Impurity profiling in pharmaceuticals-

genotoxic impurities, a review

Presented by

Goutami Perala,

JNTUH

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A seminar on

Guided by Dr.M.Ajitha,CPS, JNTUH

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Agenda1.Impurity , classifiction1.Impurity , classifiction

5.Methodology5.Methodology

b)Modern approaches b)Modern approaches

6.A review on genotoxic profiling6.A review on genotoxic profiling

2.Genotoxic impurity2.Genotoxic impurity

3.Systematic approach3.Systematic approach

4. Decision tree4. Decision tree

a)Classical approaches a)Classical approaches

7.Case studies 7.Case studies

8.Conclusion8.Conclusion

Impurity ??Presence of any foreign matter or substance

which differs from the drug substance in terms of its structure, pharmacological, and toxicological effects.

Impurity profiling – what and why?

Genotoxic impurityChemical substances capable of causing direct or indirect

damage to DNA or Chromosomes and lead to change in the

expression of “Gene” thereby leading to Mutated Gene. Which may lead to formation of defective protein Create disorder in the metabolic processes Affect the DNA repair Mechanism

e.g. Alkylating agents, diols, epoxides, nitroso groups, lead, arsenic, etc.

Genotoxic profiling?why is it important?

TTC of a genotoxic impurity :ICH guideline - Threshold of

Toxicological Concern (TTC) is 1.5 μg/day intake of a genotoxic impurity.

In terms of relative concentration limits Concentration limit (ppm) = TTC [μg/day]/dose(g/day].

Scope of genotoxicity: Mutagenesis: the formation of

mutationsCarcinogenesis: the formation of

CancersSome forms of Teratogenesis:

Damage to the DNA

Systematic approach to identify an impurity

Decision Tree: Control Strategy for Genotoxic

Decision Tree: Control Strategy for Genotoxic

Confirmed GTI*Enter Decision tree

Tree

Where, GTI is an intermediate, reagent. Observed by product or likely by-product

Is GTIintroduce d in final step?

Is GTIintroducedpenultimate step?

step

Is GTIintroduced > 4 steps from API

Provide chemicalrationale for removal

Test API and impose limit based ontoxicology assessment

Found inpenultimateat a level ofconcern

conce

Test to demonstrate absenceor rejection efficiency (orprovide chemical rationale for

Demonstrate absence and/orremoval efficiency or establishspecification for registration

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Methodology

Classical approachseparation and determination of impurities

Modern approachUsing on-line hyphenated separation/spectroscopic methods

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Classical approach HPLC (recently UPLC)TLC Capillary Electrophoresis Electrophoresis-related Chromatographic Techniques Polarography & UV

Methodology….

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Hyphenated techniques

HPLC-UV StudiesHPLC-MS StudiesGC-MS StudiesTLC-MS StudiesCE-MS StudiesMEKC-MS and CEC-MS StudiesHPLC-NMR Studies

GC MS

LC –APCI-MSLC NMR

A review on genotoxic profiling….Lee determined methyl-, ethyl- and isopropyl-

methanesulphonates in drugs using GC with flame-ionisation detection (FID) (LOQ of 5 ppm in the drug material)

Sluggett developed a GC-MS method utilising selected-ion monitoring (SIM) mode detection for the trace analysis of a genotoxic impurity carbonic acid chloromethyl tetrahydro-pyran-4-yl ester, present in a beta-lactam API with an LOQ of 10 ppm.

Another approach – chemical derivatisationLee et al. derivatised methyl-, ethyl- and

isopropyl-methanesulphonates as well as dimethyl sulphate with sodium thiocyanate to form the corresponding alkyl thiocyanates.

GC-MS determination - with high sensitivity (LOQ<1 ppm).

A new approaches for the chemically unstable analytes – MATRIX DEACTIVATION

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TLC-MS

TLC-MS Separated spots on the plate could be subjected to direct matrix-assisted-laser-desorption ionization

Time-of-flight mass spectrometry (TLC-MALDI TOF -MS) no need to remove the spots from plate

Only wetting spot with methanol required to transfer the analyte from inside the silica gel to the surface thus enhancing the MALDI-TOF-MS signal

Modern approach

TLC PLATE

MEOH

CASE STUDY 1-The Case of EMS Ethyl methly sulphonate A direct DNA damaging agent(alkylator)As an impurity in Viracept (nelfinavir

mesylate) tablets (mnfd by Roche Ltd) in june 2007 – formulation recalled from european market

maximal dose of 0.055 mg/kg/d

Hydrazine as impurity –case study2Hydrazine was observed as a by-product

reaction in a drug substance indicated for cancer.

Max’ dose of the drug - 500 mg/d,NMT 2yrsAs per USEPA a lifetime exposure of 5876

mcg – cancer risk.The acceptable daily dose for hydrazine was

8 mcg/d (5876 mcg/[365*2]). Dose of drug fixed based on this

Conclusion :Impurity profiling is very important in the field of pharmaceutical

analysisUnidentified and potentially genotoxic impurities are health hazards and

in order to increase safety, these impurities should be identified.Assessment and control of GTIs in chemical process

development is challenging, owing to the evolving nature of the synthetic process, variable points of entry of GTIs in the process, and the need for analytical measurements with adequate selectivity and sensitivity.

Impossible to eliminate all impurities & Often it is impractical to detect all those trace ones.

However it is not possible to assess the exact limit for a genotoxic impurity, just estimation. So precautions ...

References Genotoxic impurities - strategies for

Identification and Control, Edited by Andrew Teasdale

Wikipedia http://www.wiley.com/WileyCDA/WileyTitle/pr

oductCd-0470499192.htmlwww.science direct.comhttp://www.ema.europa.eu/docs/en_GB/docu

ment_library/Scientific_guideline/2009/09/WC500002903.pdf

http://www.ipapharma.org/events/IPA%20-%20EDQM%20pdf/Manjunath%20Bhanu%20-%20Impurities%20Characterization.pdf

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HYDERABAD

Thank you