Collision induced unfolding detects subtle differences in intact antibody glycoforms and associated fragments

Abstract Glycosylation has a significant impact on the effector function, immunogenicity, plasmatic clearance, and resistance towards proteases for monoclonal antibodies. Antibody glycoforms directly result in a form of structural heterogeneity due to the variety of sugar moieties and the available range of assembly states for the sugar linkages involved. Thus, the rapid characterization of antibody glycosylation is a critical yet challenging objective in the development of antibody-based therapeutics. Recently, we introduced an ion mobility-mass spectrometry approach for intact antibody analysis, which involves the collision induced unfolding of intact antibody ions. In this report, we demonstrate the use of such gas-phase unfolding analyses to differentiate subtly-different glycoforms within both intact antibody monomers and antibody Fc fragments, using minimal sample preparation and purification. We find evidence for a strong correlation between the gas-phase stabilities of antibody ions and the number of sugars attached to their sequences. We conclude by projecting the utility of our gas-phase unfolding assay in the context of antibody characterization workflows aimed at differentiating antibody glycoforms that cannot be readily resolved by MS alone.