Mutagenic Impurities

Andrew Teasdale, PhD, is a senior principal scientist with AstraZeneca and a member of ICH Q3C, Q3D, Q3E Expert working groups as well as an industry advisor to ICH M7. He received his doctorate in organic chemistry from Durham University. He is the inventor of the purge factor concept applied to risk assessment of mutagenic impurities and has authored over 30 papers on that subject.

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 3
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 Risk 21
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 Impurities 57
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 Mutagenicity 87

4 Use of Structure–Activity Relationship (SAR) Evaluation as a Critical Tool in the Evaluation of the Genotoxic Potential of Impurities 89
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 Structure–Activity Relationships ((Q)SAR) for Mutagenicity 115
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 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 139
Andrew 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 165
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 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 213
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

8.1.3 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 235
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

10 N-Nitrosamines 269
Andrew 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 321
Lucie 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 381
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

12.6 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 439
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 Products 477
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