Modulation of N-Glycosylation in Bispecific Antibody Biosimilars through Combined Modulators

Abstract To ensure a high degree of similarity between biosimilars and reference drugs, it is crucial to perform comprehensive characterization and maintain rigorous control over glycosylation processes. Here,we aimed to optimize the glycosylation profile of a biosimilar of a bispecific monoclonal antibody (Bs-mAb1) to closely resemble that of the reference drug through the synergistic use of glycosylation modulators. To identify the strongest modulators and appropriate concentration ranges, we first examined the effects of different concentrations of galactose (Gal) and manganese chloride (MnCl2) on the galactosylation rate in Shake Flask, as well as the influence of tris(hydroxymethyl)aminomethane (Tris) on the incorporation of mannose and fucose in 2 L Bioreactor. Importantly, the concurrentuse of Tris and galactose did not result in any interaction effects on N-glycan modifications and had no detrimental impact on cell growth, metabolism, antibody charge variants or purity. In conclusion, The concurrent use of 0.75 mM Tris and 8 mM galactose yields a glycosylation profile of Bs-mAb1 that is highly comparable to that of the reference drug, thereby providing an effective strategy for optimizing glycosylation in biosimilars. These findings provide significant insights into the regulation of glycosylation in the production of therapeutic monoclonal antibodies and may contribute to enhancing the consistency and therapeutic performance of biosimilars.