List of Contributors xix Preface xxi Section 1 The Development of Regulatory Guidelines for Mutagenic/Genotoxic Impurities – Overall Process 1 1 Historical Perspective on the Development of the EMEA Guideline and Subsequent ICH M7 Guideline 3Andrew 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 Risk 21Andrew 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 Impurities 57Andrew 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 Mutagenicity 87 4 Use of Structure–Activity Relationship (SAR) Evaluation as a Critical Tool in the Evaluation of the Genotoxic Potential of Impurities 89Catrin 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 Structure–Activity Relationships ((Q)SAR) for Mutagenicity 115James 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 Negative In 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 Understanding In Vitro Mechanisms 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: Aromatic N-oxides 125 5.4 Expert Knowledge 127 5.5 Future Direction 129 References 131 Section 3 Toxicological Perspective on Mutagenic Impurities 137 6 Toxicity Testing to Understand the Mutagenicity of Pharmaceutical Impurities 139Andrew Teasdale, John Nicolette, Joel P. Bercu, James Harvey, Stephen Dertinger, Michael O’Donovan, and Christine Mee 6.1 Introduction 139 6.2 In Vitro Genotoxicity 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 6.3 In Vivo Mutation Assays 148 6.3.1 In Vivo Pig-a Gene 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 Pharmaceuticals 165Joel 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 Mutagenic In vitro but not In vivo 176 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 7.5.3 N-Nitrosamines 178 7.5.3.1 Regulatory Limits for N-Nitrosamines 178 7.5.3.2 Additional Proposed Limits for N-Nitrosamines 180 7.5.3.3 N-Nitrosamine Exposure in the General Population 181 7.5.3.4 Developing a Class-specific Limit for N-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 Departure 213George 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 8.1.3 In Vitro Experimental 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 Impurities 233 9 Mutagenic Impurities – Assessment of Fate and Control Options 235Michael 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 10 N-Nitrosamines 269Andrew Teasdale, Justin Moser, J. Gair Ford, and Jason Creasey 10.1 Background 269 10.2 Generation of N-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 to N-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 Quantify N-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 Impurities 321Lucie Lovelle, Andrew Teasdale, Ian Ashworth, Adrian Clarke, and Alan Steven 11.1 Problematic Reagent Combinations per Structural Alert 323 11.1.1 N-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 [52–54] 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 [95–97] 330 11.2 Miscellaneous 331 11.2.1 B and P Based Compounds 331 11.2.2 Formation of N-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 of N-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 Impurities 381Frank 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 12.6 S-and N-mustards 398 12.6.1 Method Selection 398 12.6.2 Typical Analytical Conditions for the Analysis of N-mustards by Derivatization – SPME-GC-MS 399 12.6.2.1 Sample Preparation 399 12.6.3 Typical Results for N-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 Volatile N-nitrosamines Using SHS-GC-MS 430 12.14.4 Typical Results Obtained for N-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) Spectroscopy 439Andrew 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 Products 477Steven 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

Learn to implement effective control measures for mutagenic impurities in pharmaceutical development In Mutagenic Impurities: Strategies for Identification and Control, distinguished chemist Andrew Teasdale delivers a thorough examination of mutagenic impurities and their impact on the pharmaceutical industry. The book incorporates the adoption of the ICH M7 guideline and focuses on mutagenic impurities from both a toxicological and analytical perspective. The editor has created a primary reference for any professional or student studying or working with mutagenic impurities and offers readers a definitive narrative of applicable guidelines and practical, tested solutions. It demonstrates the development of effective control measures, including chapters on the purge tool for risk assessment. The book incorporates a discussion of N-Nitrosamines which was arguably the largest mutagenic impurity issue ever faced by the pharmaceutical industry, resulting in the recall of Zantac and similar drugs resulting from N-Nitrosamine contamination. Readers will also benefit from the inclusion of: A thorough introduction to the development of regulatory guidelines for mutagenic and genotoxic impurities, including a historical perspective on the development of the EMEA guidelines and the ICH M7 guidelineAn exploration of in silico assessment of mutagenicity, including use of structure activity relationship evaluation as a tool in the evaluation of the genotoxic potential of impuritiesA discussion of a toxicological perspective on mutagenic impurities, including the assessment of mutagenicity and examining the mutagenic and carcinogenic potential of common synthetic reagents Perfect for chemists, analysts, and regulatory professionals, Mutagenic Impurities: Strategies for Identification and Control will also earn a place in the libraries of toxicologists and clinical safety scientists seeking a one-stop reference on the subject of mutagenic impurity identification and control.