Trace Analysis of Volatile and Semi-Volatile
Genotoxic Impurities in Drug Substances and
Drug Products at Roche Palo Alto
Richard E. YoungResearch Scientist II
Analytical Research
Roche Palo Alto LLC
Agenda
Genotoxic Impurities Background
Potential Sources
Structural Alerts
Classification of Types and Regulatory Guidance
Threshold of Toxicological Concern
Examples
Conclusion and Questions
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
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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
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Structural Alerts for Mutagenicity
NA
OH
N
A
O
A
N+
O
NA
A
POR
O
SOR
O( )1,2
Aromatics Group Heteroatomic Group
X
A H
O
AN
A
OH
AN
A
NO
ANO
2
O NH2
O
A
O
A A
NH
A A
O C
O
(or S)
N N
A A
A R
A
X
A = aryl, alkyl, or H; X = halogen
ewg = e- withdrawing group (e.g., CO, CN)
Alkyl and Aryl Group
N
X
S or
ewg
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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
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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• FDA Draft Guidance - “Guidance for Industry Genotoxic and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
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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
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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”)
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Acceptable Qualification for Staged TTC of
Genotoxic and Carcinogenic Impurities
Duration of Clinical Trial Exposure
< 14 days14 days
– 1 mo
1 mo –
3 mo
3 mo –
6 mo
6 mo –
12 mo> 12 mo
Genotoxic and
Carcinogenic
Impurity Threshold
(μg/day)
120a 60a 20a 10a 5a 1.5b
“Guidance for Industry Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches” (FDA, Dec 2008)
a The probability of not exceeding 10-6 is 93%. b The probability of not exceeding 10-5 is 93%.
A Staged Threshold of Toxicological Concern approach is used where the
acceptable daily intake of the genotoxic impurity varies
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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
LC/UV & GC/FID LC/MS & GC/MSAnalytical Technique
• 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
Example 1: Isopropyl Chloride in a Drug
Substance and Drug Product for Hepatitis C
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• Isopropyl chloride could be introduced at the salt
formation step where isopropanol and hydrochloric acid
were used
• Isopropyl chloride could form on standing and be incorporated
into the drug substance’s crystal matrix
The Source of the Genotoxic Impurity
Isopropyl Chloride
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OH Cl
HCl H2O+ +
Isopropanol Isopropyl Chloride
Isopropyl Chloride Calibration Standard:
Mass Spectra
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isopropyl chloride
Cl
n-pentane (internal standard)
M+•
M+•
63
4357
43
Isopropyl Chloride Standard Chromatograms:
SIM-GC/MS and GC/FID
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m/z 43 SIM-GC/MS(EI) Chromatogram GC/FID Chromatogram
n-pentane (IS)Isopropyl chloride
n-pentane (IS) Isopropyl chloride
Temperature and Sonication Effects on
Isopropyl Chloride Recoveries
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Sample ID
Room Temp
No Sonication
Room Temp
Sonication
4 °C
No Sonication
4 °C
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.
Isopropyl Chloride Linearity:
SIM-GC/MS and GC/FID
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0
10
20
30
40
50
60
70
0 10 20 30 40 50
Dete
rmin
ed
co
nc. (p
pm
)
Actual concentration (ppm)
SIM-GC/MS
Slope = 1.39
Intercept =-0.157
r2 = 0.999
GC/FID
Slope = 0.967
Intercept =-0.686
r2 = 0.9990
10
20
30
40
50
0 10 20 30 40 50Actual concentration (ppm)
Dete
rmin
ed
co
nc. (p
pm
)
Isopropyl Chloride Spike Statistics:
SIM-GC/MS and GC/FID
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SIM-GC/MS GC/FID
Spike
Conc.
(ppm)
Determined
Mean Conc.
(ppm)
Stnd.
Dev.%RSD
Determined
Mean Conc.
(ppm)
Stnd. Dev. %RSD
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.
SIM-GC/MS Isopropyl Chloride Corrected
Percent Recovery Formula
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• 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.
SMPAMT
SLOPE=CORAMT
Isopropyl Chloride Recoveries:
SIM-GC/MS and GC/FID
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SIM-GC/MS GC/FID
Spike
Concentration
(ppm)
Determined
Mean Conc.
(ppm)*
Percent
Recovery
Determined
Mean Conc.
(ppm)
Percent
Recovery
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.
SIM-GC/MS Isopropyl Chloride Uncorrected
and Corrected Recoveries
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Spike
Conc.
(ppm)
Uncorrected
Determined
Conc. (ppm)
Uncorrected
Percent
Recovery
Corrected
Determined
Conc. (ppm)
Corrected
Percent
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
Example 2: 1-(3-Chloro-Propane-1-Sulfonyl)-4-
Methyl-Piperazine
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N
NCH
3
H
S
OOCl
Cl
N
NCH
3
S
OOCl
+
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
N
NCH
3
S
OOCl
M+•
Chromatogram and Mass Spectrum:1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine
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Extracted Ion Chromatogram
(m/z 99) of the Analyte
Mass Spectrum of the Analyte
99
Linearity & LOD/LOQ Determination:1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine
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0
1000
2000
3000
4000
5000
0 10 20 30 40 50
Peak r
esp
on
se (
are
a)
Analyte concentration (ppm)
0
100
200
300
400
500
600
0 10 20 30 40 50
Analyte concentration (ppm)
Peak s
ign
a-
to-n
ois
e r
ati
o
Slope = 105
Intercept =-89.6
r2 = 0.999
Linearity Determination
Slope = 12.5
Intercept =-18.7
r2 = 0.995
LOD/LOQ Determination
LOD = 1.66 ppm
LOQ = 2.30 ppm
Accuracy & Precission:1-(3-Chloro-Propane-1-Sulfonyl)-4-Methyl-Piperazine
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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
Example 3: 2-Chloro-1-Butene and
4-Chloro-1-Butanol
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2-Chloro-1-butene2-Chlorobutane (IS) 2-Chlorobutane (IS) & 4-Chloro-1-butanol
m/z 90 m/z 56 m/z 90
2-chlorobutane (IS)
2-chlorobutane (IS)
2-chloro-1-butene 2-chlorobutane
4-chloro-1-butanol
Mass Spectra:2-Chloro-1-Butene & 4-Chloro-1-Butanol
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2-Chlorobutane (IS)Cl
2-Chloro-1-buteneCl
M+•
4-Chloro-1-butanol
OHCl
M+•
Linearity: 2-Chloro-1-Butene
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0
5
10
15
20
25
0 5 10 15 20 25
Dete
rmin
ed
co
nc. (p
pm
)
Actual concentration (ppm)
Actual
Spike Conc.
(ppm)
Mean*
Determined
Conc. (ppm)
%RSD*
0.50 0.54 4.8
1.00 1.07 3.8
5.00 4.95 1.9
10.0 10.0 0.4
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.
Linearity: 4-Chloro-1-Butanol
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Actual
Spike Conc.
(ppm)
Mean*
Determined
Conc. (ppm)
%RSD*
0.50 0.84 5.0
1.00 0.95 9.1
5.00 4.83 3.6
10.0 9.78 3.4
25.0 20.8 8.1
0
5
10
15
20
25
0 5 10 15 20 25
Dete
rmin
ed
co
nc. (p
pm
)
Actual concentration (ppm)
Slope = 0.771; Intercept = - 0.818; r2 = 0.985 * The mean and %RSD are based on six replicate determinations.
Limits of Detection and Quantitation:
2-Chloro-1-Butene and 4-Chloro-1-Butanol
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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
Analyte NameLimit of
Quantitiation
(ppm)
Lower Limit
(ppm)
Upper Limit
(ppm)
2-Chloro-1-butene 0.8 0.7 0.8
4-Chloro-1-butanol 1.3 1.2 1.4
Limit of Detection
Limit of Quantitation
Example 4: Formaldehyde by
Headspace-GC/MS(EI)
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m/z 30 Extracted Ion Chromatogram of Formaldehyde
Drug Substance Breakdown Under GC
Conditions: Formaldehyde & Succinic Anhydride
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Total Ion Chromatogram of Formaldehyde
and Succinic Anhydride
formaldehyde
succinic anhydride
H H
O M+•
formaldehyde
succinic anhydride
O OO
M+•
Formaldehyde Linearity:
Determined vs Actual Concentrations
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Actual Spike
Conc. (ppm)
Determined
Conc. (ppm)
Percent
Recovery
9.00 8.58 95.4
15.0 17.9 119
21.0 23.3 111
30.0 30.0 100
45.0 44.5 98.9
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20 25 30 35 40 45Actual formaldehyde concentration (ppm)
Dete
rmin
ed
fo
rma
lde
hyd
e c
on
c (
pp
m)
Slope = 0.984; Intercept = 1.04; r2 = 0.991
Limits of Detection and Quantitation:
Formaldehyde
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Limit of Detection (ppm) Upper Limit (ppm)
3.8 4.8
Limit of Quantitiation (ppm) Lower Limit (ppm) Upper Limit (ppm)
12.8 11.8 13.7
Limit of Detection
Limit of Quantitation
Example 5: Sulfolane:Chromatogram and Mass Spectra
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Sulfolane Mass Spectrum
S
OO
M+•
4-Bromomethyltetrahydropyran (IS) Mass Spectrum
O
BrM+•
m/z 41 Extracted Ion Chromatogram
Sulfolane Linearity:
Determined vs Actual Concentrations
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Slope = 0.665; Intercept = 2.92; r2 = 0.999
0
50
100
150
200
250
300
350
0 100 200 300 400 500
Actual Spike
Conc. (ppm)
Mean*
Percent
Recovery
% RSD*
20.0 93.9 1.6
50.0 91.2 2.2
100 94.6 2.9
250 92.9 1.7
500 100 2.0
* The mean and %RSD are based on six replicate determinations.
Actual concentration (ppm)
Dete
rmin
ed
co
nc. (p
pm
)
Example 6:
N-(2-Iodo-Ethyl)-Methanesulfonamid
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HClClNH
2 ● N S
H
CH3
O
OCl
N S
H
CH3
O
OI
ClSO2Me
CH2Cl2
NaI
MEK
69% 48%
A B C
The source of the genotoxic impurity “Intermediate C” in a potential
incontinence drug substance
“Intermediate C” and Breakdown Product:
EI Chromatograms and Mass Spectra
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N S
H
CH3
O
OI
122
N-(2-iodo-ethyl)-methanesulfonamid
M+•
N-vinyl-sulfonamide
N-(2-iodo-ethyl)-methanesulfonamid
N-(2-iodo-ethyl)-methanesulfonamid
N-vinyl-sulfonamide
N S
H
CH3
O
O
CH2
42
79 N-vinyl-sulfonamide
N-(2-chloro-ethyl)-methanesulfonamid N-(2-chloro-ethyl)-methanesulfonamid
“Intermediate C” and Breakdown Product: CI Chromatogram and Mass Spectra
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N S
H
CH3
O
O
CH2
N S
H
CH3
O
OI
[M+H]+
[M+H]+
N-vinyl-sulfonamide
N-(2-iodo-ethyl)-methanesulfonamid
N-vinyl-sulfonamide
N-(2-iodo-ethyl)-methanesulfonamid
N-(2-chloro-ethyl)-methanesulfonamid
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)
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Questions
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