(T1230-06-33) Roadmap for Assessing Analytical Similarity: Case Study of Mab Biosimilar

Purpose: Biosimilars, being complex biologics, are not same as the innovator molecules. The differences in cell line, manufacturing process, container closure system etc used for a candidate biosimilar have an impact on multiple quality attributes. Therefore, understanding the relevance of each difference observed between the candidate biosimilar and innovator is crucial to conclude the Biosimilarity^[1]. This assessment is also crucial to design a relevant clinical study design. In this case-study of an Anti-IgE monoclonal antibody biosimilar candidate (ADL-018), the criticality of individual quality attributes was assessed before performing similarity analysis, thus pre-defining the tolerance for specific differences in quality attributes. This was followed by analyzing ADL-018 alongside the innovator batches, using a battery of orthogonal assays for each quality attribute. The clinical relevance of the differences or similarity in quality attributes was assessed based on the pre-defined criticality scores. This assessment allowed an unbiased and meaningful similarity evaluation of the candidate Biosimilar ADL-018 against the innovator product.

Methods: Fab mediated biological activity, being the most critical, was assessed by multiple orthogonal assays. Target binding ELISA was used as the primary functional assay, while β-hexosaminidase degranulation assay and histamine release assays, performed using modified cell line expressing  FcεRI receptor were used as orthogonal assays. Additional assays for low affinity receptors were also used, thus allowing for a complete characterization of Fab mediated biological activity of ADL-018 against innovator. Potential Fc activity was also assessed by CDC and ADCC assays. Lastly, FcRn binding was assessed using an ELISA method. Primary structure was assessed using multitude of methods including intact mass and reduced mass by LC-MS and amino acid sequencing by LC-MS/MS. Site-specific post translational modifications like oxidation, deamidation, isomerization, N-Terminal Pyroglutamate, deamidation etc were quantified using a unique validated multi-attribute mass spectrometry-based assay. Particles, aggregates and other size variants were analyzed by MFI, SEC, and AUC while product related substances and impurities were characterized by HIC, non-reduced CE-SDS, and CEX. N-linked glycan profile was determined by HILIC-FLR while non-glycosylated fraction was characterized by reduced CE-SDS. Finally, the higher order structure was analyzed by free thiol assay, Far UV-CD, FTIR, DSC and intrinsic fluorescence.

Results: For all bioassays performed, ADL-018 was found to exhibit similar dose response curve and binding kinetics. While all ADL-018 lots were found to fall within the quality ranges derived using innovator lots for all potency evaluations, a clear correlation between the age of products and biological activity could be drawn for both the products. The amino acid sequence of ADL-018 was found to be same as that of Innovator. Other primary structure attributes like intact and reduced mass were found to be similar between ADL-018 and innovator. Secondary structure attributes as illustrated by FTIR, Far UV-CD and DSC and tertiary structure attributes as illustrated by intrinsic fluorescence were found to be highly comparable. The level of molecular weight related variants as assessed by SEC, AUC, MFI were found to be in close agreement between both the products. Product-related substances and impurities profiles, including post-translational modifications, shown by multi-attribute mass spectrometry, size exclusion, cation exchange chromatography, hydrophobic-ion chromatography and reduced and non-reduced CE-SDS were highly similar as well. Glycosylation profiles were found to be comparable between the products, when analyzed using intact LC-MS and HILIC-FLR.

Conclusion: Assessment of quality attributes between ADL-018 and innovator based on an understanding of its criticality, pre-defined as a criticality score, allowed for an unbiased assessment of the biosimilarity. This work demonstrates the importance of using orthogonal assays for evaluation of critical attributes. Based on the analytical similarity assessment and the criticality assignment of attributes, ADL-018 was found to be similar to the innovator product for all the critical attributes evaluated.

References: Vandekerckhove, K., Seidl, A., Gutka, H. et al. Rational Selection, Criticality Assessment, and Tiering of Quality Attributes and Test Methods for Analytical Similarity Evaluation of Biosimilars. AAPS J 20, 68 (2018)

Quality attributes and criticality assignments