For a drug to be used in a human clinical trial, analytical testing must be performed to demonstrate the suitability of the drug for use. These tests ensure the identity, quality, strength, purity, and potency of an active substance and drug product or biologic and must be scientifically sound and validated to demonstrate their suitability for the defined use. Methods may be revised during the development lifecycle based on changes to synthetic route, drug product formulation, or manufacturing process; or they may be optimized for improved performance. Sponsors must ensure that these methods are sensitive enough to detect product changes over time (stability indicating) and will be used to monitor the physical and chemical stability of the drug substance and drug product over time.

Different methodologies analyze specific characteristics of a product. For example, loss on drying (LOD) or Karl Fisher (KF) are techniques used to determine water content in a drug product or a drug substance. Chromatographic methods such as high-performance liquid chromatography (HPLC) or gas chromatography (GC) are typical techniques used to show the purity of a product. For biologics, tests such as enzyme-linked immunosorbent assays (ELISA) or cell-based assays, and in rare cases even in vivo assays, are used to demonstrate potency.

Validation through the Development Process

As a product progresses from pre-IND through NDA or BLA submission, the testing to demonstrate the suitability of the analytical methods for their intended purposes becomes more prescribed moving from method qualification to full method validation, as described in ICH Q2 (R1), Validation of Analytical Procedures. Validation testing in accordance to the prescribed requirements of ICH Q2 (R1) must be performed as per an approved protocol, under GMP conditions, and includes the following assessments:

• Specificity: The ability to assess the analyte of interest without interference from impurities, degradants, or matrix components.
• Accuracy: The closeness of true results compared to theoretical values.
• Precision: The closeness of agreement between replicate analyses.
• Intermediate precision: The closeness of agreement between replicate analyses performed by different analysts using different instrumentation and run at different times.
• Sensitivity: The ability to assess the lowest amount of analyte.
  • The detection limit is the lowest amount of analyte that can be detected.
  • The quantitation limit is the lowest amount of analyte that can be quantitated with acceptable precision and accuracy.
• Linearity: The ability to obtain test results within a given range which are directly proportional to the concentration of analyte in the sample.
• Robustness: The ability to obtain reproducible results across small, deliberate changes in method parameters.

Validation information for all noncompendial methods is summarized in 3.2.S.4.3 and 3.2.P.5.3 in an NDA or BLA submission. Approved validation reports must be submitted as part of the marketing application as well.

Method Qualification

Projects in the early stages of clinical development require method qualification for the analytical methods being used. Method qualification, sometimes referred to as phase appropriate validation, may include some, but not all, of the tests associated with validation, and these tests are not required to meet the stringent requirements of ICH Q2 (R1). Typically, method qualification would include limited testing to ensure the suitability of the method for its intended purpose. For example, the qualification of an HPLC method used to determine the purity and impurity profile of a product may include testing the precision, sensitivity (e.g., limit of quantitation and limit of detection), linearity, and solution stability. As per the FDA guidance “Analytical Procedures and Methods Validation for Drugs and Biologics” (July 2015), method robustness should also be tested to ensure that slight variations in the method parameters have minimal impact on the given results. A method that is not fully validated may be used to support phase 1 and 2 clinical trials, but full validation is required for phase 3 materials.

Method Verification

Frequently, the specifications for a drug or biologic will include compendial methods as part of the analytical testing. As described in the FDA guidance document, “Analytical Procedures and Methods Validation for Drugs and Biologics” (July 2015), compendial methods are analytical methods that originate from an FDA recognized source, such as the United States Pharmacopeia (USP). Validation of compendial methods is not required in an NDA or BLA submission, instead verification should be performed for these methods. As stated in the aforementioned FDA guidance document, verification is intended to demonstrate the “suitability of a method under the actual conditions of use”. A protocol is required for verification and will describe the method and predetermined acceptance criteria of the compendial method to be verified. The data from verified compendial methods will also need to be submitted in the NDA or BLA for the product.

Rho has expertise in drafting and reporting method verification, qualification, and validation activities across all phases of drug development. If you are in the process of developing a method validation strategy for your drug product or biologic, consider Rho as your CMC consulting partner to help you with your validation activities.

Aimee Weber, Associate Director, CMC Regulatory, has more than 20 years of experience in the pharmaceutical industry, specializing in Chemistry, Manufacturing and Controls (CMC) for both drug product and drug substance. She has contributed to the development of a diverse range of drug substances covering multiple therapeutic areas and drug product formulation types, with extensive experience with semi-solid dermatological products. Ms. Weber is responsible for working with clients, providing advice for CMC regulatory strategy and authoring sections of Module 3 for all stages of clinical development. She is well versed in eCTD format and has authored a variety of CMC submissions, including INDs, IMPDs, Briefing Books and has been involved with two marketing applications. Ms. Weber earned her Bachelor of Science in Chemistry from Hope College in 1999.