Impurity analysis of Rituximab and its biosimilar

Abstract

Objective To analyze product- and process-related impurities in original Rituximab and its biosimilar. Methods Product-related impurities in Rituximab and its biosimilar were analyzed using size-exclusion high-performance liquid chromatography (SE-HPLC), capillary electrophoresis, and ion-exchange high-performance liquid chromatography (IEX-HPLC). Purity and biological activity of biosimilar charge variants were further determined. Process-related impurities in Rituximab and its biosimilar were analyzed using ELISA and quantitative PCR. Results In SE-HPLC analysis, the antibody monomers in original Rituximab and its biosimilar were 98.9% and 99.9%, and polymers were 1.0% and 0.1%, respectively. The capillary electrophoresis showed that low molecular weight impurities were 5.6% in Rituximab and 3.3% in biosimilar, while non-glycosylated heavy chains were 0.6% in both. In IEX-HPLC analysis, acidic and basic peaks were 21.0% and 10.0% for Rituximab, and 17.7% and 9.1% for biosimilar. The main peak fraction of biosimilar had higher purity and complement-dependent cytotoxicity than both acidic and basic peak fractions. The residual quantities of host cell protein and DNA, and of protein A were similar in Rituximab and its biosimilar, mostly below detection limits. Conclusion Original Rituximab and its biosimilar have similar category and level of product- and process-related impurities. Key words: Antibodies, monoclonal; Biosimilar pharmaceuticals; Drug contamination; Impurity analysis