Protein formulations typically require a shelf-life of 18-24 months. During their long periods of storage, oxidation of methionine and tryptophan residues is one of the most common forms of chemical degradation. In this study, we probed the effect of oxidation on the structure, stability, aggregation, and function of a therapeutic monoclonal antibody (mAb-8). We examined whether the extent of protein destabilization, the aggregate formation, and the loss of specific protein activity can be correlated with the site and extent of oxidation. We used three oxidants, hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BHP), and 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) commonly used in pharmaceutical industry to test the chemical stability of proteins. Both H2O2 and t-BHP preferentially oxidize methionines whereas AAPH oxidizes tryptophans in addition to methionines. The oxidized mAb by the three chemical agents did not show any noticeable changes in its secondary structure, with minor changes in its tertiary structure. All the three oxidized mAbs show a significant decrease in conformational stability compared to the un-oxidized mAb. Both H2O2 and t-BHP selectively destabilized the constant domain CH2, whereas AAPH destabilized both the CH2 as well as the variable domain of Fab. Consistently, increased mAb aggregation was found significantly more in the case of AAPH-oxidized mAb, confirming the earlier observation that the variable domain primarily controls the aggregation of this mAb. Loss of mAb effector function upon oxidation also showed a clear correlation with the site of oxidation. A significant decrease in Fc receptor binding was observed in all the three oxidized mAbs, since all the three oxidizing agents modified the CH2 domain. Since only AAPH modified the Fab region, but not H2O2 and t-BHP, a significant decrease in cell proliferation and antibody-dependent cell cytotoxicity (ADCC) activity was observed only in the case of AAPH-oxidized mAb compared to those modified by H2O2 and t-BHP. Presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation completely for H2O2 and AAPH but not for t-BHP. This study for the first time provides a complete mechanistic understanding of the effect of chemical oxidation on the stability, aggregation, and function of a mAb.

Effects of chemical oxidation on the structure, stability, aggregation, and function of an IgG1 mAb

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.