Comparative analysis of charge heterogeneity in anti-sclerostin monoclonal antibodies produced via continuous...

As critical quality attributes, post-translational modifications (PTMs) of therapeutic monoclonal antibodies in production impact their structural stability, charge heterogeneity, and clinical efficacy. This study elucidated PTM-driven charge isomer disparities in anti-sclerostin monoclonal antibodies from continuous perfusion versus fed-batch bioreactor processes. By developing an integrated analytical platform combining strong cation-exchange chromatography, size-exclusion chromatography, and non-reducing capillary electrophoresis with high-resolution mass spectrometry, we precisely characterized molecular weight variations and signature PTM profiles across distinct charge isomers. The study revealed that basic isomer 1 predominantly originated from C-terminal glycine loss and methionine oxidation, while basic isomer 2 arose from incomplete N-terminal glutamine cyclization and N-terminal truncation. The fed-batch process uniquely induced degradation at the heavy chain Met250-Ile251 site and significantly elevated low-molecular-weight species content. Crucially, antibodies from both processes exhibited minimal differences in purity and PTM profiles, robustly validating the feasibility of process transition. Biological activity assessment results showed that the relative binding activities of charge isomers from both processes ranged between 90 and 121%. The equilibrium dissociation constant values for binding to the neonatal fragment crystallizable receptor were all within (5∼9) × 10^-1 μM, indicating that FcRn function was unaffected by charge heterogeneity or the process. In summary, this study systematically compares the impact of two production processes-fed-batch and continuous perfusion-on the charge heterogeneity and biological activity of an anti-sclerostin monoclonal antibody. This work not only identifies key quality benchmarks for antibody process optimization but also establishes a PTM fingerprint-based standard for evaluating process comparability, providing critical guidance for advancing continuous biomanufacturing technologies.