Genotoxic Impurities

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Trace Analysis of Volatile and Semi-Volatile Genotoxic Impurities in Drug Substances and Drug Products at Roche Palo Alto

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  • 1. Trace Analysis of Volatile and Semi-VolatileGenotoxic Impurities in Drug Substances and Drug Products at Roche Palo AltoRichard E. Young Research Scientist II Analytical Research Roche Palo Alto LLC

2. Agenda Genotoxic Impurities Background Potential Sources Structural Alerts Classification of Types and Regulatory Guidance Threshold of Toxicological Concern Examples Isopropyl Chloride, 1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine, 2-Chloro-1-Butene, 4-Chloro-1-Butanol, Formaldehyde, Sulfolane, N-(2-Iodo-Ethyl)Methanesulfonamid Conclusion and Questions 3. Potential Sources of Genotixic Impurities Synthetic Pathway Raw Materials Intermediates Reagents, Solvents Side Reactions Degradation Products Originating from the Drug Substance Originating from the Drug Product- Originating from Microbiological Action7/18/2011Richard E. Young 3 4. Structural Alerts for Mutagenicity Aromatics Group Alkyl and Aryl GroupHeteroatomic GroupOH O ON ewg A A H A AO H OHNPA OR NNA A A A AO ( O )1,2OAS O C (or S)OR N A NO + A RXN O NO2 N N A A AANO XA A AX O NH2 S or NA = aryl, alkyl, or H; X = halogen ewg = e- withdrawing group (e.g., CO, CN)7/18/2011Richard E. Young4 5. Classification of ImpuritiesClass 1: Known to be genotoxic and carcinogenicClass 2: Known to be genotoxic Unknown carcinogenic potentialClass 3: Alerting structure unrelated to parent API Unknown genotoxic potentialClass 4: Alerting structure related to the parent APIClass 5: No alerting structure No indication of genotoxic potential7/18/2011 Richard E. Young5 6. Regulatory Guidance Documents forGenotoxic Impurities FDA Draft Guidance - Guidance for Industry Genotoxic andCarcinogenic Impurities in Drug Substances and Products:Recommended Approaches (FDA, Dec 2008) ICH Q3A(R) - New Drug Substances ICH Q3B(R) New Drug Products ICH Q3C Guideline for Residual Solvents7/18/2011Richard E. Young 6 7. Genotoxic Impurity Threshold of ToxicologicalConcern (TTC) 1.5 g/person/day: 1:100,000 lifetime risk of cancer(provided there is an expected over-riding benefit of drug) 0.15 g/person/day: 1:1,000,000 lifetime risk of cancer EPA recommends an adjustment factor for children Ages 0 to 2:10-fold exposure level decrease Ages 2 to 16: 3-fold exposure level decrease7/18/2011Richard E. Young 7 8. Genotoxic Impurity Threshold of ToxicologicalConcern (TTC)Threshold of Toxicological Concern (TTC) of 1.5 g/day forlifetime, below which a daily intake of a genotoxic impurity withunknown carcinogenic potential is unlikely to exceed a lifetimecancer risk of one additional case in a population of 100,000people.(The European Medicines Agencys Committee for Medicinal Products for Human Use (CHMP)draft Guideline On The Limits of Genotoxic Impurities)7/18/2011Richard E. Young 8 9. Acceptable Qualification for Staged TTC ofGenotoxic and Carcinogenic Impurities A Staged Threshold of Toxicological Concern approach is used where the acceptable daily intake of the genotoxic impurity varies Duration of Clinical Trial Exposure 14 days1 mo 3 mo 6 mo < 14 days > 12 mo 1 mo3 mo 6 mo12 mo Genotoxic and Carcinogenic 120a60a 20a10a 5a1.5b Impurity Threshold (g/day) a The probability of not exceeding 10-6 is 93%. b The probability of not exceeding 10-5 is 93%.Guidance for Industry Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches (FDA, Dec 2008) 7/18/2011Richard E. Young9 10. Analytical Implications for the AcceptableQualification ThresholdsDaily Dose of API (mg)0.10 1.010 1001000Concentration of Impurity 1.5%0.15% 150 ppm 15 ppm 1.5 ppm Analytical TechniqueLC/UV & GC/FID LC/MS & GC/MS7/18/2011Richard E. Young10 11. Example 1: Isopropyl Chloride in a DrugSubstance and Drug Product for Hepatitis C Potential problem of isopropyl chloride formation identifiedat beginning of the Phase I Clinical Trial Isopropyl chloride is a Class 2 impurity From where did the Isopropyl Chloride Impurity Arise? Not a starting material in synthesis of the drug substance Not a degradation product of the drug substance orexcipients Not identified as an impurity in the excipients used in thedrug product tablets7/18/2011 Richard E. Young11 12. The Source of the Genotoxic ImpurityIsopropyl Chloride Isopropyl chloride could be introduced at the saltformation step where isopropanol and hydrochloric acidwere used OHCl + HCl + H 2O IsopropanolIsopropyl Chloride Isopropyl chloride could form on standing and be incorporatedinto the drug substances crystal matrix7/18/2011Richard E. Young 12 13. Isopropyl Chloride Calibration Standard:Mass SpectraCl4343 57 63 M+M+isopropyl chloriden-pentane (internal standard)7/18/2011Richard E. Young 13 14. Isopropyl Chloride Standard Chromatograms:SIM-GC/MS and GC/FIDn-pentane (IS) n-pentane (IS) Isopropyl chloride Isopropyl chloridem/z 43 SIM-GC/MS(EI) ChromatogramGC/FID Chromatogram7/18/2011 Richard E. Young 14 15. Temperature and Sonication Effects onIsopropyl Chloride Recoveries Room TempRoom Temp4 C4 CSample ID No Sonication Sonication No Sonication SonicationUnspikedND*ND NDNDSpike (10-ppm)78.685.9 94.5 95.2Spike (50-ppm)78.082.9 87.3 96.5 * ND not detected.7/18/2011 Richard E. Young15 16. Isopropyl Chloride Linearity: SIM-GC/MS and GC/FID7060Determined conc. (ppm)50 SIM-GC/MS4030 Slope = 1.3920 Intercept =-0.15710 r2 =0.999 0 010 20304050Actual concentration (ppm)50Determined conc. (ppm)4030 GC/FID20 Slope =0.967 Intercept =-0.68610 r2 = 0.999 0 0 1020304050 Actual concentration (ppm) 7/18/2011Richard E. Young 16 17. Isopropyl Chloride Spike Statistics:SIM-GC/MS and GC/FIDSIM-GC/MS GC/FID Spike DeterminedDetermined Stnd. Conc. Mean Conc.%RSDMean Conc. Stnd. Dev. %RSD Dev. (ppm) (ppm) (ppm) 0.000.00 0.258* 4.4*0.000.315*6.6* 2.002.670.267 3.2 1.63 0.134 8.2 5.006.960.148 1.2 4.16 0.307 3.4 10.014.10.257 1.3 9.24 0.187 1.3 25.033.40.231 0.6 22.0 0.447 1.7 50.069.60.408 0.5 47.9 0.603 1.2* Isopropy chloride was present in SSS-0 at 5.80 mean ppm. The standard deviation and %RSD are based on the spiked and native amounts. 7/18/2011Richard E. Young17 18. SIM-GC/MS Isopropyl Chloride CorrectedPercent Recovery FormulaSMPAMT CORAMT= SLOPE CORAMT is the corrected amount of isopropyl chloride in thesample. SPKAMT is the amount of isopropyl chloride spiked into thesample. SLOPE is the slope of the regression line of the Spiked Sampleisopropyl chloride concentration versus isopropyl chloridesactual concentration.7/18/2011Richard E. Young18 19. Isopropyl Chloride Recoveries:SIM-GC/MS and GC/FIDSIM-GC/MS GC/FIDSpike DeterminedDeterminedPercentPercentConcentration Mean Conc.Mean Conc. Recovery Recovery(ppm) (ppm)*(ppm)2.001.92 96.01.6381.55.005.01 100 4.1683.210.010.1 101 9.2492.425.024.0 96.022.088.050.050.1 100 47.995.8 * Calculated using the SIM-GC/MS correction formula.7/18/2011Richard E. Young 19 20. SIM-GC/MS Isopropyl Chloride Uncorrectedand Corrected RecoveriesSpikeUncorrected UncorrectedCorrectedCorrectedConc.DeterminedPercent Determined Percent(ppm)Conc. (ppm)Recovery Conc. (ppm) Recovery2.002.67 1341.92 96.05.006.96 1345.01 10010.014.1 14110.1 10125.033.4 13424.0 96.050.069.6 13950.1 1007/18/2011 Richard E. Young 20 21. Example 2: 1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine O OH O OClSN NN + ClSCH3 Cl N CH3 1-(3-Chloro-Propane-1- Sulfonyl)-4-Methyl-Piperazine1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine is a potentialside reaction in the sythetic scheme for a drug for overatcive bladder5HT4 antagonist7/18/2011 Richard E. Young 21 22. Chromatogram and Mass Spectrum:1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine Extracted Ion Chromatogram (m/z 99) of the AnalyteO OMass Spectrum of the AnalyteClSN 99N CH3 M+7/18/2011 Richard E. Young22 23. Linearity & LOD/LOQ Determination:1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine5000 Linearity Determination Peak response (area)40003000 Slope =1052000 Intercept =-89.61000 r2 = 0.999 0 0 1020 30 40 50Analyte concentration (ppm) 600 LOD/LOQ Determination Peak signa- to-noise ratio 500 400 Slope =12.5 300LOD = 1.66 ppm Intercept =-18.7 LOQ = 2.30 ppm 200 100 r2 = 0.995 0 0 10 20 30 40 50Analyte concentration (ppm)7/18/2011 Richard E. Young 23 24. Accuracy & Precission:1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-PiperazineReplicate Determined Conc. (ppm)Difference Percent Difference119.5-0.5 -2.5219.2-0.8 -4.0318.7-1.3 -6.6420.70.73.4518.7-1.3 -6.3618.41.6-7.8 Mean19.2Stnd Dev 0.82% RSD4.37/18/2011Richard E. Young 24 25. Example 3: 2-Chloro-1-Butene and4-Chloro-1-Butanol2-chloro-1-butene2-chlorobutane2-chlorobutane (IS)4-chloro-1-butanol2-chlorobutane (IS)2-Chlorobutane (IS) 2-Chloro-1-butene2-Chlorobutane (IS) & 4-Chloro-1-butanolm/z 90 m/z 56m/z 907/18/2011 Richard E. Young 25 26. Mass Spectra:2-Chloro-1-Butene & 4-Chloro-1-Butanol 2-Chlorobutane (IS) Cl 2-Chloro-1-buteneCl M+ 4-Chloro-1-butanolClHO M+7/18/2011 Richard E. Young 26 27. Linearity: 2-Chloro-1-Butene 25ActualMean* Spike Conc. Determined %RSD* 20(ppm)Conc. (ppm)Determined conc. (ppm) 150.500.54 4.8 10 1.001.07 3.85 5.004.95 1.90 10.010.0 0.40510 15 20 25 Actual concentration (ppm) 25.025.5 2.2 Slope = 1.03; Intercept = - 0.483; r2 = 1.000 * The mean and %RSD are based on six replicate determinations. 7/18/2011 Richard E. Young 27 28. Linearity: 4-Chloro-1-Butanol 25 ActualMean* 20 Spike Conc. Determined%RSD*(ppm)Conc. (ppm)Determined conc. (ppm) 150.50 0.84 5.0 10 1.00 0.95 9.15 5.00 4.83 3.6010.0 9.78 3.4051015 Actual concentration (ppm)20 25 25.0 20.8 8.1 Slope = 0.771; Intercept = - 0.818; r2 = 0.985 * The mean and %RSD are based on six replicate determinations. 7/18/2011 Richard E. Young28 29. Limits of Detection and Quantitation:2-Chloro-1-Butene and 4-Chloro-1-ButanolLimit of Detection Analyte Name Limit of Detection (ppm) Upper Limit (ppm) 2-Chloro-1-butene 0.20.3 4-Chloro-1-butanol0.40.5Limit of QuantitationLimit of Lower Limit Upper Limit Analyte Name Quantitiation (ppm) (ppm) (ppm) 2