Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

This paper presents a robust strong cation-exchange high-performance liquid chromatography (SCX-HPLC) method hyphenated with mass spectrometry (MS) for the simultaneous characterization and comparison of rituximab-based biotherapeutics, specifically the originator MabThera® and its Indian copy product Reditux™. The study's primary purpose was to unravel the molecular heterogeneity arising from post-translational modifications (PTMs) that can impact the quality attributes of these complex drug products. The methodology employed a pH gradient of volatile salts, enabling direct coupling to MS, which is a significant advantage over conventional ion-exchange methods using non-volatile salts. This approach allowed for the separation of charge variants based on their isoelectric point (pI) and simultaneous mass determination, providing a comprehensive view of proteoform differences. The research revealed molecular distinctions between MabThera® and Reditux™ in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. For instance, MabThera® exhibited a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. The study also investigated forced deamidation and glycation, demonstrating the method's capability to separate and characterize these modifications. The findings underscore the importance of advanced analytical techniques like SCX-HPLC-MS for rigorous biosimilarity assessment and batch-to-batch comparison in the biopharmaceutical industry.

This paper presents a robust strong cation-exchange high-performance liquid chromatography (SCX-HPLC) method hyphenated with mass spectrometry (MS) for the simultaneous characterization and comparison of rituximab-based biotherapeutics, specifically the originator MabThera® and its Indian copy product Reditux™. The study's primary purpose was to unravel the molecular heterogeneity arising from post-translational modifications (PTMs) that can impact the quality attributes of these complex drug products. The methodology employed a pH gradient of volatile salts, enabling direct coupling to MS, which is a significant advantage over conventional ion-exchange methods using non-volatile salts. This approach allowed for the separation of charge variants based on their isoelectric point (pI) and simultaneous mass determination, providing a comprehensive view of proteoform differences. The research revealed molecular distinctions between MabThera® and Reditux™ in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. For instance, MabThera® exhibited a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. The study also investigated forced deamidation and glycation, demonstrating the method's capability to separate and characterize these modifications. The findings underscore the importance of advanced analytical techniques like SCX-HPLC-MS for rigorous biosimilarity assessment and batch-to-batch comparison in the biopharmaceutical industry. Research Background and Significance Monoclonal antibodies (mAbs) represent a cornerstone of modern biopharmaceutical therapeutics, offering targeted treatments for a range of diseases including cancers and autoi