Towards middle-up analysis of polyclonal antibodies: subclass-specificN-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

Advanced analytical strategies including top-down and middle-up HPLC-MS approaches have become powerful alternatives to classical bottom-up analysis for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up analysis of polyclonal IgGs posing additional challenges due to extensive sequence variability. The presented workflow is based on Fc/2 portions as conserved subunits of IgGs and enables global profiling of subclasses and their glycosylation patterns, both of which influence IgG effector functions. To obtain subunits of murine IgGs, we established digestion with the bacterial protease SpeB. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry allowing relative quantification of IgG subclasses and their N -glycosylation variants. In order to assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. This analysis simultaneously revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine as demonstrated for monoclonal IgGs, which may be implemented for quality control of functional antibodies.