FabULOUS Middle-Level Analysis of Murine Polyclonal Antibodies
Important advances in top-down and middle-level analytical LC-MS strategies have arisen in recent years, focused on the characterization of therapeutic monoclonal antibodies. Similar strategies to analyze polyclonal IgG in regard to subclass abundance and glycosylation patterns may provide new insight into immune regulatory processes. However, challenges associated with molecular heterogeneity due to inherent sequence variability of such samples persist.
Analysis of the Fc glycosylation pattern can be exploited as determinant of the IgG subclass and may be performed on human IgG by specific proteolytic cleavage using the FabRICATOR® (IdeS) enzyme. In this work, the scientists investigate the feasibility of middle-level analysis by FabULOUSTM (SpeB) digestion and LC-MS of polyclonal mouse IgG with extensive sequence variability. The FabULOUS enzyme was exploited for the generation of Fc/2 subunits from all murine IgG subclasses (1/1i, 2a/c, 2b/2bi, and 3), which all were proven amenable for proteolytic cleavage in 3 hours under mild reducing conditions. The obtained subunits enabled the dissociation of Fc and Fab domains needed to tackle the sequence heterogeneity.
Middle-down analysis by HPLC-MS of the Fc/2 subunits allowed the assignment of both murine IgG subclasses and isotypes, while simultaneous middle-up analysis provided quantitative information on the subclasses as well as their respective glycosylation variants. The workflow thereby permitted a global analysis of polyclonal murine IgGs with respect to subclass abundances including closely related isotypes as well as glycosylation profiles and other PTMs such as oxidation and lysine variants. Finally, the authors demonstrated the capabilities of this workflow in a pilot study where murine polyclonal IgG from mouse serum after immunization with pollen allergen.
In summary, the described middle-level workflow provides comprehensive information obtained in a single analysis involving swift sample preparation, standard LC-MS analysis, and straightforward data evaluation as an attractive extension to the toolbox of analytical strategies for antibodies.
FabULOUS Enzyme
Generation of IgG Fab fragments from several species.
FabULOUS Fab kit
Generation and purification of mouse IgG Fab fragments.