Development of a rapid RP-UHPLC-MS method for analysis of modifications in therapeutic monoclonal antibodies

- A 15 min RP-UHPLC method following IdeS digestion was developed for analyzing antibody modifications including oxidation.
- CpB treatment prior to IdeS digestion eliminated the interference of C-terminal lysine with oxidation.
- Antibody variants separated by RP-UHPLC were characterized by on-line MS.
- Oxidation levels measured by RP-UHPLC are in good agreement with those acquired by peptide mapping.
- RP-UHPLC provided high resolution and high throughput separation for antibody variants at subdomain level.

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (MAbs) that have high risk to safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic MAbs process development, thorough characterization and quantitative monitoring of CQAs requires a variety of analytical techniques. This paper describes the development of a rapid analytical method to assess modifications in MAbs, based on the analysis of subdomains with molecular weights of ∼25 kDa. These subdomains were generated by digestion with a highly specific IdeS protease, followed by disulfide bond reduction. A reversed phase UHPLC-MS method was developed that provides efficient separation and identification of the subdomains (Fc, LC, and Fd) and related variants within 10 min. Sample preparation and UHPLC instrument parameters were systematically evaluated. The methodology was applied to MAb stress panel characterization to capture the degradations induced by various stress conditions. Among the CQAs monitored by this method, Fc oxidation levels were compared with the values obtained by the more complicated and time-consuming peptide mapping method. The similar trends observed by the two methods demonstrated that the IdeS-UHPLC method is valuable as a higher throughput alternative to peptide mapping for monitoring modifications. In particular, a high-throughput methodology is preferred for analysis of the many samples associated with process development studies. Overall the method has been demonstrated as a fast, convenient and informative platform approach for analysis of therapeutic MAbs modifications including CQAs.