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 (D) cation-exchange chromatography (CEX) and subsequently collected in loops installed on a multiple heart-cutting valve prior to transfer to second-dimension (D) 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.