Hydrophobic interaction chromatography (HIC) is a widely used chromatography technique for protein purification [1], [2], [3]. It is based on hydrophobic interactions between proteins and hydrophobic ligands at or near neutral pH, which maintains the protein's native state. To prevent protein denaturation upon binding, HIC resins are designed with weak hydrophobicity, utilizing a low ligand density and a weakly hydrophobic ligand. However, this weak hydrophobicity can lead to inefficient protein binding at physiological conditions. To promote efficient protein binding under native conditions, HIC utilizes salting-out conditions, primarily by using (NH4)2SO4 at high concentrations, which belongs to the strongest salting-out class in Hofmeister series [1,[4], [5], [6], [7], [8]]. However, the effects of (NH4)2SO4 and other salting-out additives on protein binding to HIC resins are not fully understood.

Three potential mechanisms have been proposed to explain how salting-out additives promote hydrophobic interactions in HIC. The first mechanism, called “cavity theory,” is based on the effect of salts on the surface tension of water [9]. This theory suggests that a cavity is created to accommodate a protein molecule and a hydrophobic ligand. When the protein and ligand associate, the total surface area decreases, leading to a decrease in free energy of the cavity formation and thereby enhancing protein binding to the ligand. Salts that increase the surface tension of water further promote the binding of the protein to the ligand. The second mechanism is the “dehydration capacity of salts,” which suggests that salts dehydrate the hydrophobic surfaces of native proteins, exposing them to hydrophobic ligands [2,10]. The third mechanism is the “preferential exclusion of salts” [11,12]. Salting-out salts at high concentrations are excluded from the protein surface, which is thermodynamically unfavorable. When the protein molecules associate with the ligand, the unfavorable salt exclusion is reduced, promoting hydrophobic binding to the ligand.

This study was undertaken to evaluate the above three HIC mechanisms. To examine HIC behavior of three model proteins (bovine serum albumin (BSA), lysozyme, and rabbit polyclonal antibody (r-pAb)) on Phenyl Sepharose, we used salting-out (NH4)2SO4 and sodium phosphate, salting-in MgCl2, and sterically excluded amphiphilic polyethylene glycol (PEG) [[13], [14], [15]]. This study reports the results of the effects of these four additives on protein binding to the Phenyl Sepharose, which is then used to evaluate the three HIC mechanisms that had been proposed, with the goal to clarify which mechanism most adequately explains protein binding in HIC.