Application of a Stoichiometric Water Displacement Model and Property-Retention Relationships to Adsorption and Unfolding of Proteins in Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is effective in separating protein species based on interaction with a mildly hydrophobic resin. However, adsorbed proteins can unfold, reducing product yields and purity. This dissertation continues the development and application of a four-state model to describe adsorption and conformation change of proteins during HIC with a physically-based model for adsorption and surface-induced unfolding, the application of this model for detailed process modeling, and a predictive strategy using molecular property correlations.;The model treats adsorption of proteins to hydrophobic interaction chromatography resins in terms of the displacement of water molecules from hydrophobic ligands on the resin and hydrophobic residues on the protein. Adsorption data for systems consisting of several model proteins and HIC resins are successfully described. This model also leads to governing equations for the dynamics of conformational change of proteins adsorbed to HIC media.;A corresponding experimental protocol is developed that utilizes hydrogen/deuterium exchange and mass spectrometry to assess the conformational state of adsorbed proteins to determine both the kinetics and equilibria of adsorbed protein unfolding. This protocol has been used to investigate conformation behavior of model protein-resin systems exhibiting chromatographic behaviors symptomatic of unfolding.;These methodologies are also applied to study industrial purification for an Fc-fusion protein in which conformation change of monomers and aggregates was hypothesized to affect performance. The results from various experimental techniques indicate that there is some similar perturbation of Fc structure of monomers and aggregates upon adsorption. These results are applied to a detailed process model of this system.;Finally, the potential to predict adsorbed protein unfolding in HIC based on property-retention correlations is investigated. The hypothesis is tested that correlations based on adsorption of stable proteins can be extended to predict the adsorption strength of unfolded proteins, but the predicted adsorption strength of unfolded proteins is much smaller than experimental values. Theoretical analysis suggests that confinement entropy substantially affects the interaction of unfolded proteins with chromatographic resins, and must be included in future predictive methods.