Genotoxic impurities in

pharmaceuticals
Regulatory updates, Analytical Challenges
and Solutions

Ravikrishna Chebolu, Ph.D.

Pharma Segment Marketing Group

March 2015
 Disclaimer
The concepts and ideas expressed do not reflect official
company policies. The information and material contained in
this presentation is subject to change and not intended to be
exhaustive. Please always consult your regulatory team to
determine the applicability of anything contained herein.
The solutions and experimental data provided as part of this
presentation are for illustrative purposes and, depending on the
nature of each project, the approach and degree of
experimentation for a particular product or process may vary.

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Agenda

Impurities & Genotoxic impurities - fundamentals

ICH M7 guidance

Analytical challenges and solutions

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ICHQ11 Development and Manufacture of Drug Substances

Adopted November 2012

Manufacturing process development should

include the list of potential CQAs (Critical Quality
Attributes) properties that affect identity, purity,
biological activity and stability.

Impurities are an important class of potential

CQAs
Organic (including potential genotoxic)
impurities
Inorganic/elemental impurities
Residual solvents

ICH Q3A: Impurities in New Drug

Substances
ICH Q3B: Impurities in New Drugs
ICH Q3C: Residual solvents
ICH Q3D: Elemental Impurities
ICH : The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use

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Impurities in Pharmaceuticals

Raw Material (RM) /

Intermediate (IM) Drug Substance (DS)
Starting Material (SM)

SM and IM residues
Genotoxic impurities: Impurities in SM
Reagents, solvents and
Not addressed by generic ICH guidelines catalysts
Reaction by-products
(Q3A/B/C) because of exceptionally high
Degradation products
toxicity Excipient API interactions
Drug substance synthesis often requires Container closure interactions
the use of reactive materials which have Metabolites
the capacity to interact with human DNA
to cause mutations and cancer
Normal organic impurity in drug substance daily dose 2 g/day)
Level > 0.05%: reporting
Level > 0.10% or 1 mg/day (whichever is low): Identification
Level > 0.15% or 1 mg/day (whichever is low): qualification

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Genotoxic impurities - Guidelines

Guidance Milestone

EMA 2002 Reduce to as low as technically feasible

as low as technically feasible replaced by as low

EMA 2004
as reasonably practical

EMA 2004 TTC Concept introduced

Dr. Ludwig Huber
Dr. Ravikrishna Chebolu
Concept of flexible limits based on duration, but no
EMA 2004
numbers
PhRMA 2006 Published numbers for Staged TTC
Guidelines for five impurity classifications and
PhRMA 2006
controls
5991-1876EN
EMA 2006/7 Decision Tree Flow Charts

ICH M7 2014 Harmonization of guidelines (FDA, EMA)

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Genotoxicity?

Genotoxicity: potential to interact with genetic material, measurable DNA*

alteration or damages

DNA reactive
Clastogenicity: Breakage in
chromosomes leading to loss or
rearrangement of chromosome
segments
Mutagenicity: potential to change
genetic properties, causing Aneugenicity: Change in the number
heritable, genetic mutations (gain or loss) of chromosomes)

*DNA: Deoxyribonucleic acid-molecule that encodes the genetic instructions used in the development and functioning of all known living organisms
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ICH M7 Assessment and control of Mutagenic impurities

Implementation in ICH
Regions US, Europe and
Japan expected by
January 2016

Jun 2014

Nov 2012
2010
Formal ICH Procedure
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ICH M7 - Scope

.is about DNA-reactive (mutagenic) impurities only (genotoxicity due to other reasons not discussed)

Scope:
Recommends use
ALL New Drug Applications for small of science and risk
molecules after November 2015 based approach as
described in ICH
Changes in manufacturing, formulation etc. to Q10
CURRENT (marketed) small molecule drugs
that could affect impurities.

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ICH M7 - Scope

Not Covered but principles may apply

Biologicals, peptides, oligonucleotides, radiopharmaceuticals, fermentation products,
herbal products

Not applicable to
Drug substances and products for advanced cancer indications
Genotoxic drug substances

Excluded
Excipients used in existing marketed products and flavoring agents
Leachables associated with drug product packaging (safety risk assessment
principles may apply)

Always apply
principles of
Quality and Risk
management

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ICH M7 applicability to marketed products
Marketed Products need to be evaluated if:

New impurities or increased acceptance

Changes to drug substance synthesis
criteria for existing impurities

Changes in formulation, composition of New impurities or increased acceptance

manufacturing process criteria for existing impurities

Changes in indication or dosing Changes in levels of acceptable cancer

procedures risk levels

For Drug substance evaluation:

No need to re-examine the whole synthesis;
Change is assessed from the registered starting material onwards
For Drug product evaluation:
If no changes to drug substance, re-evaluation of drug substance not required

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ICH M7 Hazard assessment
Advocates the same approach as current guidelines:

Observed above ICH Q3A

reporting thresholds

Initial risk assessment based

Actual on literature and SAR
evaluation
Potential Classification of impurities
into 5 classes
Impurities

From knowledge and experiments

No need to attempt to identify every possible impurity at a TTC like level !

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Structural Alerts (genotoxic) for Impurities - Examples

Halides and haloalkenes (R-X)

Mustards (-S-)

Sulphonate esters

Aldehydes (RCHO)

Epoxides

Arylamines and aminopyridines

Boronic Acids

Aziridines

Michael acceptors

Hydrazines

Agilent Publication Number: 5990-5732EN

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ICH M7 Hazard assessment

Class Definition Proposed Action for Control

Experimental evidence for known Control at or below compound-specific
1
mutagenic carcinogens. acceptable limit
Known mutagens with
Control at or below acceptable limits
unknown carcinogenic potential
2 (generic or adjusted TTC)
(bacterial mutagenicity positive*, no
rodent carcinogenicity data)
Control at or below acceptable limits
Alerting structure, unrelated to the (generic or adjusted TTC) or do bacterial
3 structure of the drug substance; mutagenicity assay;
no mutagenicity data. If non-mutagenic = Class 5
If mutagenic = Class 2
Alerting structure, same alert in drug
4 substance which has been tested and is Treat as non-mutagenic impurity
non-mutagenic
No structural alerts, or alerting structure Treat as non-mutagenic impurity
5 with sufficient data to demonstrate lack
of mutagenicity

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ICH M7 Risk assessment

For Clinical trials and marketed products:

Acceptable intakes for an individual impurity

Acceptable intakes for multiple impurities

Only applies to impurities that are specified on drug substance specifications

Doesnt apply to degradants
Impurities with compound specific limits also treated individually

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Decision Tree for Assessment of Potential Genotoxic Impurities
Identify compounds of potential concern: No Indication of GIs.
Review of synthetic route, structural alerts, Treat as normal impurities, calculate
genotoxic data from experiments and literature PDE and reduce to safe levels.

Indication of Genotoxic impurities (GI) Yes

Can the GI be avoided by changing the Use Alternate synthetic route..
synthetic route or materials?

No
Reduce the level of GIs to as low as
reasonably practical by purification or other
modern techniques.

Determine if intake levels of GI exceed Within TTC limits:

Threshold of Toxicological Concern (TTC) Negligible risk.
of 1.5 g/day with suitable analytical methods.

Exceed TTC limits:

Risk/benefit analysis to justify restricting or
rejecting proposed use. Several Regulatory Clarifications issued
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ICH M7 Control of mutagenic impurities

4 options for Control Strategy:

(testing for impurity using an appropriate analytical procedure)

Raw Material (RM) /

Intermediate (IM) Drug Substance (DS)
Starting Material (SM)

Test in DS at or
Option 1: below acceptance
level

Option 2: Test in RM or SM or IM at or below acceptance level

Scientific
Option 3: Test in RM or SM or IM above acceptance level justification
for absence/
lower levels
Option 4: No testing required ! (Reactive/unstable) later in the
process
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Genotoxic impurities Analytical challenges
To be controlled at significantly lower levels; analytical
procedure should allow detection limits : 1-5 ppm (0.0001-
0.0005% w/w).
Require more sensitivity and high selectivity
Interference of large amounts of API with low levels of
impurities

Varied functional groups and come from different sources

Multiple analytical techniques/procedures may be
required

Reactive nature of genotoxic impurities makes sampling

difficult
Special precautions necessary to ensure integrity of
analytes

Depending on the nature and quantity of the genotoxic impurity being

investigated, an appropriate analytical technique (or a combination) needs
to be selected early in drug development
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Genotoxic impurities Analytical solutions

Analytical techniques used in genotoxic

impurity analysis
ICP-
Others OES/MS

MS/MS
GC

HPLC

* Current estimates (continuously changing)

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Genotoxic impurities Analytical solutions

Impurity Detection

CE
LC
GC

Impurity Separation

ICPMS SFC

Impurity isolation
SQ LC/MS
Prep LC/MS

Impurity identification
FTIR
GC/MS
QTOF LC/MS/MS

Impurity Quantification
QQQ LC/MS/MS
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Genotoxic impurities GC and GC/MS analysis

Halides, Haloalkenes,
sulphonate esters: GC is
the preferred technique

Examples:
Chloromethane , bromomethane
Benzyl halides
2-Bromoethanol, 2-iodoethanol
3-bromo-2-methyl-acrylonitrile

Reaction mechanisms for EMS formation from methane

sulphonic acid and ethanol (above) and EMS
Agilent GC- MSD system derivatisation reaction with pentafluorothiophenol (below).

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Genotoxic impurities (U)HPLC analysis

1290 Infinity II series LC

Most commonly used impurity analysis technique

in QC labs
High resolution is particularly helpful when using
LC/UV analysis for impurity detection, because all
impurities can be identified with less chance of
error.
HDR and 2D LC : very useful for complex impurity
analysis

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Genotoxic Impurity analysis HDR-DAD Impurity Analyzer System
Identification and quantification of trace level impurities in the presence of large amounts of drug
substances (in a single run)

Provides a 30-fold increase in linear dynamic range

compared with a traditional diode array detector.

Combines the signals from two diode array detectors

with different path length Max-Light flow cells.
1200 Infinity Series HDR-DAD Impurity Analyzer System

Agilent application
note 5991-4613EN
Omeprazole N-oxide impurity

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Quantitation of trace level genotoxic oxidative impurity
DAD1 A, Sig=280,4 Ref=off 60mm Flow cell API Blank 5L Without HDR.D
# Time Area Area% 5L injection volume
mAU API 12.771 Without HDR
No HDR 1200 1 5.148 2.2 0.008
mAU N-oxide Observation:
5L 0.3 Impurity 2 12.312 42 0.125 1) Main peak : Not
N-oxide 33643.
800 0.2 saturated
Impurity 0.1 3 12.681 6 99.869 2) Impurity: Trace, hard
400 0.0

12.310
5.148
to integrate
4 6 8 10 min
0
5 10 15 20 25 min
DAD1 A, Sig=280,4 Ref=off 60mm flow cell API Blank 20L Without HDR.D
mAU API 12.774 N-oxide Impurity # Time Area Area%
20L injection volume
No HDR 3000 Peak mAU 1 5.150 6.6 0.012 Without HDR
20L Saturation 0.6
2 12.317 136.3 0.239 Observation:
2000 N-oxide 0.2 1) Main peak : Saturated
0 3 12.774 56925.2 99.75
Impurity 2) Impurity: Clear
1000
12.317

-0.2
5.150

4 6 8 10 min
0
5 10 15 20 25 min
HDR1 A, Sig=280,4 Ref=off API Blank 20L With HDR
mAU/cm API 12.771 N-oxide # Time Area Area% 20L injection volume
800 mAU/cm Impurity
5.150 With HDR
With HDR 1 5.150 1 0.009
0.6 Observation:
600 N-oxide 2 12.311 18.3 0.151
20L 0.2 3 12.76 12106.6 99.841
1) Main peak : Not
400 Impurity Saturated
12.317

0 2) Impurity: Clear, Easy to

200
5.150

Integrate
4 6 8 10 min
0
5 10 15 20 25 min
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Genotoxic impurity analysis Quantitation by LC/MS/MS

1290 Infinity LC and 6460 QQQ MS/MS

Quantitation of nine arylamine and aminopyridine

potential genotoxic impurities at trace levels
(well below 1 ppm relative to the API)

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Genotoxic impurity analysis combination of techniques

Genotoxic impurities from degradation of

Agilent application
note 5991-4404EN

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Monitoring (genotoxic) degradants (U)HPLC
3% H2O2
Atorvastatin Degradation mixture
40oC, 24 hrs

1290 infinity LC

Agilent application note

5991-4404EN
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Identification of (trace) genotoxic degradants QToF MS/MS
3% H2O2
Atorvastatin Degradation mixture
40oC, 24 hrs

Structural Identification
using MSMS pattern and
MSC software

6530 QTOF LC/MS/MS

Impurities (I, II, and III) having alerting structures

(epoxide and hydro peroxide functional groups)
found in atorvastatin oxidation degradation
sample. Agilent application note
5991-4404EN
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Identification of (trace quantities of) genotoxic degradants:

Structural
Identification
using MSMS
pattern and MSC
software

Fragmentation pattern comparison of newly found alerting Impurity III having M: 465.4702 (bottom trace) with
known alerting Impurity II, M: 449.4708 (top trace) in negative mode.
Agilent application note
5991-4404EN
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Isolation of genotoxic degradants Prep LC/MS

3% H2O2
Atorvastatin Degradation mixture
40oC, 24 hrs

Isolation of
genotoxic
impurities of
interest
1290 Infinity LC + 6510 SQ with fraction
collection

Agilent application note

5991-4404EN

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Summary
1. Genotoxic impurities and ICHM7 guidance for 3. Analytical Challenges and solutions for
Hazard and Risk assessment genotoxic impurity analysis

2. Control Strategy for genotoxic impurities as per

ICHM7

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Agilent publications impurity analysis

5990-8620EN 5991-0090EN

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Agilent publications impurity analysis

Dr. Ludwig Huber and Dr. Ravi Chebolu

5991-1876EN

5991-2796EN

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