HCIC – Ypatent

See also “purification schemes” under Antibody Purification. See also “mixed mode chromatography” under Chromatography

Classification:

HCIC is a type of mixed mode chromatographic process in which the protein of interest in the mixture binds to a dual mode (i.e., there is one mode for binding and another mode for elution), ionizable ligand through mild hydrophobic interactions in the absence of added salts (Arunakumari, US 2012/0165511 A1). Hydrophobic charge induction chromatography (HCIC) is a subset of HIC (EP2004052807). HIC is also a type of mixed mode chromatographic process in which the protein of interest in the mixture binds to a dual mode (i.e., there is one mode for binding and another mode for elution), ionizable ligand. HCI is useful for the separation of biological molecules such as proteins based on the pH dependent behavior of ionizable dual-mode ligands. At netural pH, the ligand is uncharged and binds a protein of interest via mild non-specific hydrophobic interaction. As pH is reduced during a buffer gradient, the ligand becomes positively charged and hydrophobic binding is disrupted by electrostatic charge repulsion.

HCIC was developed for protein separation based on the use of dual mode ligands so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligad becomes ionically charged resulting in the deorption of the protein (Guerrier Bioseparation 9: 211-221, 2000).

Types of resins:

HCIC employs a hydrophobic charge induction chromatography material which comprises chromatographical function groups which can in one pH range form hydrophobic bonds to the substance to be separated and which are charged either positively or negatively in other pH ranges (i.e., HCIC uses ionizable hydrophobic groups as chromatographical functional group). A hydrophobic charge induction chromatogrophy resin contains a ligand which has the combined properties of thiophilic effect (i.e., utilizing the properties of thiophilic chromatography), hydrophbicity and an ionizable group for its separation capability. The term “thiophilic” refers to the selectivity that proteins have for sulfone groups that lie in close proximity to theioether groups. Thiophilic absorption chromatogrpahy is based on electron donor-acceptor properties and is distinct from chromatography based on hydrophobicity.

MEP-HyperCel®: Mercapto-ethyl-pyridine (MEP Hypercell) is a common HIC resin. MEP HyperCel, carries a ligand dervied from 4-mercaptoethylpyridine (4-MEP). Such nitrogen hererogycles have been shown to have particular selectivity for immunoglobulins. The pK, of 4-MEP is 4.8. Thus, under near neutral or alkaline conditions, the ligand is uncharged and behaves much like a phenyl group to bind antibody by hydrophobic interaction. However, the distinct structure of the ligand provides the antibody selective characteritics for which this sorbent was designed. The pKa of the ligand also provides sorbent characteristics that support antibody recovery under mild conditions. Desorption is achieved by reducing the pH of the mobile phase to confer a net positive charge on both ligand and antibody. At pH values in the range of 4-4.5, the ligand and most antibodies carry a predominant positive charge. Thus, desorption is achieved under conditions significantly milder than those typically employed during affintiy chromatorgpahy on protien A sorbents (Schwartz J. Chromatog, 908 (2001) 251-263).

Conditions/Parameters:

General Principles:

Generally the polypeptide is bound to the hydrophobic charge induction material under neutral pH conditions and recovered afterwards by the generation of charge repulsion by a change of the pH value. Thus at one pH range, the ligand is predominantly uncharged and binds a protein of interest via mild non-specific hydrophobic interaction. As pH is reduced, the ligand acquires charge and hydrophobic binding is disrupted by electrostatic charge repulsion towards the solute due to the pH shift.

Thus HCIC is based on the pH dependent behavior of dual-mode ionizable ligands. When pH of the mobile phase is reduced, both the ionizalbe ligand and the target molecule take on a net positive electrostatic charge. The resulting electrostatic charge repulsion overcomes hydrophobic binding interactions, and desorption of the biomolecule occur (Schwartz, J. Chromatogr. 908 (2001) 251-263).

The pKa of the ligand for HCIC is carefully chosen so that the ligand will be uncharged at neutral pH where absorption can be achieved solely by hydrophobic interactions at physiological conditions. By adjustment of the mobile phase pH, the ligand can take on charges with the same polarity as the protein, and elution is achieved by electrostatic repulsion. Although the adsorption process in HCIC is similar to that in single mode hydrophobic interaction chromatography (HIC), the charge facilitated elution mode can offer extra advantages of salt independent adsorption and facile elution.

Burton (5,945,520) discloses mixed mode resins which have a hydrophobic character at the pH of binding of the target compound and a hydrophilic and/or electrostatic character at the pH of desorption of the target compound from the resin. These resins overcome the problem of typical mixed mode chromatographic resins of the prior art where binding efficiencies of less hydrophobic target compounds to the resin is not very high unless a high salt concentration is employed.The resins described comprise a solid support matrix and ionizable ligands that have an ionizable functionality and a spacer arm covalently attaching the ligand to the solid support matrix. The ionizable functionality is partically electrostatically charged at the pH of binding of the compound to the resin and is either further charged or charged at a different polarity at the pH of desorption of the compound from the resin.

Purification of particular Proteins

Antibodies: See also Antibody purification –HCIC

MEP Hypercell was specifically designed to capture immunoglobulins. In contrast to HI chromatography, adsorption of antibodies form cell culture supernatants on HCIC resin is accomplished without the need of any pH or ionic strength adjustment. At neutral pH, hydrophobic capture of antibody occurs by both an aliphatic-hydrophobic spacer and a neutral (uncarged) pyridine ring. Once th pH is lowered from pH 7.2 to pH 4, the pyridine ring in the resin and the bound antibodybecome positively charged, resulting in charge repulsion. The immunoglobulins detaches and elutes form the column (WO2005/014621).

Non-immunoglobulin molecules:

Although MEP Hypercell is specifically designed for the purificaiton of antibodies, Schwarts-Burd (WO 2005/014621 A1) discloses that HCIC resins such as MEP Hypcercell can also be used for purifying non-immunoglobulin protines which have Ig-like domains such as IL-18BP. For washing out unbound contaminants, solutions which have the same pH as the loaded material (e.g., PBS 7.2) and/or neutral pH and/or even acidic (e.g., pH 3-6.8) can be used. Elution is carried out with organic solvent such as isopropyl alcohol, propylene glycols or polyalcohols such as glycerol, and polyethylene glycol (e.g., between about 25-50%). In contrast to immunogolbulins, IL-18BP is highly acidic (isoelectri point of about 3) and pH of 4.5 is not acidic enough to induce IL-18BP to become positively charged. Thus, in order to elute the IL-18BP from the column, an organix solvent such as propylene glycol, which can weaken hydrophobic interaction between proteins can be used.

In combination with other types of purification techniques

HCIC-AEX:

Kinoshita (WO02/15927) discloses a method purifying a high mannose glucocerebrosidase (hmGCB) which includes HCIC in combination with one or more ion exchange steps such as AEX.

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

HCIC-CEX:

Arunakumari (US8,129,508) discloses a method of purifying a target protein from a mixure by subjecting the mixture to a CEX and a HCIC in either order wherein the elution buffer for the HCIC or CEX elutes the host cell proteins differentially in relation to the target protein and wherein the tailing of the ABS280 peak of the target protein is not collected at less than 20% agove the baseline as measured by ABS280 relative to the maximum of the peak height.

Kinoshita (WO02/15927) discloses a method purifying a high mannose glucocerebrosidase (hmGCB) which includes HCIC in combination with one or more ion exchange steps such as CEX.