Kinetics and Thermodynamics of Protein Unfolding On Hydrophobic Interaction Chromatography Media Investigated by Global and Local Hydrogen Exchange

It is well known that protein unfolding during hydrophobic interaction chromatography (HIC) can lead to loss of product activity, irreversible binding, and poor selectivity. We utilize hydrogen-deuterium exchange to investigate the conformational state of proteins adsorbed to HIC resins of various ligand types and densities showing a variety of peak shapes in isocratic elution chromatography. Kinetic modeling of global exchange behavior at multiple labeling times reveals equilibrium conformation distributions and unfolding and refolding kinetics on the surface. Exchange behavior of selected systems is also investigated at the peptide level by proteolytic digestion of labeled protein after elution, providing evidence of protein regions where protection is changed by conformational changes upon adsorption. Comparisons are made with computer calculations of local stability of adsorbed proteins.