Multidimensional LC-MS with ¹D multi-method option and parallel middle-up and bottom-up MS acquisition for in-depth...

Monoclonal antibodies (mAbs) are large and highly heterogeneous species typically characterized using a plethora of analytical methodologies. There is a trend within the biopharmaceutical industry to combine several of these methods in one analytical platform to simultaneously assess multiple structural attributes. Here, a protein analyzer for the fully automated middle-up and bottom-up liquid chromatography-mass spectrometry (LC-MS) analysis of charge, size and hydrophobic variants is described. The multidimensional set-up combines a multi-method option in the first dimension (¹D) (choice between size exclusion - SEC, cation exchange - CEX or hydrophobic interaction chromatography - HIC) with second dimension (²D) on-column reversed-phase (RPLC) based desalting, denaturation and reduction prior to middle-up LC-MS analysis of collected ¹D peaks and parallel on-column trypsin digestion of denatured and reduced peaks in the third dimension (³D) followed by bottom-up LC-MS analysis in the fourth dimension (⁴D). The versatile and comprehensive workflow is applied to the characterization of charge, hydrophobic and size heterogeneities associated with an engineered Fc fragment and is complemented with hydrogen-deuterium exchange (HDX) MS and FcRn affinity chromatography - native MS to explain observations in a structural/functional context.