Affinity Capillary Electrophoresis–Mass Spectrometry as a Tool to Unravel Proteoform-Specific Antibody–Receptor Interactions

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 K D 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.

Affinity capillary electrophoresis – mass spectrometry permits direct binding assessment of IgG and FcγRIIa in a glycoform-resolved manner

“…We have recently developed a novel approach based on affinity capillary electrophoresis – mass spectrometry (CE-MS) that allows monitoring the specific binding of antibody proteoform mixtures (including glycosylated variants) to the FcRn receptor ( 24 ). Mobility shift affinity capillary electrophoresis is an approach able to determine binding affinities of specific proteoforms by monitoring the change of their electrophoretic mobility after addition of the interacting partner to the background electrolyte ( 25 ).…”

Studying molecular interactions via capillary electrophoresis and microscale thermophoresis: A review

“…The examples of conventional ms‐ACE applications are summarized in Table 1 as reported in Refs. [14, 30, 45, 52, 54, 66, 69, 71–80, 83–125]. Moreover, the conventional ms‐ACE was modified using external hydrodynamic pressure applied during an electrophoretic run and achieve faster analysis.…”

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

“…ACE utilizes the specific interactions between ligands, receptors, and/or antibodies and analytes as a separation mechanism and is used for selective analysis of proteins in complex mixtures, and especially for studies of protein interactions with variable ligands under native conditions via determination of the binding or dissociation constants of the formed complexes [199,200]. ACE was successfully applied for charge heterogeneity profiling of biopharmaceuticals [201] and binding of mAb proteoforms to Fc receptors also in combination with MS detection [202,203].…”

“…Gstöttner et al [202,203] developed an approach based on mobility shift-ACE−MS to determine the binding of coexisting mAb proteoforms to Fc receptors. The approach required only low microgram amounts of antibody and receptor and coupling through a sheathless interface allowed functional characterization of mAbs with a high sensitivity and dynamic range.…”