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• FDA Draft Guidance - “Guidance for Industry Genotoxic and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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