Impact factor: 3.958/ICV: 4.10 ISSN:

Transcription

1 Impact factor: 3.958/ICV: 4.10 ISSN: Pharma Science Monitor 8(2), Apr-Jun 2017 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: DETERMINATION OF GENOTOXIC IMPURITY BY CHROMATOGRAPHIC METHOD Megha Rajput*, Nilesh Patel, Urvi Chotaliya, Ashok Patel, Ajay Patel, Amit Vyas Department of Quality Assurance, B. K. Mody Government Pharmacy College, Polytechnic campus, Near Aji dam, Rajkot , Gujarat, India. ABSTRACT Impurity occurs essentially in all drug substance and drug product. Impurities have the potential to cause adverse effect. Hence, there is a need to ensure the level of impurity which is administered to human for safety. Therefore, impurity profiling is of prime importance. Routine impurity analysis in pharmaceuticals generally requires identification at levels of 0.05 percent to 0.2 percent depending on the daily dose. However, genotoxic impurities can be difficult to detect due to their presence at low ppm levels. This review focuses on the regulations and analytical technologies used to detect and quantitate impurities (genotoxic) in pharmaceuticals. Genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, that may lead to cancer. The data set usually available for genotoxic impurities is quite variable and is the main factor that dictates the process used for the assessment of acceptable limits. In the absence of data usually implementation of a generally applicable approach Threshold of Toxicological Concern (TTC) is proposed. This review focuses on assessment and control of DNA reactive impurities by implementing ICH M7 guideline. Analytical methods are suggested on how to determine genotoxic impurity. KEYWORDS: Threshold of toxicological concern (TTC), genotoxicity, carcinogen, mutagen, Drug substance, Drug product, GC, HPLC, GC/MS. INTRODUCTION Any component of the new drug substance that is not the chemical entity is defined as impurity. A description of the identified and unidentified impurities present in a new drug substance is termed impurity profile Classification of impurities [1] Impurities can be classified into the following categories: Organic impurities (process- and drug-related) Inorganic impurities Residual solvents

2 Impact factor: 3.958/ICV: 4.10 ISSN: Organic impurities can arise during the manufacturing process and/or storage of the new drug substance. They can be identified or unidentified, volatile or non-volatile, and include starting materials, by-products, intermediates, degradation products, reagents, ligands and catalysts. Inorganic impurities can result from the manufacturing process. They are normally known and identified and include heavy metals or other residual metals, inorganic salts; other materials (e.g., filter aids, charcoal). Residual solvents in pharmaceuticals are defined here as organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products. Genotoxic Impurity [2-3] Genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, that may lead to cancer. The genotoxic substance causes damage to the genetic material in the cells through interactions with the DNA sequence and structure. For example, the transition metal chromium interacts with DNA in its high valent oxidation state so as to cause DNA lesions leading to carcinogenesis. Any impurity which has the property of genotoxicity is called genotoxic impurity. Genotoxic impurity can lead to serious consequences, one example being residues of the alkylating agent 1, 4-butane sulfone in the excipient, sulfo butyl ether betacyclodextrin sodium (SBECD). This excipient is employed as part of the formulation of Vfend (voriconazole) and concerns were raised in 2001 as part of the EU assessment process about the toxic potential of this alkylating agent. Ultimately, setting a limit of < 1 ppm 1, 4-butane sulfone in SBECD was considered sufficient to enable the authorization of Vfend. Guidelines related to genotoxic impurity [4-6] Guideline related to control of genotoxic impurity: ICH M7: Assessment and Control of DNA Reactive (Mutagenic ) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, This document is under development and may replace existing EMA and FDA guidelines. EMEA: Guideline on the Limits of Genotoxic Impurities. It introduced the concept and values for the threshold of toxicological concern (TTC). FDA Guidance for Industry (Draft): Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches (2008). Generally aligned with the EMA guideline. Guideline related to genotoxicity testing:

3 Impact factor: 3.958/ICV: 4.10 ISSN: ICH S2: Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. This document combines previous guidelines ICH S2A (1996) and ICH S2B (2007), and is the global document for genotoxicity testing. EMA: Guideline on the Assessment of Genotoxicity of Herbal Substances/Preparations (2008). This guideline describes a general framework and practical approaches for testing the potential genotoxicity of herbal substances/preparations, as well as how to interpret the results ICH GUIDELINE M7 [4] Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk A TTC value of 1.5 μg/day intake of a genotoxic impurity is considered to be associated with an acceptable risk (excess cancer risk of <1 in 100,000 over a lifetime) for most pharmaceuticals. From this threshold value, a permitted level in the active substance can be calculated based on the expected daily dose. Higher limits may be justified under certain cases. The concentration limits in ppm of genotoxic impurity in drug substance derived from the TTC can be calculated based on the expected daily dose to the patient using equation stated below. Concentration limit (ppm) = TTC [μg/day] Dose (g/day] Acceptable Total Daily Intakes for an Individual Impurity (during clinical development and at marketing) Duration of 1 month >1-12 >1-10 years >10 years to treatment months lifetime Daily Intake ( μg/day) Acceptable Total Daily Intakes for Multiple Impurities* Duration of 1 month >1-12 >1-10 years >10 years to treatment months lifetime Daily Intake ( μg/day)

4 Impact factor: 3.958/ICV: 4.10 ISSN: EMEA GUIDELINE [5] Limit genotoxic impurities in DS and DP must be to levels associated with negligible risk Threshold of Toxicological Concern (TTC) Maximal daily intake of a genotoxic impurity at which negligible increased risk for cancer exists Generic limit based on database of several hundred genotoxic rodent carcinogens At marketing TTC = 1.5 μg/day For pharmaceuticals, risk factor = 1 x 10-5 Clinical development Staged limits based on duration of treatment Risk factor of 1 x 10-6 and additional safety factor of 2 FDA GUIDELINE [6] Generally aligned with EMEA guidance Exception Level of 120 μg/day acceptable for less than 14 days rather than just for single dose Methods for assessing genotoxicity Identification of genotoxicity in drug molecule Identification of genotoxic characteristic can be done by knowing its structure and with the help of software Software which are helpful in determining genotoxic behavior are: Toxtree software Toxnet database Derek software Leadscope software

5 Impact factor: 3.958/ICV: 4.10 ISSN: Structure alerts which are responsible for genotoxicity are: Classification of genotoxic impurity Classification Class 1: known genotoxic carcinogens Compound-specific limit Class 2: genotoxic but with unknown carcinogenic potential Limit to staged TTC (Threshold of Toxicological Concern) Class 3: alerting structure unrelated to API and of unknown genotoxic potential Further evaluation. Limit as appropriate. Class 4: alerting structure related to API Treat similarly as API Class 5: No alerting structure or indication of genotoxic potential Treat as routine impurity Mechanism including genotoxicity It is recommended to use information on mechanism including genotoxic activity as one element in conjunction with the other elements. Genotoxic chemicals are defined in the EU Guidance mutagens (positive evidence obtained from experiments in mammals and/or in some cases from

6 Impact factor: 3.958/ICV: 4.10 ISSN: in vitro experiments, obtained from somatic cell mutagenicity tests in vivo, in mammals or other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays). Lack of genotoxic activity in appropriate, well-performed tests may indicate a lower carcinogenic potency and may thus move a chemical near the potency borders to the next lower potency group, normally from intermediate to low. Option for controlling genotoxic impurity Control Options 1.Monitor the impurity in the drug substance Acceptance criterion below the TTC 2. Monitor the impurity in intermediate, starting material or in-process control Acceptance criterion below the TTC 3. Monitor the impurity in intermediate, starting material or in-process control Acceptance criterion above the TTC, with demonstrated understanding of fate and purge and associated process controls 4. Design robust process controls to reduce the risk of impurity level above the TTC to negligible Chromatographic method for determination of genotoxic impurity Gas chromatography(gc) The following steps are performed: Selection of column Selection of carrier gas Selection of diluent Optimization of column oven program Optimization of injector type, temperature and injection volume Using this development method and by performing various trial and error, optimized method is selected. For the given purpose the limit is decided with the help of TTC. TTC is calculated with respect to drug dose and its factor as mentioned in ICH M7 guideline. According to limit the sample and standard concentration are decided and injected to bring the molecule in limit. Thereby, the molecules which possess the characteristic of genotoxicity are brought to desired level by optimizing the above mentioned condition.

7 Impact factor: 3.958/ICV: 4.10 ISSN: High performance liquid chromatography (HPLC) The following steps are performed: Selection of Wavelength Selection of Mobile Phase Selection of diluent Selection of column Using this development method and by performing various trial and error, optimized method is selected. For the given purpose the limit is decided with the help of TTC. TTC is calculated with respect to drug dose and its factor as mentioned in ICH M7 guideline. According to limit the sample and standard concentration are decided and injected to bring the molecule in limit. Thereby, the molecules which possess the characteristic of genotoxicity is brought to desired level by optimizing the above mentioned condition. Hyphenated Technique GC-MS Genotoxic impurities are analysed by GC-MS by deciding the limit through TTC. TTC is calculated with respect to drug dose and its factor as mentioned in ICH M7 guideline. According to limit the sample and standard concentration are decided and injected to bring the molecule in limit. The method is performed by following a pretreatment procedure i.e. solvent extraction. Then, chromatographic method is optimized and method is validated. LC-MS/MS [11] LC-MS/MS can be used for separation and quantification of potential genotoxic impurities. The method applies quadrupole analyzer with electrospray ionization technique. It was operated in MRM ( multiple reaction monitoring ) for enhancement of sensitivity.lc-ms/ms can be used in pharmaceutical analysis. CONCLUSION The ICH M7 guideline provides a framework for assessing DNA reactive impurities and describes how these impurities may be controlled. This framework is currently being implemented across the pharmaceutical industry and international regulatory agencies. This review focuses on determination of genotoxic impurity and bringing them to the limit with the help of chromatographic method. The methods discussed GC and HPLC are reliable in determining genotoxic impurity.

8 Impact factor: 3.958/ICV: 4.10 ISSN: REFERENCES 1. ICH, Q3A (R2) Impurities in New Drug Substances: Text and Methodology, International Conference on Harmonization, (2006), IFPMA, Geneva, Switzerland. 2. Genotoxic Impurities Part 1: General Overview- Pharmaceutical Outsourcing, the Journal of Pharmaceutical & Biopharmaceutical Contract Services (7 October 2016) neraloverview/ 3. Genotoxic Impurities Part 2: Toxicological Overview Pharmaceutical Outsourcing the Journal of Pharmaceutical & Biopharmaceutical Contract Services (7 October 2016) xicologicaloverviewintroduction/ 4. ICH M7, Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, in International Conference on Harmonization Tripartite Guideline, Step 1 working draft, 11th November EMEA CHMP. Guideline on the limits of genotoxic impurities, Q&A Document, 2009 (8 October 2016) 6. FDA Draft Guidance. Genotoxic and carcinogenic impurities in drug substances and products: Recommended approaches (8 October 2016) ances/ucm pdf 7. Hennes C, Incorporating potency into EU classification for carcinogenicity and reproductive toxicity, Regulatory Toxicology and Pharmacology, (2014); 70: Alexander A, Principles and procedures for implementation of ICH M7 recommended (Q) SAR analyses, Regulatory Toxicology and Pharmacology, (2016); 77: Romualdo B, Structural Alerts of Mutagens and Carcinogens, Current Computer-Aided Drug Design, (2006); 2(2): Analysis of potential genotoxic impurities in active pharmaceutical ingredients (5 April 2017) Vijaya B.: A selective and sensitive LC-MS/MS method for the simultaneous determination of two potential genotoxic impurities in celecoxib. Journal of Analytical Science and Technology 2014; 5:18.