Purpose: Rituximab is a chimeric (human/murine) monoclonal antibody (mAb) that targets CD20 located on the surface of B cells, and is administered for the treatment of non-Hodgkin’s lymphoma. As the patent of rituximab has expired, many pharmaceutical companies have been developing biosimilars to rituximab. However, mAb biosimilars are challenging to produce because of their complex structure and heterogeneity. Accordingly, extensive characterization is required to ensure the safety and efficacy of a biosimilar compared with its respective innovator.
In this study, we demonstrate how various MS workflows address structural similarities and differences between innovator rituximab (Rituxan®) and its biosimilar (Acellbia®). Furthermore, we correlate glycan profiling with biological activity (Antibody-dependent Cell Cytotoxicity - ADCC) of rituximab via reporter gene assay.
Methods: Intact antibodies were characterized 3 ways through a Q Exactive mass spectrometer (ThermoFisher) - native, deglycosylated, and deglycosylated and reduced states. Ion mobility mass spectrometry (IM-MS) of intact mAbs was conducted on a Synapt G2 (Waters). Hydrogen exchange was conducted with an H/DX PAL robot (LEAP technologies), with deuteration levels determined by QTOF mass spectrometry (Agilent 6530), which was interfaced with LC (Agilent 1260). Rituximab was reduced, alkylated and digested by AccuMAP™ low pH protein digestion kit (Promega). Peptide mapping was also carried out on a Q Exactive HF mass spectrometer (ThermoFisher). Post-translational modifications (PTMs) such as glycosylation, oxidation, deamidation and N-terminal pyroglutamate (Pyroglu) conversion were quantified by Byonic software (Protein Metrics Inc.). N-glycan profiling was performed using fluorescence (FL) labelling and FL-UPLC (Waters). ADCC activity of FcγRIIIa V (high affinity FcγRIIIa) and F (low affinity FcγRIIIa) variants were measured through an ADCC Reporter Assay kit (Promega).
Results: Intact mass characterization revealed that both innovator and biosimilar had similar glycoforms, except for the presence of Man5/Man5 (dual mannosylation) of the innovator rituximab. The only other difference detected by intact characterization was the presence of a single C-terminal lysine for biosimilar rituximab. When comparing biosimilar to innovator rituximab by HDX-MS, there was no statistically significant difference found regarding the dynamics of deuterium uptake. Innovator rituximab and its biosimilar showed similar stability based on IM-MS generated collision induced unfolding (CIU) fingerprints. Peptide sequencing by tandem MS showed 100% sequence coverage. In addition, both showed comparable levels of oxidation and deamidation. Key differences were found for N-terminal pyroglu levels and N-terminal glycan profiles. Biosimilar rituximab showed a higher percentage of N-terminal pyroglu and afucosylated glycans when compared with the innovator. Glycan profiles were confirmed by FL-UPLC analysis, showing the same tendency of afucosylated glycan levels as shown by tandem MS. When comparing ADCC activity, the Rituximab biosimilar showed a lower EC50, regardless of FcγRIIIa V and F variants, corresponding to the results of glycan analysis.
Conclusion: In this study, we have demonstrated the utility of multiple-attribute method (MAM) and various mass spectrometry-based workflows using innovator rituximab (Rituxan®) and its biosimilar (Acellbia®). These analysis provide rapid and robust analytical information vital for biosimilar development. In addition, both showed similar higher order structure and levels of oxidation and deamidation. Key differences that we detected were the presence of C-terminal lysine on biosimilar, glycan profiles and ADCC activity via FcγRIIIa.
Sang Yeop Kim– Graduate student, University of Michigan, Ann Arbor, Michigan
Daniel Vallejo– Graduate student, University of Michigan
Tyler Hageman– Graduate student, University of Kansas
Ilker Sen– Principle Scientist, Protein Metrics Inc.
Michael Ford– Co-Founder, MS Bioworks
Sergei Saveliev– R&D Project leader, Promega Corporation
Brandon Ruotolo– Professor, University of Michigan
David Weis– Associate professor, Uniiversity of Kansas
Anna Schwendeman– University of Michigan