Abstract
Monoclonal antibody (mAb) pharmaceuticals consist of a plethora of different proteoforms with different functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their individual assessment. Current binding techniques do not distinguish between coexisting proteoforms requiring tedious production of enriched proteoforms. Here, we have developed an approach based on mobility shift-affinity capillary electrophoresis-mass spectrometry (ACE-MS), which permitted us to determine the binding of coexisting mAb proteoforms to Fc receptors (FcRs). For high-sensitivity MS analysis, we used a sheathless interface providing adequate mAb sensitivity allowing functional characterization of mAbs with a high sensitivity and dynamic range. As a model system, we focused on the interaction with the neonatal FcR (FcRn), which determines the half-life of mAbs. Depending on the oxidation status, proteoforms exhibited different electrophoretic mobility shifts in the presence of FcRn, which could be used to determine their affinity. We confirmed the decrease of the FcRn affinity with antibody oxidation and observed a minor glycosylation effect, with higher affinities for galactosylated glycoforms. Next to relative binding, the approach permits the determination of individual KD values in solution resulting in values of 422 and 139 nM for double-oxidized and non-oxidized variants. Hyphenation with native MS provides unique capabilities for simultaneous heterogeneity assessment for mAbs, FcRn, and complexes formed. The latter provides information on binding stoichiometry revealing 1:1 and 1:2 for antibody/FcRn complexes. The use of differently engineered Fc-only constructs allowed distinguishing between symmetric and asymmetric binding. The approach opens up unique possibilities for proteoform-resolved antibody binding studies to FcRn and can be extended to other FcRs and protein interactions.