List of Contributors xix
Preface xxi
Section 1 The Development of Regulatory Guidelines for Mutagenic/Genotoxic Impurities Overall Process1
1 Historical Perspective on the Development of the EMEA Guideline and Subsequent ICH M7 Guideline3
Andrew Teasdale
1.1 Introduction 3
1.1.1 CPMP Position Paper on the Limits of Genotoxic Impurities 2002 4
1.1.1.1 Scope/Introduction 4
1.1.1.2 Toxicological Background 4
1.1.1.3 Pharmaceutical (Quality) Assessment 4
1.1.1.4 Toxicological Assessment 4
1.1.2 Guideline on the Limits of Genotoxic Impurities Draft June 2004 5
1.1.3 PhRMA (Mueller) White Paper 6
1.1.4 Finalized EMA Guideline on the Limits of Genotoxic Impurities June 2006 8
1.1.4.1 Issues Associated with Implementation 9
1.1.4.2 Control Expectations for Excipients 11
1.1.4.3 Control Expectations for Natural/Herbal Products 12
1.1.4.4 Identification of Potential Impurities 12
1.1.4.5 The Principle of Avoidance 12
1.1.4.6 The ALARP Principle 14
1.1.4.7 Overall 14
1.1.5 SWP Q&A Document 14
1.1.5.1 The Application of the Guideline in the Investigational Phase and Acceptable Limits for GIs Where Applied to Studies of Limited Duration 14
1.1.5.2 Application of the Guideline to Existing Products 15
1.1.5.3 Avoidance and ALARP 17
1.1.5.4 ICH Identification Threshold and its Relation to MI Assessment 17
1.1.6 FDA Draft Guideline 17
1.1.7 Other Relevant Guidance 17
1.1.7.1 Excipients 18
1.1.8 Herbals 18
1.1.9 ICH S9 18
1.1.10 Conclusions 19
References 19
2 ICH M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk21
Andrew Teasdale and Raphael Nudelman
2.1 Introduction 21
2.2 ICH M7 22
2.2.1 Introduction 22
2.2.2 Scope 22
2.2.2.1 Established Products 22
2.2.2.2 Anticancer Treatments 23
2.2.2.3 Nature of Therapeutic Agent/Excipients 23
2.2.3 General Principles 24
2.2.4 Considerations for Marketed Products 25
2.2.4.1 Post-approval Changes to Drug Substance, Chemistry, and Manufacturing Controls 26
2.2.4.2 Post-approval Changes to Drug Product Chemistry, Manufacturing, and Controls 26
2.2.4.3 Changes to the Clinical Use of Drug Products 26
2.2.5 Other Considerations for Marketed Products 27
2.2.6 Drug Substance and Drug Product Impurity Assessment 27
2.2.6.1 Synthetic Impurities 28
2.2.6.2 Degradation Products 28
2.2.7 Hazard Assessment 29
2.2.8 Risk Characterization 32
2.2.8.1 Acceptable Intakes Based on Compound-specific Risk Assessments 32
2.2.8.2 Acceptable Intakes for Class 2 and Class 3 Compounds 33
2.2.8.3 Multiple Impurities 34
2.2.8.4 Exceptions and Flexibility in Approaches 35
2.2.9 Control Strategy 35
2.2.9.1 Considerations for Control Approaches 37
2.2.9.2 Considerations for Periodic Testing 37
2.2.9.3 Control of Degradation Products 38
2.2.10 Lifecycle Management 38
2.2.11 Documentation 38
2.2.11.1 Clinical Trail Applications 38
2.2.11.2 Common Technical Document (Marketing Application) 39
2.2.12 Other Aspects 39
2.2.12.1 Relationship Between ICH M7 and ICH Q3A 39
2.3 Conclusions 40
2.4 Commentary on ICH M7 Questions and Answers 40
2.4.1 Section 1 Introduction 41
2.4.1.1 Question 1.1 41
2.4.1.2 Question 1.2 42
2.4.1.3 Question 1.3 42
2.4.1.4 Question 1.4 42
2.4.2 Section 2 Scope 43
2.4.2.1 Question 2.1 43
2.4.3 Section 3 General Principles 43
2.4.3.1 Question 3.1 44
2.4.3.2 Question 3.2 44
2.4.4 Section 4 Considerations for Marketed Products 44
2.4.4.1 Question 4.1 45
2.4.5 Section 5 Drug Substance and Drug Product Impurity Assessment 45
2.4.6 Section 6 Hazard Assessment Elements 45
2.4.6.1 Question 6.1 45
2.4.6.2 Question 6.2 46
2.4.6.3 Question 6.3 47
2.4.6.4 Question 6.4 48
2.4.7 Section 7 Risk Characterization 48
2.4.7.1 Question 7.1 48
2.4.7.2 Question 7.2 49
2.4.7.3 Question 7.3 49
2.4.7.4 Question 7.4 50
2.4.7.5 Question 7.5 51
2.4.8 Section 9 Documentation 53
References 55
3 Control Strategies for Mutagenic Impurities57
Andrew Teasdale, Michael Burns, and Michael Urquhart
3.1 Introduction 57
3.2 Assessment Process 58
3.2.1 General 58
3.2.2 Step 1 Evaluation of Drug Substance and Drug Product Processes for Sources of Potentially Mutagenic Impurities 60
3.2.3 Step 2 Structural Assessment 61
3.2.4 Step 3 Classification 61
3.2.5 Step 4 Assessment of Risk of Potential Carryover of Impurities 63
3.2.6 Overall Quantification of Risk 63
3.2.6.1 Predicted Purge Factor 64
3.2.6.2 Required Purge Factor 65
3.2.6.3 Purge Ratio 66
3.2.6.4 High Predicted Purge 67
3.2.6.5 Moderate Predicted Purge 67
3.2.6.6 Low Predicted Purge 67
3.2.6.7 ICH M7 Control Option 1, 2, or 3 67
3.2.6.8 Step 5 Further Evaluation 67
3.2.6.9 Safety Testing 67
3.2.7 Quantification of Level Present 68
3.3 Step 6 Overall Risk Assessment 69
3.4 Further Evaluation of Risk Purge (Spiking) Studies 70
3.5 Conclusion 70
3.6 Case Studies 71
3.6.1 Case Study 1 GW641597X 71
3.6.1.1 Ethyl Bromoisobutyrate 2 73
3.6.1.2 Hydroxylamine 74
3.6.1.3 Alkyl Chloride 8 75
3.6.1.4 Additional Evidence for the Purging of Ethyl Bromoisobutyrate and Alkyl Chloride 8 76
3.6.2 Proposed ICH M7-aligned Potential Mutagenic Control Regulatory Discussion 78
3.6.3 Case Study 2 Candesartan 78
References 84
Section 2 In Silico Assessment of Mutagenicity87
4 Use of StructureActivity Relationship (SAR) Evaluation as a Critical Tool in the Evaluation of the Genotoxic Potential of Impurities89
Catrin Hasselgren and Glenn Myatt
4.1 Introduction 89
4.2 (Q)SAR Assessment 90
4.2.1 Looking-up Experimental Data 90
4.2.2 (Q)SAR Methodologies 91
4.2.2.1 Overview 91
4.2.2.2 OECD Validation Principles 91
4.2.3 Expert Rule-Based Methodology 92
4.2.4 Statistical-Based Methodology 95
4.2.5 Applying (Q)SAR Models 97
4.2.6 Expert Review 98
4.2.6.1 Overview 98
4.2.6.2 Refuting a Statistical-Based Prediction 100
4.2.6.3 Mechanistic Assessment 101
4.2.6.4 Assessing Lack of Chemical Reactivity 101
4.2.7 Class Assignment 103
4.2.7.1 Overview 103
4.2.8 Documentation 109
4.3 Discussion 109
4.4 Conclusions 110
Acknowledgments 111
References 111
5 Evolution of Quantitative StructureActivity Relationships ((Q)SAR) for Mutagenicity115
James Harvey and David Elder
5.1 Introduction 115
5.2 Pre ICH M7 Guideline 116
5.3 Post ICH M7 117
5.3.1 Evolution of (Q)SAR Platforms 117
5.3.2 Robust NegativeIn Silico(Q)SAR Predictions 118
5.3.3 Development of Composite (Q)SAR Models 119
5.3.4 Expansion of Training Data Sets to Enhance the Predictive Power of (Q)SAR Tools 120
5.3.5 Focused Data Sharing Initiatives on Specific Chemical Classes 120
5.3.5.1 UnderstandingIn VitroMechanisms Leading to Mutagenicity 121
5.3.5.2 Shared Data, Shared Progress 122
5.3.6 Novel Data Mining Approaches 125
5.3.6.1 Case Study: Primary Aromatic Amines (PAAs) 125
5.3.6.2 Case Study: AromaticN-oxides 125
5.4 Expert Knowledge 127
5.5 Future Direction 129
References 131
Section 3 Toxicological Perspective on Mutagenic Impurities137
6 Toxicity Testing to Understand the Mutagenicity of Pharmaceutical Impurities139
Andrew Teasdale, John Nicolette, Joel P. Bercu, James Harvey, Stephen Dertinger, Michael ODonovan, and Christine Mee
6.1 Introduction 139
6.2In VitroGenotoxicity Tests 141
6.2.1 Background 141
6.2.2 Bacterial Reverse Mutation or Ames Test 142
6.2.3 Modifications to the Standard Ames Test 145
6.2.3.1 Six-well Ames Assay 146
6.2.4 Test Strategy 146
6.3In VivoMutation Assays 148
6.3.1In Vivo Pig-aGene Mutation Assay 148
6.3.2 Rodent Micronucleus Test 152
6.3.3 Rodent Comet Assay 155
6.3.4 Transgenic Rodent (TGR) Mutation Assay 155
6.4 Conclusions 158
Glossary 159
References 160
7 Compound-and Class-Specific Limits for Common Impurities in Pharmaceuticals165
Joel P. Bercu, Melisa J. Masuda-Herrera, Alejandra Trejo-Martin, David J. Snodin, Kevin P. Cross, George E. Johnson, James Harvey, Robert S. Foster, David J. Ponting, and Richard V. Williams
7.1 Introduction 165
7.2 Monograph Development 167
7.2.1 Exposure to the General Population 167
7.2.2 Mutagenicity/Genotoxicity 170
7.2.3 Noncarcinogenic Effects 170
7.2.4 Carcinogenic Effects 170
7.2.5 Mode of Action (MOA) and Assessment of Human Relevance 171
7.2.6 Toxicokinetics 171
7.2.7 Regulatory/Published Limits 171
7.3 Derivation of the Compound-specific Limit 171
7.3.1 PoD Selection 172
7.3.2 Limited Data Sets 172
7.3.3 PDE Development 172
7.3.4 AI Development 172
7.3.5 Class-specific Limit 173
7.3.6 Less than Lifetime (LTL) AIs 173
7.4 Examples of Published Compound-specific Limits 173
7.4.1 Mutagenic Carcinogens 173
7.4.2 Nonmutagenic Carcinogens 176
7.4.3 Mutagenic Noncarcinogens 176
7.4.4 Nonmutagenic Compounds 176
7.4.5 MutagenicIn vitrobut notIn vivo176
7.4.6 Route of Administration-specific Limits 177
7.5 Class-specific Limits 177
7.5.1 Alkyl Chlorides 177
7.5.2 Alkyl Bromides 178
7.5.3N-Nitrosamines 178
7.5.3.1 Regulatory Limits forN-Nitrosamines 178
7.5.3.2 Additional Proposed Limits forN-Nitrosamines 180
7.5.3.3N-Nitrosamine Exposure in the General Population 181
7.5.3.4 Developing a Class-specific Limit forN-Nitrosamines 182
7.5.4 Arylboronic Acids and Esters 193
7.6 EMS Case Study and Updated Toxicity Analysis 196
7.6.1 Potential for Human Exposure 196
7.6.2 Mutagenicity/Genotoxicity 196
7.6.3 Noncarcinogenic Effects 198
7.6.4 Carcinogenicity 199
7.6.5 Regulatory and/or Published Limits 199
7.6.6 Permitted Daily Exposure 199
7.7 Extractables and Leachables 202
7.8 Lhasa AI/PDE Database for Impurities 203
7.9 Conclusions and Future Directions 203
Acknowledgments 204
References 204
8 Genotoxic Threshold Mechanisms and Points of Departure213
George E. Johnson, Shareen H. Doak, Gareth J.S. Jenkins, and Adam D. Thomas
8.1 Introduction to Genotoxic Dose Responses 213
8.1.1 The Linear Default Position for Genotoxic Carcinogens 213
8.1.2 Theoretical Evidence for Rejecting the Linear Approach 214
8.1.3In VitroExperimental Evidence for Threshold Mechanism 215
8.1.4 In Vivo Evidence for Genotoxic Thresholds 218
8.2 Threshold Mechanisms 221
8.2.1 Statistical Assessment of Dose Response Data Sets 224
8.2.2 Extrapolation from One Chemical to Another 224
8.2.3 Extrapolation of Threshold Mechanisms and PoDs to Populations 225
8.3 Conclusions 227
References 227
Section 4 Quality Perspective on Genotoxic Impurities233
9 Mutagenic Impurities Assessment of Fate and Control Options235
Michael W. Urquhart, Andrew Teasdale, and Michael Burns
9.1 Introduction/Background 235
9.2 Reactivity 236
9.2.1 Reactivity Classification 238
9.3 Solubility Isolated Stages 238
9.4 Recrystallization 239
9.4.1 Solubility Liquid/Liquid Partitioning 239
9.5 Volatility 241
9.6 Chromatography 241
9.7 Other Techniques 242
9.7.1 Activated Charcoal 242
9.7.2 Scavenger Resins 242
9.8 Overall Quantification of Risk 243
9.9 Alignment to ICH M7 Control Options 244
9.10 Control Option Selection 247
9.10.1 Predicted Purge Factor 248
9.10.2 Required Purge Factor 249
9.10.3 Purge Ratio 249
9.10.4 High Predicted Purge 250
9.10.5 Moderate Predicted Purge 250
9.10.6 Low Predicted Purge 250
9.10.7 ICH M7 Control Option 1, 2, or 3 251
9.10.8 Representative Data to be Supplied in Regulatory Submission Under an ICH M7 Control Strategy 251
9.10.9 Summary of PMI Purging Across the Synthetic Route 251
9.10.10 Details of Individual Impurity Purging Through the Subsequent Downstream Chemistry 253
9.10.11 Development of a Knowledge Base Expert In Silico System 254
9.10.12 Experimental Work to Assess Reactivity 257
9.11 Utilizing Mirabilis for a Purge Calculation 259
9.11.1 Utility of In Silico Predictions 260
9.11.1.1 Case Study Camicinal [38] 260
References 266
10N-Nitrosamines269
Andrew Teasdale, Justin Moser, J. Gair Ford, and Jason Creasey
10.1 Background 269
10.2 Generation ofN-Nitrosamines 270
10.3 Article 31 273
10.4 Further Issues Cross Contamination and Ranitidine 275
10.4.1 Article 5(3) and Associated Q&A Document 276
10.5 How to Assess the Risk Posed in Pharmaceuticals 278
10.5.1 Drug Substance 278
10.5.1.1 Where do Nitrites Come Within Drug Substance Come From? 278
10.5.1.2 What Other Sources Are There? 278
10.5.1.3 Other Factors Associated with Drug Substance Synthesis 280
10.5.2 Process to Assess Drug Substance-Related Risk 280
10.5.3 Drug Product-Related Risk 282
10.5.3.1 Related Risks of Contamination and Formation in Drug Products 282
10.5.4 Container Closure Systems 289
10.5.5 Elastomeric Components 291
10.5.6 Nitrosamine Impurities in Biologics 293
10.5.6.1 Active Substance 293
10.5.6.2 The Water Used in Formulation Is Depleted in Nitrosating Agents 295
10.5.6.3 Bioconjugated or Chemically Modified Products 295
10.5.6.4 Excipients 296
10.6 Regulatory Guidance Pursuant toN-Nitrosamines and its Implications 297
10.6.1 Article 31 Process and Outcomes 297
10.6.1.1 Article 31 Request 297
10.6.2 Sartans Lessons Learnt Report 298
10.6.2.1 Reflection on the Initial Section of the EMA Report 299
10.6.3 Article 5(3) Report 299
10.6.3.1 Quality 299
10.6.3.2 Consideration for Analytical Method Development to Identify and QuantifyN-Nitrosamines in Drug Substances and Medicinal Products 300
10.6.3.3 Safety 301
10.6.3.4 Conclusions 305
10.6.4 EMA Question and Answer Document [6] 305
10.6.4.1 Further Revision of the EMA Question and Answer Document 310
10.6.5 FDA Guideline 310
10.6.5.1 Introduction and Background 310
10.6.5.2 Recommendations 310
10.6.5.3 Acceptable Intakes (section III.A) 313
10.6.5.4 Quality/Chemistry and Controls 314
10.7 Way Forward 315
Acknowledgments 316
References 317
11 Conditions Potentially Leading to the Formation of Mutagenic Impurities321
Lucie Lovelle, Andrew Teasdale, Ian Ashworth, Adrian Clarke, and Alan Steven
11.1 Problematic Reagent Combinations per Structural Alert 323
11.1.1N-Nitroso Compounds (COC) 323
11.1.1.1 Amines and Nitrosating Agents [10] 323
11.1.1.2 Amine Derivatives and Nitrosating Agents 324
11.1.1.3 Other 324
11.1.2 Alkyl-azoxy Compounds (COC) 325
11.1.2.1 Reduction [5254] 325
11.1.2.2 Oxidation 325
11.1.2.3 Others 325
11.1.3 Other N-O Compounds 326
11.1.3.1 Reduction of Nitro Groups 326
11.1.3.2 Oxidation of Amines and Hydroxylamines 326
11.1.4 Nitration 326
11.1.5 Other N-N Compounds [59, 60] 326
11.1.6 Aflatoxin-like Compounds [62] (COC) 327
11.1.7 Dioxin-like Compounds (Including Polychlorinated Biphenyls = PCBs) [63] 327
11.1.8 Alkyl and Acyl Halides 327
11.1.8.1 ROH + HCl RCl + H2O 327
11.1.8.2 Ether Opening with Halides 328
11.1.9 Methyl Sulfoxides and Pummerer Rearrangement 328
11.1.10 Acyl Chlorides Formation [82] 329
11.1.11 Halogenation of Unsaturated Compounds 329
11.1.12 Ammonium Salts (Hofmann Elimination) 329
11.1.12.1 Alkyl Sulfonates [90] 329
11.1.13 Epoxides and Aziridines [9597] 330
11.2 Miscellaneous 331
11.2.1 B and P Based Compounds 331
11.2.2 Formation ofN-Methylol 331
11.2.3 Acetamide 332
11.2.4 Quinones and Quinone Derivatives 332
11.2.5 Anilines [100] 332
11.2.6 Michael Acceptors 333
11.2.7 Others 333
11.3 Mechanism and Processing Factors Affecting the Formation ofN-nitrosamines 333
11.3.1 Introduction 333
11.3.2 Mechanisms of Amine Nitrosation 333
11.3.2.1 Nitrosation of Secondary Amines 333
11.3.2.2 Aqueous Nitrosation 334
11.3.2.3 Nitrosation in Organic Solvents 336
11.3.3 Nitrosation of Tertiary Amines 337
11.3.3.1 Nitrosation of Quaternary Amines 337
11.3.3.2 Nitrosation of Amine Oxides 338
11.3.4 Sources of Nitrosating Agents 338
11.3.4.1 Process Water 338
11.3.4.2 Nitric Acid 339
11.3.4.3 Atmospheric Sources 339
11.3.4.4 Excipients Used in Drug Product Manufacture 340
11.3.4.5 Nitrocellulose 340
11.3.4.6 Nitrosating Agent Scavengers 340
11.3.4.7 Removal of Nitrosamines 341
11.4 Formation, Fate, and Purge of Impurities Arising from the Hydrogenation of Nitroarenes to Anilines 341
11.4.1 Primary Reaction Mechanism 341
11.4.2 Mass and Heat Transfer Effects 342
11.4.3 Condensation Chemistry 344
11.4.4 Factors Affecting Aryl Hydroxylamine Accumulation 346
11.4.5 Aryl Hydroxylamine Control 347
11.4.5.1 Use of Cocatalysts 347
11.4.5.2 Physical Adsorption 348
11.4.5.3 Kinetic Understanding Around Formation and Consumption 349
11.4.5.4 Holistic Control of Impurity Profile 349
11.4.6 Controlling Residual Nitroarene 351
11.4.7 Specific Considerations of Alkyl Nitro Reductions 353
11.4.8 Closing Comments on Hydrogenation of Nitroarenes to Anilines 353
11.5 Mechanism and Processing Parameters Affecting the Formation of Sulfonate Esters Summary of the PQRI Studies 353
11.5.1 Introduction 353
11.5.2 Reaction Mechanism 355
11.5.3 Experimental Results 357
11.5.3.1 Experimental Results from Study of the Ethyl Methanesulfonate (EMS) System 357
11.5.3.2 Other Methanesulfonic Acid Systems 359
11.5.3.3 Experimental Results from Study of the Isopropyl Methanesulfonate (IMS) System 360
11.5.4 Experimental Results from Study of Toluenesulfonic (Tosic) Acid Systems 361
11.5.4.1 Experimental Results from Study of the Ethyl Tosylate (ETS) System 362
11.5.4.2 Kinetic Modeling 363
11.5.4.3 Key Learnings and Their Implications for Process Design 365
11.5.4.4 Processing Rules 366
11.5.5 What About Viracept? 366
11.5.6 What About Other Sources of Sulfonate Esters? 367
11.5.7 Potential for Ester Formation in the Solid Phase 368
11.5.8 Conclusions 369
References 369
12 Strategic Approaches to the Chromatographic Analysis of Mutagenic Impurities381
Frank David, Gerd Vanhoenacker, Koen Sandra, Pat Sandra, Tony Bristow, and Mark Harrison
12.1 Introduction 381
12.2 Method Development and Validation 384
12.3 Analytical Equipment for Mutagenic Impurity Analysis 385
12.4 Alkyl Halides and Aryl Halides 388
12.4.1 Method Selection 388
12.4.2 Typical Conditions Used for Alkyl-and Aryl Halide Analysis by SHS-GC-MS and SPME-GC-MS 390
12.4.2.1 Sample Preparation 390
12.4.2.2 GC-MS Parameters 391
12.4.3 Typical Results Obtained for Alkyl-and Aryl Halide Analysis by SHS-GC-MS and SPME-GC-MS 391
12.5 Sulfonates 393
12.5.1 Method Selection 393
12.5.2 Typical Conditions Used for Sulfonate Analysis by Derivatization SHS-GC-MS 394
12.5.2.1 Sample Preparation 395
12.5.2.2 Synthesis of Deuterated Internal Standards 395
12.5.2.3 GC-MS Parameters 395
12.5.3 Typical Results Obtained Using Derivatization SHS GC-MS 395
12.5.4 Confirmation Analysis by PTV-GC-MS 396
12.6S-andN-mustards 398
12.6.1 Method Selection 398
12.6.2 Typical Analytical Conditions for the Analysis ofN-mustards by Derivatization SPME-GC-MS 399
12.6.2.1 Sample Preparation 399
12.6.3 Typical Results forN-mustards by Derivatization SPME-GC-MS 399
12.7 Michael Reaction Acceptors 400
12.7.1 Method Selection 400
12.7.2 Typical Analytical Conditions for Michael Reaction Acceptors 400
12.7.2.1 Sample Preparation 401
12.7.2.2 Parameters for SHS-GC-MS 401
12.7.2.3 Parameters for Liquid Injection and GC-MS with Back-flush 402
12.7.3 Typical Results Obtained for Trace Analysis of Michael Reaction Acceptors 402
12.7.3.1 SHS with PTV 402
12.7.3.2 Liquid Injection GC-MS 403
12.8 Epoxides 404
12.8.1 Method Selection 404
12.8.2 Typical Analytical Conditions for the Analysis of Volatile Epoxides by SHS-GC-MS 406
12.8.2.1 Sample Preparation 406
12.8.2.2 SHS-GC-MS Parameters 406
12.8.3 Typical Results Obtained for Volatile Epoxides Using SHS-GC-MS 407
12.9 Haloalcohols 407
12.9.1 Method Selection 407
12.9.2 Analytical Conditions for Trace Analysis of Halo-alcohols by Derivatization and Liquid Injection - 2DGC-MS 409
12.9.2.1 Sample Preparation 409
12.9.2.2 2D-GC-MS Parameters 410
12.9.3 Typical Results for Analysis of Halo-alcohols by Derivatization and Liquid Injection - 2DGC-MS 410
12.10 Aziridines 411
12.10.1 Method Selection 411
12.10.2 Typical Analytical Conditions for RPLC-MS and HILIC-MS Analysis of Aziridines 412
12.10.2.1 Sample Preparation 412
12.10.2.2 RPLC-MS Method Parameters 413
12.10.2.3 HILIC-MS Method Parameters 413
12.10.3 Typical Results Obtained for Aziridine Analysis Using RPLC and HILIC 413
12.11 Arylamines and Amino Pyridines 414
12.11.1 Method Selection 414
12.11.2 Typical Analytical Conditions for Arylamines and Aminopyridines by RPLC-MSD 415
12.11.2.1 Sample Preparation 415
12.11.2.2 HPLC-MS Parameters 416
12.11.3 Typical Results for Arylamines and Aminopyridines by RPLC-MSD 417
12.12 Hydrazines and Hydroxylamine 419
12.12.1 Method Selection 419
12.12.2 Analytical Conditions for the Analysis of Hydrazines Using Derivatization and HPLC-MS 420
12.12.2.1 Sample Preparation 421
12.12.2.2 HPLC-MS Parameters 421
12.12.3 Typical Results Obtained for Hydrazines Using Derivatization LC-MS 421
12.13 Aldehydes and Ketones 423
12.13.1 Method Selection 423
12.13.2 Typical Analytical Conditions for Analysis of Aldehydes and Ketones by DNPH Derivatization, Followed by LC-MS Analysis 423
12.13.2.1 Sample Preparation 424
12.13.2.2 Derivatization Reagent Solution 425
12.13.2.3 HPLC-MS Parameters 425
12.13.3 Typical Results Obtained for Aldehyde Analysis by DNPH Derivatization LC-MS 426
12.14 Nitrosamines 426
12.14.1 Method Selection 426
12.14.2 Sample preparation for SHS-GC-MS Analysis (according to ref [85]) 428
12.14.2.1 SHS-GC-MS Analysis [85] Sample Preparation 428
12.14.2.2 GC-MS (HRAM-MS) Conditions 428
12.14.2.3 UHPLC-MS Analysis 429
12.14.2.4 Sample Preparation for Hydrophilic Samples (e.g. Metformin) 429
12.14.2.5 Sample Preparation for Hydrophobic Matrices 430
12.14.2.6 UHPLC Conditions 430
12.14.2.7 HRAM-MS and MS/MS Conditions 430
12.14.3 Typical Results Obtained for VolatileN-nitrosamines Using SHS-GC-MS 430
12.14.4 Typical Results Obtained forN-nitrosamines Using LC-MS 431
12.15 Nontarget Analysis of PMI/MIs 434
12.16 Conclusions 435
Acknowledgements 436
References 436
13 Analysis of Mutagenic Impurities by Nuclear Magnetic Resonance (NMR) Spectroscopy439
Andrew R. Phillips and Stephen Coombes
13.1 Introduction to NMR 439
13.2 Why Is NMR an Insensitive Technique? 439
13.2.1 Nuclear Spin 439
13.2.2 Boltzmann Distribution 440
13.3 How Could NMR Be Used for Trace Analysis? 440
13.3.1 Generating an NMR Spectrum 440
13.3.2 Chemical Shift 442
13.3.3 Scalar Coupling 443
13.3.4 The Quantitative Nature of NMR 444
13.3.5 Relaxation 445
13.3.6 Summary 446
13.4 What Can Be Done to Maximize Sensitivity? 446
13.4.1 System Performance 447
13.4.1.1 Field Strength 447
13.4.2 Probe Performance 447
13.4.2.1 Probe Design 447
13.4.2.2 Probe Diameter 448
13.4.2.3 Cryogenically Cooled Probes 448
13.4.3 Substrate Concentration 449
13.4.4 Molecular Weight Ratio 451
13.4.5 Acquisition Time and Signal Averaging 451
13.4.6 Number of Protons and Linewidth 453
13.4.7 Resolution 455
13.4.8 Dynamic Range 455
13.4.8.1 Selective Excitation 458
13.4.8.2 Shaped Pulses 458
13.4.8.3 Quantification Using Selective Pulses 460
13.4.8.4 Excitation Sculpting 461
13.4.9 Limit Tests 461
13.4.9.1 Method Development 462
13.4.9.2 Validation 463
13.4.9.3 Unresolved Signals 463
13.4.9.4 Rapid Analysis 464
13.4.10 Expanded Use of MI NMR Methodology 464
13.4.11 Summary 464
13.5 Case Studies 464
13.5.1 Case Study 1 An Aldehyde Functionalized MI 464
13.5.2 Case Study 2 Use of 19F NMR 466
13.5.3 Case Study 3 Epoxide and Chlorohydrin MIs 468
13.5.4 Case Study 4 Sulfonate Esters 469
13.5.5 Case Study 5 Limit Test for Poorly Resolved Signals 470
13.5.6 Case Study 6 Using NMR MI Methodology for Cleaning Validation 472
13.6 Conclusion 473
References 475
14 Addressing the Complex Problem of Degradation-Derived Mutagenic Impurities in Drug Substances and Products477
Steven W. Baertschi and Andrew Teasdale
14.1 Introduction 477
14.1.1 Background 477
14.2 Working Definitions 478
14.3 Challenges Associated with the Assessment of Risk Posed by (Potentially) Mutagenic Degradation Products 479
14.4 Risk Assessment Process for Mutagenic Degradants 479
14.4.1 Stability-Related MRA Process Overview 479
14.4.2 Stress Studies 480
14.4.3 Accelerated Stability Studies 480
14.4.4 Long-term ICH Stability Studies 481
14.4.5 Deciding Which Products to Include in the MRA 481
14.4.6 In Silico Tools for the Prediction of Potential Degradation Products 482
14.5 Using Stress Testing to Select Degradation Products for Identification 482
14.5.1 Approach 1: Criteria for Structure Identification After Observation in Accelerated and Long-term Stability Studies 483
14.5.2 Approach 2: Criteria for Structure Identification Through Use of an Algorithm in Stress Testing Studies 483
14.5.3 Approach 3: Structure Identification Through Use of Kinetic Equivalence and Scaled ICH Q3B Thresholds 485
14.5.3.1 Kinetic Equivalence 485
14.5.3.2 Scaled ICH Q3B Thresholds 486
14.6 Development Timeline Considerations 487
14.6.1 Drug Discovery Stage 487
14.6.2 Preclinical to Phases 1/2 487
14.6.3 Phase 3 to New Drug Application (NDA) Regulatory Submission 488
14.6.4 Post-marketing/Line Extensions 488
14.7 Developing Control Strategies for (Potential) Mutagenic Degradation Products 488
14.7.1 Determining Relevancy of Potential Degradation Products and Developing Control Strategies for Actual Degradation Products 488
14.7.2 Accelerated Stability (40 °C/75% RH Six months) or Kinetic Equivalent 489
14.7.3 Photostability Studies 489
14.7.4 Degradation Chemistry Knowledge 490
14.8 Risk Assessment Process Illustrated 491
14.8.1 Case Study #1: Molecule A 491
14.8.2 Case Study #2: Galunisertib 492
14.8.3 Case Study #3: Naloxegol 494
14.8.4 Case Study #4: Selumetinib Side Chain 496
14.9 Significance of the Risk of Forming Mutagenic Degradation Products 498
14.9.1 Frequency of Alerting Structures in Degradation Products 498
14.10 Degradation Reactions Leading to Alerting Structures in Degradation Products 499
14.10.1 Frequency of Alerting Structures Giving Rise to Ames Positive Tests 503
14.10.2 Mutagenic Degradation Products: Overall Predicted Frequency 503
14.11 N-Nitrosamines: Special Considerations 503
14.11.1 Evaluation of Potential Formation of N-Nitrosamines in Drug Product 504
14.12 Conclusions 506
References 507
Index 513
