Mahesh Bhalgat

Detection and quantitation of process-related impurities is an essential part of quality control in pharmaceutical and biopharmaceutical manufacturing. Process-related impurities can significantly impact the quality, safety, and efficacy of a pharmaceutical product. These impurities may be generated during the manufacturing process, arise from starting materials and reagents, develop during storage, or start from by-products of the process. If not properly detected and purified to acceptable levels, these impurities would lead to unacceptable product quality, potentially resulting in adverse effects on patients.

One such classification of impurities is called nitrosamines. All human medicinal products should be free of nitrosamine impurities, and if present, the nitrosamine impurities should be at a low concentration. In collaboration with regulatory counterparts, FDA has set internationally recognized acceptable daily intake limits for nitrosamines (1). This requirement applies irrespective of the marketing status or the type of product, including generics and over-the-counter products (2), because nitrosamines may increase cancer risk if patients are exposed to them above acceptable levels and over long periods (3). If drugs containing levels of nitrosamines above the acceptable daily intake limits, are released into the market, FDA requires the manufacturer to recall the drugs. Detection of nitrosamines in several commercial drugs has resulted in manufacturing batch recalls followed by a review of the APIs’ synthesis processes by marketing authorization holders (MAHs).

Nitrosamine impurities are probable human carcinogens in pharmaceuticals. In collaboration with regulatory counterparts around the world, FDA issued guidance to API and drug product manufacturers on appropriate actions they need to take to detect and prevent unacceptable levels of nitrosamine impurities in pharmaceutical products.

Root causes for nitrosamines contamination

There are several ways that the undesirable contaminants can be created. Formation of nitrosamines may be due to contaminated starting materials and intermediates, recycled materials from non-qualified vendors, contaminated equipment, cross-contamination from different processes on the same product line, or degradation of drug substance during the final finished product formulation (4). Additionally, impurities in raw materials, fresh solvents, recycled solvents, reagents, catalysts, water, excipients, or processing aids to produce the finished drug product may result in nitrosamines creation. Impurities in the container-closure system for the finished drug product, may also contain amines and are potential sources of a nitrosating agent (e.g., nitrile, nitrocellulose).

N-nitroso compounds (NOCs) are listed as Class 1 mutagens in International Council for Harmonisation (ICH) M7 guidance “Assessment and Control of DNA reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk” (5). ICH M7 also includes NOCs in the cohort of concern, a designation with a recommendation to control the impurities at or below the acceptable risk. In addition, some NOCs are listed as Class 2 compounds. As a result of the significant potential toxicity associated with these impurities, it is recommended to take steps to control and limit their presence in pharmaceutical materials.

In acidic conditions, secondary or tertiary amines react with nitrites to form nitrosamines. There are several pathways by which nitrosamines can be introduced into or generated as impurities in pharmaceutical drug products. There is evidence that despite processing and purification steps, reactive species, whether intentionally added to or formed during the process or reaction sequence (e.g., nitrites and secondary amines in the presence of acidic conditions), can carry over to subsequent steps. Special attention should be given to the formation of nitrogen-containing heterocycles by employing azide. It should be followed by quenching with nitrile to remove the excess azide. The drug may degrade under some conditions resulting in the formation of nitrosamines dialkyl amines (6).

It is recommended that manufacturers should consider the allowable intake (AI) limits established by different agencies such as the European Medicines Agency (EMA), FDA, Swissmedic, the Brazilian Health Regulatory Agency (ANVISA), and Health Canada when determining specification limits for nitrosamine impurities in API and drug products (7,8).

All regulatory agencies expect that existing methods based on certain matrices would be modified for new matrices/interferences, and the new methods must be validated for detecting other nitrosamines in the drug product and drug substance stages. Different instruments are used to estimate the trace level impurities, which is about 0.03 ppm or lower. The initial estimation started with the analysis of nine nitrosamine impurities, and today, there are 30 possible nitrosamine impurities that need to be estimated in products.

This adds to the challenges concerning specificity, sensitivity, limit of detection (LOD), and limit of quantification (LOQ) with acceptable recovery in a good manufacturing practice (GMP) environment.Given the significant work involved here, contract research organizations-contract development manufacturing organizations (CRO-CDMOs) have developed specialized capabilities to support analytical method development, validation and testing of routine batches.

The regulatory agencies may also update their published methods to improve their detection and quantitation limits. If updated, the regulatory agencies expect that manufacturers will update their methods to achieve comparable limits and apply the new limits, if any, in making decisions about batch suitability.

A three-step decision tree-based approach has been published by various regulatory agencies with timelines to comply with the requirements, which is useful for manufacturers to follow. Current available regulatory guidance for nitrosamines is available for the three steps: risk evaluation, confirmatory testing, and making changes to market authorization (9).

In-silico tools offer a promising approach for the detection of nitrosamines, providing a computational method to predict their presence. These tools leverage advanced algorithms and databases to analyze chemical structures to identify potential nitrosamine impurities. EMA’s guideline states that an Ames assay or mammalian cells test can only be used for mutagenic potential assessment. Carcinogenicity assessment using cancer bioassays in rodents takes a long time for evaluation and involves a significant cost (10).

Transgenic rodent bioassays are relatively insensitive to low-dose exposure, and extensive studies would be needed to enable a robust calculation of benchmark doses. Additional studies would be required for all nitrosamine impurities that are relevant.

 tools for mutagenicity prediction of pharmaceutical impurities are accepted by regulatory agencies as indicated in guideline ICH M7-R (5). However, a similar approach has yet to be reported for nitrosamines as a contaminant. The safety assessment team at Syngene preferred to use in-silico analysis to develop a screening assay for qualitative prediction. Derek Nexus 6.1.0 or Derek Knowledge Base 2020 1.0 and Sarah Nexus 3.1.0 (Lhasa Limited, UK) were employed to study the mutagenicity and carcinogenicity of nitrosamines. At Syngene, 40 nitrosamines in silico prediction results for mutagenicity and carcinogenicity were compared with in-vitro and in-vivo data compiled from the public domain (11,12). The ICH M7-R analysis using the tools together accurately predicted mutagenicity and carcinogenicity of nitrosamines. Thus, although these findings require further validation from other research groups working in this domain, the approach of using in silico analysis to predict the mutagenicity and carcinogenicity appears promising.

The presence of process-related impurities, particularly nitrosamines, can have significant impacts on product quality, efficacy, and patient safety if present above acceptable limits. A comprehensive and systematic approach along with strong analytical capabilities helps to ensure control, and prevention of nitrosamine impurities, thereby safeguarding the quality and safety of pharmaceutical products.

FDA. Information about Nitrosamine Impurities in Medications. FDA.gov/drugs (Sep. 1, 2020).

EMA. Questions and Answers for Marketing Authorisation Holders/Applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 Referral on Nitrosamine Impurities in Human Medicinal Products (March 30, 2023).

WHO. Information Note Update on Nitrosamine Impurities. WHO.int, Medical Product Alert, Nov. 20, 2019.

Agilent Technologies. Nitrosamine Impurity Application Guide, Confidently Detect and Quantify Mutagenic Impurities in APIs and Drug Products. Agilent.com, Application Guide (Sep. 14, 2020).

ICH. M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, Step 4 Version (2017).

FDA. Guidance for Industry, Control of Nitrosamine Impurities in Human Drugs (CDER, February 2021).

AZIERTA Life Sciences & Health Consulting. Nitrosamines EU/FDA/ANVISA. eBook (2022).

Health Canada. Nitrosamine Impurities in Medications: Guidance. Canada.ca/en/health-canada (last updated April 17, 2023).

Rao B. Nitrosamine Impurities—Current Regulatory Status. Spinco Biotech- Cutting Edge J online, December 2021.

EMA. Assessment Report: Procedure Under Article 5(3) of Regulation EC (No) 726/2004 Nitrosamine Impurities in Human Medicinal Products (June 25, 2020)

NIH National Library of Medicine. Carcinogenic Potency Database. Nlm.nih.gov/databases/download/ (accessed Oct. 21, 2022).

Lhasa Ltd. Carcinogenicity Database. carcdb.lhasalimited.org (2022).

Mahesh Bhalgat is chief operating officer at Syngene International Ltd.

When referring to this article, please cite it as Bhalgat, M. Understanding Nitrosamine Impurities in the Pharmaceutical Industry. Pharmaceutical Technology 47 (7) 2023.

Articles

Detection of nitrosamines in several commercial drugs has resulted in manufacturing batch recalls followed by a review of the APIs’ synthesis processes by MAHs. 