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 ∼25kDa. 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 10min. 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.
Keywords: CQAs; Fc oxidation; High resolution; IdeS; MAbs; Mass spectrometry; Monoclonal antibodies; Peptide mapping; Process characterization and process validation (PC/PV) studies; UHPLC.
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