Multimodal mass spectrometric characterization of structural microheterogeneity in rituximab reference and biosimilars

초록

Three biosimilars to Rituxan® (rituximab)—Truxima®, Ruxience®, and Riabni™—have received FDA approval. Monoclonal antibodies exhibit inherent heterogeneity due to post-translational modifications, and defining acceptable ranges of such variability remains a critical challenge in biosimilar development. While pairwise comparisons between a biosimilar and its reference product are extensively studied, comprehensive analyses across multiple products are limited. In this study, we systematically characterized Rituxan® and its three FDA-approved biosimilars using advanced mass spectrometry (MS)-based techniques. Native intact MS assessed molecular weight variations, LC-FLR-MS glycan profiling evaluated glycoform distributions, and LC-MS/MS peptide mapping examined sequence integrity and modifications. Notably, the greatest glycan-related variation was observed between biosimilars—between Ruxience® and Truxima® in glycoform distribution, and between Ruxience® and Riabni™ in total afucosylated glycan levels—which correlated with slight differences in in vitro antibody-dependent cellular cytotoxicity activity. However, overall heterogeneity between biosimilars and the reference product was relatively small compared to that observed among biosimilars, with results consistent across analytical methods. Since commercially available biosimilars were analyzed, the observed variations reflect real-world analytical variability that does not impact clinical performance. These findings, which offer detailed analytical comparisons of biosimilars, provide insights into acceptable structural variation and support efforts to refine regulatory assessment practices.

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