2D-CEX–FcRn–MS to Study Structure/Function Relation of mAb Charge Variants

The automated elucidation of the interplay between monoclonal antibody (mAb) structure and function using two-dimensional liquid chromatography–mass spectrometry (2D-LC–MS) is reported. Charge variants, induced through forced degradation, are resolved by first-dimension (¹D) cation-exchange chromatography (CEX) and subsequently collected in loops installed on a multiple heart-cutting valve prior to transfer to second-dimension (²D) neonatal crystallizable fragment receptor (FcRn) affinity chromatography coupled with MS. As such, binding affinity of the latter mAb variants can elegantly be assessed and a first glimpse of identity provided. To maximize MS sensitivity, charge variants are unfolded upon eluting from the ²D affinity column by postcolumn addition of a denaturing solution. Further structural details, i.e., modification sites and chain distribution, are unraveled by a multidimensional LC–MS (mD-LC–MS) setup incorporating ¹D CEX and parallel online middle-up and bottom-up LC–MS analysis in the subsequent dimensions. Identified charge variants could be ranked according to their affinity for FcRn. Binding is predominantly impacted by heavy chain (HC) M₂₅₃ oxidation and to a lesser extend, M₄₂₉ oxidation. Oxidation of both HCs more drastically affects FcRn interaction compared to single-chain oxidation, and the more oxidation, the less binding. Other modifications, such as HC glycosylation, HC N_385/390, and N₃₂₆ deamidation or HC C-terminal processing, are not shown to affect binding. The streamlined platform is challenged against the established workflow involving offline collection of charge variants and structural and functional assessment by, respectively, LC–MS and enzyme-linked immunosorbent assay (ELISA). A decent correlation is demonstrated between the binding affinity measured with ELISA and ²D FcRn affinity chromatography. In addition, throughput is improved (7-fold), material requirements are substantially reduced (2 orders of magnitude), and sample preparation artifacts and loss are minimized. With the simultaneous determination of mAb structure and function, the current study takes the concept of multiattribute analysis to the next level, thereby contributing to the future development of safer and more effective antibody therapeutics.