Physicochemical and Biological Characterization of RTXM83, a New Rituximab Biosimilar.

Abstract

RTXM83 is a rituximab biosimilar with proven clinical safety and efficacy. It is the first rituximab biosimilar developed and approved in South America and is currently marketed in several Latin American, Middle Eastern and African countries. The aim of this study was to present the physicochemical and biological characterization studies utilized to demonstrate the similarity between RTXM83 and its reference product. Primary and higher order protein structures were analysed using peptide mapping with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), fluorescence spectroscopy and circular dichroism, and micro-differential scanning calorimetry, among other techniques. Charge variants were determined by cation-exchange chromatography (CEX)and capillary isoelectric focusing(cIEF). Glycosylation and glycoforms distribution were analysed using MS, normal phase high-performance liquid chromatography (NP-HPLC) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Size variants were evaluated by size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV-AUC), dynamic light scattering (DLS), and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Biological characterization included binding assays for complement C1q, CD20, and several Fc receptors (FcRs), as well as potency determination for in vitro apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). RTXM83 and the reference product showed identical primary sequences and disulfide bridge patterns, and similarity at higher order protein structures, post-translational modification profiles (amino acid modifications, charge variants, and glycosylation) and levels of purity and process-related impurities. Functional studies demonstrated that RTXM83 is similar to the reference product regarding the three known mechanisms of action of rituximab: CDC, ADCC, and apoptosis induction. Binding affinities to CD20, complement component C1q, and different FcRs were also equivalent. RTXM83 is similar to its reference product in all critical quality attributes.