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

1. Trace Analysis of Volatile and Semi-Volatile
Genotoxic Impurities in Drug Substances and
Drug Products at Roche Palo Alto
Richard 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 Action
7/18/2011 Richard E. Young 3

4. Structural Alerts for Mutagenicity
Aromatics Group Alkyl and Aryl Group Heteroatomic Group
OH O O
N ewg
A A H A A
O
H
OH N P
A OR
N
N A A A A A
O ( O )1,2
O A S
O C (or S)
OR
N
A NO
+ A R X
N
O NO2 N N
A A
A
A
N O X A
A
A X
O NH2
S or N A = aryl, alkyl, or H; X = halogen
ewg = e- withdrawing group (e.g., CO, CN)
7/18/2011 Richard E. Young 4

5. Classification of Impurities
Class 1: Known to be genotoxic and carcinogenic
Class 2: Known to be genotoxic
Unknown carcinogenic potential
Class 3: Alerting structure unrelated to parent API
Unknown genotoxic potential
Class 4: Alerting structure related to the parent API
Class 5: No alerting structure
No indication of genotoxic potential
7/18/2011 Richard E. Young 5

6. Regulatory Guidance Documents for
Genotoxic Impurities
• FDA Draft Guidance - “Guidance for Industry Genotoxic and
Carcinogenic 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 Solvents
7/18/2011 Richard E. Young 6

7. Genotoxic Impurity Threshold of Toxicological
Concern (TTC)
• 1.5 μg/person/day: 1:100,000 lifetime risk of cancer
(provided there is an expected over-riding benefit of drug)
• 0.15 μg/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 decrease
7/18/2011 Richard E. Young 7

8. Genotoxic Impurity Threshold of Toxicological
Concern (TTC)
“Threshold of Toxicological Concern (TTC) of 1.5 μg/day for
lifetime, below which a daily intake of a genotoxic impurity with
unknown carcinogenic potential is unlikely to exceed a lifetime
cancer risk of one additional case in a population of 100,000
people.”
(The European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP)
draft “Guideline On The Limits of Genotoxic Impurities”)
7/18/2011 Richard E. Young 8

9. Acceptable Qualification for Staged TTC of
Genotoxic 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 days 1 mo – 3 mo – 6 mo –
< 14 days > 12 mo
– 1 mo 3 mo 6 mo 12 mo
Genotoxic and
Carcinogenic
120a 60a 20a 10a 5a 1.5b
Impurity Threshold
(μg/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)
7/18/2011 Richard E. Young 9

10. Analytical Implications for the Acceptable
Qualification Thresholds
Daily Dose of API (mg) 0.10 1.0 10 100 1000
Concentration of Impurity 1.5% 0.15% 150 ppm 15 ppm 1.5 ppm
Analytical Technique LC/UV & GC/FID LC/MS & GC/MS
7/18/2011 Richard E. Young 10

11. Example 1: Isopropyl Chloride in a Drug
Substance and Drug Product for Hepatitis C
• Potential problem of isopropyl chloride formation identified
at 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 or
excipients
– Not identified as an impurity in the excipients used in the
drug product tablets
7/18/2011 Richard E. Young 11

12. The Source of the Genotoxic Impurity
Isopropyl Chloride
• Isopropyl chloride could be introduced at the salt
formation step where isopropanol and hydrochloric acid
were used
OH Cl
+ HCl + H 2O
Isopropanol Isopropyl Chloride
• Isopropyl chloride could form on standing and be incorporated
into the drug substance’s crystal matrix
7/18/2011 Richard E. Young 12

13. Isopropyl Chloride Calibration Standard:
Mass Spectra
Cl 43
43
57
63
M+•
M+•
isopropyl chloride n-pentane (internal standard)
7/18/2011 Richard E. Young 13

14. Isopropyl Chloride Standard Chromatograms:
SIM-GC/MS and GC/FID
n-pentane (IS) n-pentane (IS) Isopropyl chloride
Isopropyl chloride
m/z 43 SIM-GC/MS(EI) Chromatogram GC/FID Chromatogram
7/18/2011 Richard E. Young 14

15. Temperature and Sonication Effects on
Isopropyl Chloride Recoveries
Room Temp Room Temp 4 °C 4 °C
Sample ID No Sonication Sonication No Sonication Sonication
Unspiked ND* ND ND ND
Spike (10-ppm) 78.6 85.9 94.5 95.2
Spike (50-ppm) 78.0 82.9 87.3 96.5
* ND – not detected.
7/18/2011 Richard E. Young 15

16. Isopropyl Chloride Linearity:
SIM-GC/MS and GC/FID
70
60
Determined conc. (ppm)
50
SIM-GC/MS
40
30 Slope = 1.39
20 Intercept =-0.157
10
r2 = 0.999
0
0 10 20 30 40 50
Actual concentration (ppm)
50
Determined conc. (ppm)
40
30
GC/FID
20
Slope = 0.967
Intercept =-0.686
10
r2 = 0.999
0
0 10 20 30 40 50
Actual concentration (ppm)
7/18/2011 Richard E. Young 16

17. Isopropyl Chloride Spike Statistics:
SIM-GC/MS and GC/FID
SIM-GC/MS GC/FID
Spike Determined Determined
Stnd.
Conc. Mean Conc. %RSD Mean Conc. Stnd. Dev. %RSD
Dev.
(ppm) (ppm) (ppm)
0.00 0.00 0.258* 4.4* 0.00 0.315* 6.6*
2.00 2.67 0.267 3.2 1.63 0.134 8.2
5.00 6.96 0.148 1.2 4.16 0.307 3.4
10.0 14.1 0.257 1.3 9.24 0.187 1.3
25.0 33.4 0.231 0.6 22.0 0.447 1.7
50.0 69.6 0.408 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.
7/18/2011 Richard E. Young 17

18. SIM-GC/MS Isopropyl Chloride Corrected
Percent Recovery Formula
SMPAMT
CORAMT = 
SLOPE
• CORAMT is the corrected amount of isopropyl chloride in the
sample.
• SPKAMT is the amount of isopropyl chloride spiked into the
sample.
• SLOPE is the slope of the regression line of the Spiked Sample
isopropyl chloride concentration versus isopropyl chloride’s
actual concentration.
7/18/2011 Richard E. Young 18

19. Isopropyl Chloride Recoveries:
SIM-GC/MS and GC/FID
SIM-GC/MS GC/FID
Spike Determined Determined
Percent Percent
Concentration Mean Conc. Mean Conc.
Recovery Recovery
(ppm) (ppm)* (ppm)
2.00 1.92 96.0 1.63 81.5
5.00 5.01 100 4.16 83.2
10.0 10.1 101 9.24 92.4
25.0 24.0 96.0 22.0 88.0
50.0 50.1 100 47.9 95.8
* Calculated using the SIM-GC/MS correction formula.
7/18/2011 Richard E. Young 19

20. SIM-GC/MS Isopropyl Chloride Uncorrected
and Corrected Recoveries
Spike Uncorrected Uncorrected Corrected Corrected
Conc. Determined Percent Determined Percent
(ppm) Conc. (ppm) Recovery Conc. (ppm) Recovery
2.00 2.67 134 1.92 96.0
5.00 6.96 134 5.01 100
10.0 14.1 141 10.1 101
25.0 33.4 134 24.0 96.0
50.0 69.6 139 50.1 100
7/18/2011 Richard E. Young 20

21. Example 2: 1-(3-Chloro-Propane-1-Sulfonyl)-4-
Methyl-Piperazine
O O
H O O Cl S
N
N
N + Cl S
CH3 Cl N
CH3
1-(3-Chloro-Propane-1-
Sulfonyl)-4-Methyl-Piperazine
1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine is a potential
side reaction in the sythetic scheme for a drug for overatcive bladder
5HT4 antagonist
7/18/2011 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 Analyte
O O Mass Spectrum of the Analyte
Cl S
N
99 N
CH3
M+•
7/18/2011 Richard E. Young 22

23. Linearity & LOD/LOQ Determination:
1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine
5000
Linearity Determination
Peak response (area)
4000
3000 Slope = 105
2000
Intercept =-89.6
1000
r2 = 0.999
0
0 10 20 30 40 50
Analyte concentration (ppm)
600
LOD/LOQ Determination
Peak signa- to-noise ratio
500
400
Slope = 12.5
300 LOD = 1.66 ppm
Intercept =-18.7 LOQ = 2.30 ppm
200
100
r2 = 0.995
0
0 10 20 30 40 50
Analyte concentration (ppm)
7/18/2011 Richard E. Young 23

24. Accuracy & Precission:
1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine
Replicate Determined Conc. (ppm) Difference Percent Difference
1 19.5 -0.5 -2.5
2 19.2 -0.8 -4.0
3 18.7 -1.3 -6.6
4 20.7 0.7 3.4
5 18.7 -1.3 -6.3
6 18.4 1.6 -7.8
Mean 19.2
Stnd Dev 0.82
% RSD 4.3
7/18/2011 Richard E. Young 24

25. Example 3: 2-Chloro-1-Butene and
4-Chloro-1-Butanol
2-chloro-1-butene 2-chlorobutane
2-chlorobutane (IS)
4-chloro-1-butanol
2-chlorobutane (IS)
2-Chlorobutane (IS) 2-Chloro-1-butene 2-Chlorobutane (IS) & 4-Chloro-1-butanol
m/z 90 m/z 56 m/z 90
7/18/2011 Richard E. Young 25

26. Mass Spectra:
2-Chloro-1-Butene & 4-Chloro-1-Butanol
2-Chlorobutane (IS)
Cl
2-Chloro-1-butene
Cl
M+•
4-Chloro-1-butanol
Cl
HO
M+•
7/18/2011 Richard E. Young 26

27. Linearity: 2-Chloro-1-Butene
25 Actual Mean*
Spike Conc. Determined %RSD*
20
(ppm) Conc. (ppm)
Determined conc. (ppm)
15
0.50 0.54 4.8
10 1.00 1.07 3.8
5 5.00 4.95 1.9
0 10.0 10.0 0.4
0 5 10 15 20 25
Actual concentration (ppm) 25.0 25.5 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
Actual Mean*
20
Spike Conc. Determined %RSD*
(ppm) Conc. (ppm)
Determined conc. (ppm)
15
0.50 0.84 5.0
10 1.00 0.95 9.1
5 5.00 4.83 3.6
0
10.0 9.78 3.4
0 5 10 15
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. Young 28

29. Limits of Detection and Quantitation:
2-Chloro-1-Butene and 4-Chloro-1-Butanol
Limit of Detection
Analyte Name Limit of Detection (ppm) Upper Limit (ppm)
2-Chloro-1-butene 0.2 0.3
4-Chloro-1-butanol 0.4 0.5
Limit of Quantitation
Limit of
Lower Limit Upper Limit
Analyte Name Quantitiation
(ppm) (ppm)
(ppm)
2-Chloro-1-butene 0.8 0.7 0.8
4-Chloro-1-butanol 1.3 1.2 1.4
7/18/2011 Richard E. Young 29

30. Example 4: Formaldehyde by
Headspace-GC/MS(EI)
m/z 30 Extracted Ion Chromatogram of Formaldehyde
7/18/2011 Richard E. Young 30

31. Drug Substance Breakdown Under GC
Conditions: Formaldehyde & Succinic Anhydride
succinic anhydride
O M+•
H H
formaldehyde
O O O
formaldehyde succinic anhydride
M+•
Total Ion Chromatogram of Formaldehyde
and Succinic Anhydride
7/18/2011 Richard E. Young 31

32. Formaldehyde Linearity:
Determined vs Actual Concentrations
45
Actual Spike Determined Percent
Determined formaldehyde conc (ppm)
40
35 Conc. (ppm) Conc. (ppm) Recovery
30
25 9.00 8.58 95.4
20
15.0 17.9 119
15
10 21.0 23.3 111
5
30.0 30.0 100
0
0 5 10 15 20 25 30 35 40 45 45.0 44.5 98.9
Actual formaldehyde concentration (ppm)
Slope = 0.984; Intercept = 1.04; r2 = 0.991
7/18/2011 Richard E. Young 32

33. Limits of Detection and Quantitation:
Formaldehyde
Limit of Detection
Limit of Detection (ppm) Upper Limit (ppm)
3.8 4.8
Limit of Quantitation
Limit of Quantitiation (ppm) Lower Limit (ppm) Upper Limit (ppm)
12.8 11.8 13.7
7/18/2011 Richard E. Young 33

34. Example 5: Sulfolane:
Chromatogram and Mass Spectra
Br
M+•
O
4-Bromomethyltetrahydropyran (IS) Mass Spectrum
O O
S
M+•
m/z 41 Extracted Ion Chromatogram Sulfolane Mass Spectrum
7/18/2011 Richard E. Young 34

35. Sulfolane Linearity:
Determined vs Actual Concentrations
350
Mean*
300 Actual Spike
Percent % RSD*
Conc. (ppm)
Recovery
Determined conc. (ppm)
250
200 20.0 93.9 1.6
150
50.0 91.2 2.2
100
100 94.6 2.9
50
0 250 92.9 1.7
0 100 200 300 400 500
Actual concentration (ppm) 500 100 2.0
* The mean and %RSD are based on six replicate determinations.
Slope = 0.665; Intercept = 2.92; r2 = 0.999
7/18/2011 Richard E. Young 35

36. Example 6:
N-(2-Iodo-Ethyl)-Methanesulfonamid
ClSO2Me NaI
H O H O
CH2Cl2 MEK
NH2 ● HCl N S CH3 N S CH3
Cl 69% 48%
Cl O I O
A B C
The source of the genotoxic impurity “Intermediate C” in a potential
incontinence drug substance
7/18/2011 Richard E. Young 36

37. “Intermediate C” and Breakdown Product:
EI Chromatograms and Mass Spectra
N-(2-iodo-ethyl)-methanesulfonamid
N-vinyl-sulfonamide
N-(2-iodo-ethyl)-methanesulfonamid
N-vinyl-sulfonamide
N-(2-chloro-ethyl)-methanesulfonamid N-(2-chloro-ethyl)-methanesulfonamid
79 N-vinyl-sulfonamide H O
H O N S CH3
I O
H 2C N S CH 3
122
O
N-(2-iodo-ethyl)-methanesulfonamid
42
M+•
7/18/2011 Richard E. Young 37

38. “Intermediate C” and Breakdown Product: CI
Chromatogram and Mass Spectra
H O [M+H]+
N-(2-iodo-ethyl)-methanesulfonamid
H 2C N S CH 3
O
N-vinyl-sulfonamide
N-vinyl-sulfonamide
H O N-(2-iodo-ethyl)-methanesulfonamid
N S CH3
I O
[M+H]+
N-(2-chloro-ethyl)-methanesulfonamid
7/18/2011 Richard E. Young 38

39. Acknowledgements
The Roche Palo Alto Genotoxic Impurities Assessment Group
 Keshab Sarma, Gary Cooper, Colin Beard (Process Chemistry)
 Yvonne Walbroehl (Process Analytical Support)
 Michael Brandl, Fujun Li, Tom Alfredson (Pharmaceutics)
 Sushmita Chanda, Stefan Platz, Kyle Kolaja (Toxicology)
 Amid Salari, Richard Young, Joe Pease (Analytical Research)
 Richard Daley, Paul Kopeck (Regulatory Affairs)
 Al Holstein (Quality Assurance)
7/18/2011 Richard E. Young 39

40. Questions
7/18/2011 Richard E. Young 40