Monitoring oxidation in recombinant monoclonal antibodies at subunit level through two-dimensional liquid chromatography coupled with mass spectrometry

Abstract

Monoclonal antibodies (mAbs) are highly susceptible to chemical/post-translational modifications (PTMs), a crucial concern for chemical and biological safety during mAb production and development. Modifications in different mAb domains have different consequences on mAb stability and activity. Hence, characterization and monitoring of domain-specific modification in mAbs is of significant interest so as to be able to assure consistency in product quality. Generally, mAb samples are enzymatically digested and analyzed with traditional reversed-phase chromatography for domain-specific characterization. However, conventional analytical methods do not provide complete resolution and identification of mAb modifications. Here, a novel workflow of two-dimensional liquid chromatography with mass spectrometry interfacing has been proposed to overcome the limitations of conventional one-dimensional analytical methods and provide complete information on oxidized mAb domains. The proposed method successfully analysed the oxidized mAb samples and the control sample at the subunit level, simultaneously separating oxidized variants that are otherwise not detected by one-dimensional methods. The proposed 2D WCX-RPC-MS setup uses 2D multiple heart-cut method that identified 10 oxidation variants in mAb A, 6 in the Fab fragment and 4 in the Fc fragment, of which 3 were exclusive and not identified by the conventional 1D analysis. Further the 2D method takes only 7–13 h in sample preparation and analysis to anticipate oxidation in mAb fragments. Hence, the proposed workflow offers a faster and more convenient platform for routine monitoring and domain-specific mAb characterization with mass spectrometry coupling.