Simultaneous Monitoring Of Monoclonal Antibody Variants By Strong Cation-Exchange Chromatography Hyphenated To Mass Spectrometry To Assess Quality Attributes Of Rituximab-Based Biotherapeutics

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera(R) and its Indian copy product Reditux (TM). The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera(R) and Reditux (TM) displayed differences at the molecular level. MabThera(R) showed a higher degree of galactosylated and sialylated glycoforms, while Reditux (TM) showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux (TM) but not in MabThera(R). This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.