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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) M253oxidation and to a lesser extend, M429oxidation. 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 N385/390, and N326deamidation 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.

Hydrophobic interaction chromatography (HIC) is one of the historical analytical methods applied for the separation and purification of proteins. In the first part of this series, the elution and separation mechanisms were discussed. In this second part, some practical considerations such as method development, selection of the phase system, combined salt systems, and possibilities to combine HIC with other chromatographic modes are explained. Applications are critically discussed and the hyphenation to mass spectrometry (MS) briefly tackled., Historically, a kosmotropic salt--typically 1.5-2 M ammonium-sulfate--has been used as a mobile phase additive in hydrophobic interaction chromatography (HIC) (as well as 50-100 mM phosphate buffer). However, various salts can [...]