김수아 (성신여자대학교)

고병준 (성신여자대학교)

김수아 (성신여자대학교)

김지현 (성신여자대학교)

박시영 (성신여자대학교)

고병준 (성신여자대학교)

Biopharmaceuticals are susceptible to oxidative stress during manufacturing, storage, and in vivo administration. Oxidative stress can induce post-translational modifications (PTMs), which are widely recognized as a major contributing factor to protein instability. In particular, monoclonal antibody therapeutics are vulnerable to site-specific oxidation, potentially resulting in structural alterations and undesired aggregation.

This study aimed to investigate oxidation-induced PTMs in Avastin, a representative monoclonal antibody drug, and to propose amino acid substitution strategies for enhancing its structural stability. To induce oxidative stress, Avastin samples were treated with hydrogen peroxide (H₂O₂) at concentrations of 0%, 1%, 2%, and 5%, followed by incubation at 37°C for 24 hours. Aggregated fractions were isolated using size-exclusion chromatography (SEC-HPLC), digested with trypsin, and subjected to peptide mapping via Orbitrap-based LC-MS/MS to identify oxidation-associated PTMs.

The analysis revealed a positive correlation between H₂O₂ concentration and aggregation levels. Comparative sequence analysis of native and oxidized samples indicated the presence of distinct oxidative PTMs localized within aggregation-prone regions. LS-MS/MS profiling identified susceptible amino acid residues and PTM hotspots that were specifically enriched in the aggregated fractions. Notably, the data suggest a causal relationship between certain oxidation-specific PTMs and aggregation propensity.

Based on these findings, we identified PTM-prone residues likely responsible for reduced structural stability and proposed targeted amino acid substitutions to mitigate aggregation. This study presents a practical strategy for enhancing the long-term physicochemical stability of therapeutic antibodies through rational molecular design.​