(compare hydrophobic charge induction chromatography under “mixed mode chromatography”); See also antibody purificaiton by HIC

HIC is useful for the purification and separation of molecules such as proteins based on differences in their surface hydrophobicity. Hydrophobic groups of a protein interact non-specifically with hydrophobic groups coupled to the chromatography matrix. Differences in the number and nature of protein surface hydrophobic groups results in differential retardation of proteins on an HIC column and, as a result, separation of proteins in a mixture of proteins. HIC was first developed following the observation that proteins could be retained on affinity gels which comprised hydrocarbon spacer arms but lacked the affinity ligand. 

Applications: Hydrophobic interactions are strongest at high ionic strenght. Thus this form of separation is conveniently performed following salt preciptiations or ion exchange procedures (Shadle, US5429,746). A description of the general principles of HIC can be found in US 3,917,527 and 4,000,098. The application of HIC to the purificaiton of specific proteins include human growth hormone (US4,332,717), toxin conjugates (US4771128), antihemolytic factor (4,743,680), TNF (4,894,439), IL-2 (US4,908,434), human lymphotoxin (US4,920,196). HIC steps are generally performed to remove protein aggregates, such as antibody aggregates and process related impurities. 

Mechanism of Action: Whereas ion exchange chromatography relies on the charges of the antibodies to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of the antibodies. Hydrophobic groups on the antibody interact with hydrophobic groups on the column. The more hydrophobic a protein is the stronger it will interact with the column.

Types of HIC Resins/Ligands

In general, HIC resins contain a base matrix (e.g., cross-linked agarose or synthetic copolymer) to which hydrophobic ligdands (e.g., alkyl or aryl groups) are ocupled. Examples include Phenyl SEPHAROSE 6 FAST FLOW (Pharmacacis LKB Biotechnology, AB, Sweden), Phenyl SEPHAROSET high performance, Octyl SEPHAROSET High performance, Fractogel EMID Propyl or fRACTOGEL EMD Phenyl (Merck), MACRO-PREP Methyl or MACO_Prep t-Butyl (Bio-Rad, CA), WP HI-Propyl (C3) (J.T. Baker, NJ), TOYPEARL either phenyl or butyle (TosoHaas, PA) and Tosoh-Butyl-650M (Tosoh Corp. Tokyo). (Welsh, US 20230077205)

HIC column normally comprises a base matrix (e.g., cross-lined agaros or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. Suitables column include an agarose resin substituted with phenyl groups (e.g., a Phenyl SepharoseTM column). Many HIC column are commercially available (Hickman, US 12/582556). The most extensively used chromatogrpahic supports for HIC are agarose, silica and organic polymer or co-polymer resins. Useful hydrophobic ligands include groups having from about 2-8 carbon atoms such as butyl, propyl, or octyl or aryl groups such as phenyl. Conventional HIC products for gels and columns may be obtained commercially from suppliers such as Pharamcia LKB AB, Upsala, Sweden (butyl-SEPHAROSE), phenyl or butyl-SEPHAROSE CL-4B, butyl-SEPHAROSEFF, octyl-SEPHAROSE FF and phenyl-SEPHAROSE FF or Tosoh Corporation, Tokyo Japon as TOYOPEARL either 650, phenyl 650 or butyl 650 (Factogel); Miles-YEda, Rehovot, Israel as aklyl-agarose, wherein the alkyl group contains from 1-10 C atoms and J.T. Baker, Phillipsburg, N.J. as Bakerbond WP-HI-propyl. (Shadle, WO 95/22389) It is also possible to prepare the desired HIC column using conventional chemistry. (Biochem.Biophys. Res. Comm, 49:383 (1972)) 

A suitable column is one whose stationary phase comprises hydrophobic groups such as a phenyl Sepharose column. US 2010/0075375 A1 lists common HIC resins. 

Membrane HIC: Cellulose membranes bonded with four commonly used hydrophobic lgiands, octyl, butyl, pheny and polyethylene glycol have been evaluated for protein and enzyme purification, reportedly resulting in faster purification. Both polyvinylidene fluoride (PVDF) and paper-PEG membranes have also been examined for purificaiton for mAbs. For mAb purification it is advantageous to carry out the membrane HIC in flow through mode so that the majority of the product of interest will flow through, leaving the limited capacity for absorption of aggregates and other impurities. Since mAb aggregates show higher hydrophobicity and elute later than the monomer species, it is conceivable that membrane HIC can be designed to be carried out in flow through mode for aggregate removal. (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)).

While HIC has been presented as an efficient mode of removing dimers and higher MW aggregates when used as a polishing step in mAb purification, the flow rate and diffusion limitation associated with packed-bed HiC can increase the risk of protein denaturation due to the long contact time on the hydrophobic surface and high concentration of lyotropic salt. The use of a convective chromatography technique such as membrane chromatography reduces these problems by allowing much faster processing time.  In this respect, Phenyl™ membrane adsorber has been developed for large scale purificaiton of biomolcules based on HIC principles. (Kuczewski, Biotechnology & Bioengineering, 105(2), 2010).

Conditions/parameters  see outline

In Combination with Other Purification Techniques

HIC-IEX (CEX or AEX): Daniel (US7,138,262) teaching a method of purifying high mannose glucocerbrosidase (hmGCB) by subjecting the hmGCB to HIC such as MEP Hypercel and further purification by at least one ion exchange chromatography step such as AEX or CEX.