Abstract
The oxidation of the complementarity-determining region (CDR) in monoclonal antibodies (mAbs) is a critical quality attribute that can affect the clinical efficacy and safety of recombinant mAb therapeutics. In this study, a robust hydrophobic interaction chromatography (HIC) method was developed to quantify and characterize CDR oxidation variants in mAb-A by using a Proteomix Butyl-NP5 column. The HIC analysis revealed oxidation variants that eluted earlier than the main species with weaker hydrophobicity. It was found that Met(105) in the CDR was more susceptible to oxidation. Additionally, it was noted that the oxidation of Met(105) on a single heavy chain resulted in elution at a distinct position compared to the oxidation on two heavy chains. This observation led to the fractionation and enrichment of the oxidized variants for further evaluation of their biofunction. The study also demonstrated that the oxidation of Met(105) did not impact the antigen-binding capacity but significantly reduced the PD-1/PD-L1 blockade activity of mAb-A. The HIC method, which was employed to quantify CDR oxidation, underwent validation and was subsequently utilized for stability studies as well as for assessing the similarity between mAb-A and its reference product.