High-throughput identification of protein binding domains to elucidate selectivity in multimodal chromatography

A plate-based method was developed to identify protein preferred binding domains for multiple chromatographic conditions in a single parallel experiment. This approach employs covalent labeling with diethylpyrocarbonate (DEPC) followed by enzymatic digestion and mass spectrometry analysis to compare the labeling of proteins in the in-solution and resin-bound state. The differences observed in the labeling data are then used to identify potential binding patches and the results are compared with electrostatic potential and spatial aggregation propensity maps. For proof of concept, the binding of 11 proteins with a range of physicochemical properties on Capto MMC at pH 6 is evaluated. The charge and hydrophobic characteristics of the identified potential binding regions are then examined to classify the chromatographic retention behavior of these proteins. The results of this analysis indicate that, in addition to the charge patches, varying contributions of the hydrophobic patches in these binding regions can play an important role in elucidating and classifying the retention behavior of this protein set. The ability to experimentally determine preferred binding domains of proteins interacting with chromatographic resins using this approach may facilitate the application of this method for a wide range of protein resin systems.