES......Page 473
INDEX......Page 478
color plate......Page 512