1. 신약 승인을 위한 Genotoxicity 불순물 관리 및 분석 김 훈 주 ( hoonjoo@cj.net)

2. 2 Contents  불순물 종류  일반불순물 관리 (ICH Q3A, Q3B,Q3C)  Genotoxic Impurities(GTIs) 관련 Guideline 의 역사  Genotoxic Impurities 관리 방법  Genotoxic Impurities 관리를 위한 분석방법  Genotoxic Impurities 관리를 위한 분석방법 개발 예

3. 3 Classification of Impurity  Organic Impurity Organic impurities can arise during the manufacturing process and/or storage of drug substance and drug product.  Inorganic Impurity Inorganic impurities can result from the manufacturing process.  Residual Solvent  Others

4. 4 Organic Impurity  Starting Material  By-Product  Intermediates  Degradation Products  Reagents  Ligands  Catalysts  포장제 추출물  발효 원료 물질  소포 제, 금속제거제  Detergents  세척제  포장제 추출물  Deamidation Protein  Oxidation Protein  Aggregation Protein

5. 5 Residual Solvent  Solvents are inorganic or organic liquids used as vehicles for the preparation of solutions or suspensions.  Since these are generally of known toxicity, the selection of appropriate controls is easily accomplished ( ICH Q3C)

6. 6 Enantiomeric Impurity  한 Isomer 만 독성이 있는 경우, 한쪽 Isomer 만이 효능을 지닌 경우가 존재.  Enantiomeric Impurity 분석을 Spec. 으로 하지 않더라도 초기에는 분석.  Routine 하게 분석하지 않는 근거를 마련하여 문서화.  분석법 개발이 중요.

7. 7 Others Impurity  Extraneous Contaminants : GMP Issues. 교차 요염 방지 대책, 세척, Cleaning Validation  Polymorphism XRD 등으로 분석, 안정성과 Dissolution 등에 영향  대사 체 비임상 시료에서의 대사 체와 임상 시료의 대사체가, 동일한 경우와 다른 경우

8. 8 Reporting 방법 /Organic impurity  Each specified identified impurity  Each specified unidentified impurity  Any unspecified impurity  Total impurities

9. 9 Drug substance Thresholds (ICH Q3A) Maximum Reporting Identification Qualification Daily Dose Threshold¹ Threshold² Threshold ≤2 g/day 0.05 % 0.10 % or 0.15 % or 1.0 mg 1.0 mg > 2g/day 0.03 % 0.05 % 0.05 % 1.Higher reporting thresholds should be scientifically justified 2.Lower thresholds can be appropriate if the impurity is unusually toxic

10. 10 Drug substance Thresholds  Qualification The process of acquiring and evaluating data that establishes the biological safety of an individual impurity or a given impurity profile at the level(s) specified

11. 11 Drug Product Reporting Thresholds (ICH Q3B) Maximum Daily Dose 1 Thresholds 2,3 < 1 g0.1 % > 1 g0.05 % 1.The amount of drug substance administrated per day 2.Thresholds for degradation products are expressed as a percentage of the drug substance or as total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the degradation product is unusually toxic 3.Higher thresholds should be scientifically justified.

12. 12 Drug Product Identification Thresholds (ICH Q3B) Maximum Daily Dose 1 Thresholds 2,3 < 1 mg1.0 % or 5 ug TDI, which is lower 1 mg ~ 10 mg0.5 % or 20 ug TDI, which is lower > 10 mg ~ 2 g0.2 % or 2 mg TDI, which is lower > 2 g0.10 %

13. 13 Drug Product Qualification Thresholds (ICH Q3B) Maximum Daily Dose 1 Thresholds 2,3 < 10 mg1.0 % or 50 ug TDI, which is lower 10 mg ~ 100 mg0.5 % or 200 ug TDI, which is lower > 10 mg ~ 2 g0.2 % or 3 mg TDI, which is lower > 2 g0.15 %

14. Genotoxic 불순물 관리 및 분석 I. GTI Regulation 역사 II.GTI 관리 방법 III.GTI 관리 사례 IV.GTI 분석 방법 및 사례

15. Genotoxic Impurity?  Compounds that have the potential to damage DNA at any level of exposure and that such damage may lead/contribute to turmour development.  Thus for genotoxic carcinogens it is prudent to assume there is no discernible threshold and that any level exposure carries a risk 15

16. 16 Genotoxic Impurity 관련 Guideline 역사  ICH S2A, Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (JUL. 1996)  Before genotoxcity became an ICH topic in 1992, a meeting of the European Federation of Pharmaceutical Industry Association (EFPIA) Genotoxicity Working Party identified more than 60 strategic and technical issues that differed in substance between the regulatory authorities of the USA, EU and Japan. Ref.) David J.Tweats, Impact of ICH guidelines on genotoxicity testing, PSTT, Vol. 1, No 5, 1998, 181~183

17. 17 Genotoxic Impurities Guideline  Guideline 예고  2002. Discussion in the safety working group  2006.11 AAPS, RS Section  Guidelines on the limits of genotoxic impurities  EMEA, 2006.06., 2007.01  FDA,2008.12.  Compounds that have been demonstrated to induce genetic mutations, chromosomal breaks, and/or chromosomal rearrangements are considered genotoxic and have the potential to cause cancer in humans.  Exposures to even low levels of these impurities may be of significant concern. Therefore, the identification limits provided in ICHQ3A and Q3B may not be acceptable for genotoxic impurities.

18. Genotoxic Impurity 관리 방법 Guided by existing genotoxicity data or the presence of structual alerts, potential genotoxic impurities should be identified. 1.Genotoxic compound with sufficient evidence for a threshhod related mechanism For compounds with clear evidence for threshold genotoxicity, exposure levels which are without appreciable risk of genotoxicity can be established according to the procedure as outlined for class 2 solvents in Q3C This approach calculates a “Permitted Daily Exposure” (PDE), which is deroved from the NOEL, or the lowest-observed effect level (LOEL) in the most relevant (animal) study using “uncertainty factor” 18

19. Genotoxic Impurity 관리 방법 2.Genotoxic compound without sufficient evidence for a threshhod related mechanism ① Pharmaceutical assessment A rationale of the proposed formulation/manufacturing strategy should be provided based on available formulation options and technologies The applicant should highlight, within the chemical process and imprutiy profile of active substance, all chemical substances, used as reagents, intermediates, or side-products, known as genotoxic and/or carcinogenic (potential, alerting structure) 19

20. 20 Genotoxic Impurity 관리 방법 2.Genotoxic compound without sufficient evidence for a threshhod related mechanism ① Pharmaceutical assessment Potential alternatives which do not lead to genotoxic residue in the final product, should be used if available A justification needs to be provided that no viable alternative exists, including alternative routes of synthesis or formulations, different starting material. If a genotoxic impurity is considered to be unaviadable in a drug substance, technical efforts(e.g. purification steps) shoud be undertaken to reduce the content of the genotoxic residues in the final products in complance with safety needs or to a level as low as reasonably practical (ALARP)

21. 21 Genotoxic Impurity 관리 방법 2.Genotoxic compound without sufficient evidence for a threshhod related mechanism ② Application of a Threshold of Toxicological Concern (TTC) A Threshold of Toxicological Concern (TTC) has been developed to difine a common exposure level for unstudied chemical that will not pose a risk of significant carcinogenicity ot other effects (Munro et al. 1999, Kroes and Kozianowski 2002)

22. 22 Genotoxic Impurity 관리 방법 2.Genotoxic compound without sufficient evidence for a threshhod related mechanism ② Application of a Threshold of Toxicological Concern (TTC) This TTC value was estimated to be 1.5 µg /person/day The TTC, originally developed as a “threshold of regulation” at the FDA for food-contact material (Rullis 1989, FDA 1995) was established based on the analysis of 343 carcinogens from a carcinogenic potency database (Gold et al 1984) The TTC was repeated confirmed by evaluations expanding the database to more than 700 carcinigens (Munro 1990, Cheeseman et al.1999, Kroes et al. 2004)

23. 23 Thresholds of Toxicological Concern (TTC)  Certain impurities with structural alerts suggesting particularly high genotoxic and carcinogenic potential would not be appropriate for this general threshold approach and would need to be evaluated on a case-by- case basis  0.15 µg per day may be more appropriate for chemicals with structural alerts for potential genotoxicity. Certain impurities containing certain functional group (N-nitoso-, aflatoxin-like-, and azoxy- ) that have extremely high carcinogenic potency are excluded from threshold approach. Ref) Kreos, Structure-based TTL, Food Chem Toxicol, 42, 2004, 65-83.

24. 24 Limit of genotoxic impurities  Concentration limits of genotoxic impurity in DS Concentration limit (ppm) = TTC (µg /day) / dose (g/day)  The TTC concept should not be applied to carcinogens where adequate toxicity data are available

25. 25 Genotoxic impurity Sufficient evidence for threshold-related mechanism of genotoxicity Test data indicate concern ( Ames test, structure) Presence of GTI unavoidable? Level as low as reasonably practical? Does intake exceed TTC of 1.5 µg /day ? Intake level > 1.5 µg /day acceptable? Calculate PDE: Safe exposure? Use alternative w/o genotoxic impurity Reduce to as low as resonably practical Negligible risk Restrict or reject applied use No further action Reduce to safe level Yes Negligible/accepta ble risk Yes No

26. 26 Recommended Approaches Based on Development Development Stage Recommended Approach IND Evaluate identified impurities for genotoxic and carcinogenic risk via SAR assessment Conduct assay for the presence of genotoxic and carcinogenic impurities If impurity with genotoxic and carcinogenic potential is identified:  Modify synthetic pathway to eliminate the impurity, if possible Or  Conduct genotoxicity assays to characterize the genotoxic potential if not already known AND/OR  Set spec. to that associated with a potential daily impurity exposure supported by compound-specific risk assessment or relevant TTL NDA, BLA or ANDA Evaluate identified impurities for genotoxic and carcinogenic risk via SAR assessment If impurity with genotoxic and carcinogenic potential is identified:  Conduct genotoxicity assays to characterize the genotoxic potential if not already known AND/OR  Set spec. to that associated with a potential daily impurity exposure supported by compound-specific risk assessment or 1.5 µg /day

27. 27 Acceptable Levels during Clinical Development Duration of Clinical Trial Exposure < 14 days 14 days ~ 1 mo. 1 mo ~ 3 mos 3 mos ~ 6mos 6 mos ~ 12mos > 12 mos Genotoxic and carcinogenic impurity threshold (µg /day ) 12060201051.5

28. 28 Structures of commonly encountered GTIs  Alkyl halides  Alkyl sulfonates  Hydrazine  Epoxide

29. 29 Structures of commonly encountered GTIs

30. 30 Perspective on existing pharmaceutical excipients for GTIs  Many existing expients(700 cpds) are foods, food additives or GRAS substances already approved for use by regulatory authorities.  There have been suggestions that existing excipients should also be subjected to the same procedures as API. Ref.) David J.Brusick, A pespective on testing of existing pharmaceutical excipients for GTIs, Regulatory Toxicology and Pharmacology, 55, 2009, 200~204.

31. 31 Perspective on existing pharmaceutical excipients for GTIs Table. Examples of the average daily intake of genotoxic/carcinogenic substances in the human diet FoodContaminantAverage Daily IntakeTest Potato chipsAcrylamide2.7 µgAmes CoffeeFurfural2.1 mgAmes MushroomHydrazinobenzoate28 µgAmes White breadFurfural500 µgChromosome aberrations Ref.) David J.Brusick, A pespective on testing of existing pharmaceutical excipients for GTIs, Regulatory Toxicology and Pharmacology, 55, 2009, 200~204.

32. 사례  Submission of the dossier: Acino Phatma GmbH on 19 Feb 2009 an application for MA to EMA for Chlopidogrel Hexal 75 mg film coated tablet, in accordance with CP.  Pharmacetical Development The potential risk of occurrence of contamination with benzene sulphonic acid methyl ester in chlopidogrel besilate was further elucidated in the documentation. A GC/MS method has been established and LOD of benzene sulphonic acid methyl ester is far below the TTC value of 1.5 µg /day intake of a genotoxic impurity. Results of batch analysis provided on several batches of chlopidogrel besilate from both active manufacturing sites, and the possible GTI in question was below LOD. 32

33. Genotoxic 불순물 분석 I. GTI 분석방법 ( Separation, 유도체 ) II.GTI 분석 사례 (GSK )

34. 34 Separation Method  GC: Volatile  Direct injection: large amount of non-volatile API can accumulate in the injector liner or on the head of GC column which can cause deterioration in the method performance (peak tailing, recovery, sensitivity)  Headspace injection: the sample is dissolved in a high boiling point solvent (water, DMSO) in a closed headspace vial. Upon heating, volatile analytes partition into the vial headspace during an incubation period and headspace vapor is sampled and injected into a GC  HPLC

35. 35 유도체법 ( Alkyl sulfonates)

36. 36 유도체법 (a)Derivatized with heptafluorobutyrylchloride. This led to the increase of the volatility of analyte as well as ECD sensitivity. (b) 2-Iodoethanol can react with trimethyl amine to produce a quaternary amine derivative, which can easily separated from API and detected by MS(0.1 ppm) (c) Aromatic amines are not retained on C18. The analyts were converted to hexylcarbamate derivatives (d) Chloroformates are very reactive and moisture sensitive cpds. It was observed that strong basic derivatization reagents caused decompostion. A less basic nucleophile, 2-mercaptopyridine could serve a proton sink to drive the reaction to completion.

37. 37 유도체법 (hydrazines) (a) HPLC/UV (b) HS-GC/MS

38. 38 유도체법 (epoxides) (a) VII was converted to a dimethyl amine derivative. The high affinitive derivative is an excellent candidate for ESI-MS analysis. LOD: 1 ng/ml (b) VIII, multiple products could be generated following aminolysis due to its multiple electrophilic site. Its multiple proximate oxygen atoms make it good candidate for ion spray MS method. It was found that analyte gave [M+K] + as the strongest species.

39. 39 GTI 분석 사례  A pracrical derivatization LC/MS approach for determination of trace level alkyl sulfonates genotoxic impurities in drug substances  Jianguo, J. Pharmaceutical and Biomedical Analysis, 48(2008), 1006~1010  GSK, Pharmaceutical Research & Development.

40. 40 GTI 분석 사례  Methanesulfonic acid(MSA) are commonly used acids for salt formation of API or employed as reagent in synthesis.  Methanol, ethanol, IPA are frequently used as solvents for crystallization or purification. Interacation between MSA and the alcohol could lead to the formation of their corresponding alkyl esters.  The presence of trace level of corresponding alkyl esters of MSA in drug substance or drug product is of genotoxicity.

41. 41 GTI 분석 사례

42. 42 GTI 분석 사례  Threshold of toxicological concern (TTC): 1.5 µg/day.  GC/MS For ppm-level detection, injection of very high concentration is unavoidable; thus direct injection GC methods suffer severe contamination.  LC/MS

43. 43 GTI 분석 사례  LC/MS: ionization

44. 44 GTI 분석 사례

45. 45 GTI 분석 사례

46. 46 GTI 분석 사례

47. 47 신약 승인을 위한 Genotoxicity 불순물 관리 및 분석 고맙습니다 !