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

• Published in: International journal of molecular sciences Vol. 22; no. 16; p. 9072
• Main Authors: Di Marco, Fiammetta, Berger, Thomas, Esser-Skala, Wolfgang, Rapp, Erdmann, Regl, Christof, Huber, Christian G
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 23.08.2021; MDPI
• Subjects: Antibodies, Monoclonal - chemistry; Antineoplastic Agents, Immunological - chemistry; Biological products; biosimilar; Biosimilar Pharmaceuticals - chemistry; Cation exchanging; Cation-exchange chromatography; Cations - chemistry; Chromatography; Chromatography, Affinity - methods; Chromatography, High Pressure Liquid - methods; Glycosylation; Heterogeneity; High performance liquid chromatography; Immunotherapy; Ions; Lymphoma; Lysine; MabThera; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Monoclonal antibodies; Peptides; Pharmaceutical industry; Physicochemical properties; Post-translation; post-translational modifications; Proteins; Quality assessment; Quality management; Reditux; Retention; Rituximab; Rituximab - chemistry; Salts; Scientific imaging; Spectroscopy; Storage conditions; strong cation-exchange chromatography; MabThera; charged variant separation; pH gradient; Reditux; strong cation-exchange chromatography; mass spectrometry; post-translational modifications; biosimilar; monoclonal antibodies
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22169072

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 and its Indian copy product Reditux™. 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 and Reditux™ displayed differences at the molecular level. MabThera showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ 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™ but not in MabThera . 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.