Assay Validation in Biologics and Biosimilars Development

Rigorous bioanalytical assay development and validation are critical in biopharmaceutical development. A comprehensive assay development, qualification, and validation initiative ensures product safety, quality, and regulatory compliance in biologics and biosimilars. The primary goal of assay validation is to demonstrate that bioanalytical assays provide reproducible and reliable data, ensuring they are fit for purpose to support decision-making and regulatory submissions. 

Assay Validation incorporates performance characteristics, including accuracy, precision, specificity, and robustness, critical for biologics and biosimilar development. This article discusses the regulatory requirements, validation methodologies, and approaches for assay validation in biologics and biosimilars. 

Biologics and biosimilar development

Biologics are active drug compounds produced by living cells or obtained from living organisms such as animals, yeast, bacteria, or human cell lines. Biosimilars are versions of original biologics that are developed post-expiry of patent and exclusivity. Data shows that the biosimilar market is growing slowly, but at a steady pace. However, a push from the global market may soon increase the demand for biosimilar products.

Compared to traditional generic drug products, biosimilar development presents challenges. Unlike small-molecule products, biologics are highly complex. The 3D biologic structure is largely based on the production process, such as cell type, purification, fermentation, storage, formulation, and packaging. Hence, any minor changes in these processes may generate larger variations in the macromolecular structure of the biologic drug. These modifications may have severe implications, such as changes in impurity levels or post-translational modification, which may generate undesirable immunogenic responses or loss of efficacy.

Although few biosimilars have been approved, studies demonstrate significant heterogeneity between biosimilars developed in different countries. For example, recombinant human erythropoietin used to treat anemia associated with cancer therapy and chronic disease conditions exhibited variability in concentration, composition, structure, and activity marketed in different countries. Additionally, these studies did not show comparability between the biologic and biosimilar drug. Clinical studies also failed to demonstrate equivalent efficacy and safety. Hence, meaningful clinical and preclinical comparative studies are critical for establishing equivalency between biologics and biosimilars.

Comparing and assessing structural and physicochemical elements of a biologic to a biosimilar have unique challenges. The EMA guidelines suggest applying extensive state-of-the-art characterization studies to biosimilar and original biologics in parallel to exhibit high-level assurance in the quality and comparability of the biosimilar drug product. Similar guidelines by the US FDA suggest designing studies to establish parameters that define the product’s purity, potency, quantity, identity, and consistency, and compare them between the biosimilar and the biologic.

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Regulatory framework and assay validation principles

Assay validation in biologics and biosimilar development complies with the regulatory guidelines laid by the US FDA and ICH guidelines. These regulatory bodies govern qualification, development, and validation to ensure data integrity and accuracy throughout pharmaceutical development initiatives.

The US FDA has released guidelines for drug developers and companies conducting method development and validation required by regulatory bodies to ensure the reliability and quality of generated data. These guidelines include public comments suggested after the first draft was published in 2013. Notably, the guidelines propose that drug developers can change the recommendations depending on the bioanalytical method. However, these modifications will need justifications. To develop and validate analytical assays, the FDA recommends addressing the following key concerns:

• What is the assay sensitivity?
• Does the assay measure the target analyte?
• Is the assay selective or specific?
• What is assay precision and accuracy?
• Does the data have variability?
• Is data reliability influenced by sample handling, collection, and storage?

The final US FDA guidance also contains a section on bioanalytical method validation for ligand binding assays and chromatographic assays. The method validation guidance includes recent research and practices in method validation, new analytical platform applications, research on incurred sample reanalysis, biomarker validation, and repurposing of diagnostic kits.

Biologics and biosimilar research includes assays such as in vivo and in vitro assay development. Similar to bioanalytical method validation for pharmaceutical drug products, comprehensive validation parameters required for biologics and biosimilar assay validation include precision, accuracy, selectivity, linearity, specification, and detection and quantitation limits. Establishing the performance of these parameters ensures the assays are fit for intended applications and comply with regulatory requirements.

Assay validation protocols help demonstrate assay robustness, assess matrix effects, and stability indicating capabilities. However, drug developers should address the critical aspects of statistical analysis, acceptance criteria, and validation documentation to support GLP compliance and the regulatory approval process for clinical studies. A thorough and rigorous implementation of validation standards ensures regulatory acceptance and analytical data quality while supporting the development of complex biosimilars and biologic products.

Conclusion

Bioanalytical method development and validation are crucial for generating reliable assay data and complying with regulatory recommendations in biologics and biosimilar development. A consistent and rigorous assay validation is critical in advancing biosimilar and biologics development through comprehensive analytical testing and compliant methodologies. A robust assay development supports capabilities such as large molecule bioanalytics and Neutralizing Antibody Assays, for successful regulatory submissions and patient safety. However, understanding the value of adequate assay validation is necessary to support drug development and ensure therapeutic success.