Simultaneous monitoring of monoclonal antibody variants by strong cation-exchange chromatography hyphenated to mass spectrometry to assess biosimilar-ity of rituximab-based biotherapeutics

The increasing importance of biosimilars in the biopharmaceutical market leads to high demands for enhanced protein characterization methods. Different manufacturing processes can lead to a significant variability of biotherapeutics arising from chemical and enzymatic post translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. Thus, biosimilarity to the originator product must be proven rigorously. Among these PTMs, N-terminal pyroglutamate formation, C-terminal lysine clipping, glycosylation, glycation, and deamidation lead to differences in the net charge of the protein, resulting in charge variants (CV). To unravel the heterogeneity of these proteoforms, Strong Cat-ion eXchange (SCX) High-Performance Liquid Chromatography (HPLC) is routinely used. However, the use of non-volatile salts makes the technique incompatible for hyphenation to mass spectrometry (MS). Recently, an approach employing volatile salts and a pH gradient was applied for CV analysis, opening the era of SCX-HPLC-MS approaches. Here, we apply an already established SCX-HPLC-MS approach by Füssl et al. to characterize two Rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products and constitutes the basis for biosimilarity characterization using a fast SCX-HPLC-MS approach.