Hydrophobic interaction chromatography (HIC) is a powerful tool for the process purification of biomolecules. The technique utilizes the accessible hydrophobic regions located on protein surfaces and their interactions with a weakly hydrophobic stationary phase. HIC is an excellent complement to ion exchange and size exclusion chromatography particularly when protein isoforms exist or when feedstock impurities are of similar isoelectric point or molecular weight. The selectivity differences exploited by HIC can also be used after affinity separations in which closely related proteins with similar recognition sites are not distinguishable by the affinity ligand.
Proteins and other molecules with hydrophobic surfaces are attracted to the hydrophobic ligands of both reversed phase (RPC) and HIC resins. RPC resins have higher surface coverage and/or more hydrophobic ligand compared to HIC resins. Because of this, in a RPC separation the target binding readily occurs in an aqueous solution, and desorption is promoted by the addition of an increasing amount of organic solvent. In HIC, proteins are bound to the resin by employing an aqueous high salt mobile phase. The salt conditions contribute to a lyotropic effect which allows the proteins to bind to the lower surface coverage of a hydrophobic ligand. Proteins are eluted by the simple technique of decreasing the salt concentration. Most therapeutic targets are eluted in a low salt or a no salt buffer. During elution the energy of interaction for a HIC step is less than that of a RPC step. One means of gauging the relative binding energy between the two techniques is to measure the surface tension of the two sets of binding and elution conditions. Since HIC
separations are done with under milder eluting conditions, biological activity is typically retained.
Tosoh Bioscience offers five HIC ligands featuring different degrees of hydrophobicity and selectivity. The hydrophobicity of HIC resins increases through the ligand series: Ether, PPG (polypropyleneglycol), Phenyl, Butyl, and Hexyl.