See also HIC and HCIC under “affinity chromatography” 

Hydrophobic charge induction chromatography (HCIC) is a type of mixed mode chromatography which as advatnages of mild elution conditionk high capacity and salt-toelrance. The binding of HCIC resin and target proteins can be acheived vai hydrophobic interactions at neutral pH, and then prtoeins can be eluted via electrostatic repulsion between proteins and charge dligands under acidic conditions. (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

Advantages of HCIC for the purification of antibodies: 

A sorbent that employes HCIC does not require feedstock preconditioning, such as concentration, dilution, pH adjustment or addition of specific sals, to bind antibodies optimally. Proteins are absorbed directly from a variety of sources under physiological conditions and elution is achieved by simply lowering the pH.(Guerrier, 755 (2001) 37-46).

CIC shows specific advantages over traditional methods for antibody purification. Sample preparation is primarily limited to the clarificaiton of feedstocks, which can be loaded onto the column without adjustment of ionic strength or pH. IgG dynamic binding capacity ranging from 25-35 mg/ml-1 of sorbent at pH708 and a 10% breakthorugh, are routinely acheived even for murine IgG1. Concomitant purification and concentration effects are acheived in a single step. Several parameters contribute to acheive the sorbent proerties for IgG capture, such as the structure of the ligand as a whole and the ligand density attached to the porous matrix. Although most of the investigations to date have been focused on the separation of antibodies of IgG class, preliminary trials have also been performed to separate IgA. Specificity was not extensively investigated, but binding capacity determinations showed significantly lower numbers than for IgG.  (Boschetti “Antibody spearation by hydrophobic charge induction chromatography” Trends in Biotechnology 20(8) 2002).

Conditions: 

Over a pH range 6-9, the ligand is uncharged. Under these conditions, the ligand and the spacer arm behave much like a hydrophobic site and bind the protein by hydrophobic association. When pH of the mobile phase is modified, the ligand takes a net ionic charge. Under these conditions, the antibody also carries a similar net positive charge, therefore desorption occurs on the basis of electrostatic repulsion between the solid phase sorbent and the protein. In contrast to hydrophobic interaction chromatography or thiophilic chromatography, hydrophobic charge induction chromatography is controlled on the basis of pH rather than salt concentration (Guerrier, Bisoseparation, 9, 211-221, 2000). 

Hydrophobic charge induction chromatography provides for antibody capture without need for adjustment of pH or ionic strengh of typical feedstocks. Like bioaffinity chromatography on Protein A/G, capture is accomplished under near physiological conditions. HICIC is based on the pH dependent behavior of dual-mode, ionizalbe ligands (e.g., pyridine derivative). Over a pH range 6-9, the ligand is uncharged. Under these conditions, the ligand the spacer arm behave much like a hydrophobic stie and bind the protein by hydrophobic association. The sorbent is designed so that binding occurs without need for addition of lyotropic salt. In contrast to HIC or thiophilic chromatography, hydrophogic charge induction chromatography is controlled on the basis of pH rather than salt concentraiton. When pH is modified, the ligand takes a net ionic charge and the antibodies also carries a similar net positive charge and thus desorption occurs. (Guerrier Bioseparation 9: 211-221, 2000)

Washing:

–Caprylic acid is a saturated fatty acid which is commonly used to precipitate protein impurities and separate IgG from plasma, serum and ascites. Tong used caprylate as the albumin selective additive to enhance adsorption selectivity of MEP Hypercell. The reustls showed that the addition of 50 mM adoium caprylate in the laoding ufer and 75 mM in teh washing buffer could greatly improve IgG purity when separating form BSA/IgG mixtures. (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

—ethylene glycol + Inorganic salt: 

Falkenstein (US13/883243, published as US Paent No: 9422329; see also US Patent Application No: 15/216099, published as US Patent 10377794 and US Patent Application 16/778,128, published as US 20200165297) discloses washing an antibody from a multimodal weak CEX such as Capto MMC or Streamline CST using ethylene glycol and an inorganic salt such as sodium chloride, potassium chloride and ammonium chloride. In some embodiments, a binding buffer comprising an inorganic salt and denaturant such as gaunidinium hydrochloride or urea is applied to the multimodal weak CEX.

Elution:

—ethylene glycol + Inorganic salt: 

Falkenstein (US13/883243, published as US Paent No: 9422329; see also US Patent Application No: 15/216099, published as US Patent 10377794 and US Patent Application 16/778,128, published as US 20200165297) discloses eluting an antibody from a multimodal weak CEX such as Capto MMC or Streamline CST using ethylene glycol and an inorganic salt such as sodium chloride, potassium chloride and ammonium chloride. In some embodiments, a binding buffer comprising an inorganic salt and denaturant such as gaunidinium hydrochloride or urea is applied to the multimodal weak CEX.

Particular Resins Used

MEP HyperCell: 

MEP HyperCel is the most widely used HCIC resin whcih was commercialied by Pall. The funcitonal ligand of MEP HyperCel is 4-mercaptoethyl-pyridine (MEP) which contains a pyridine structure and a hydrophobic long chain with a sulfur atom. It has a pKa of 4.8 that enables adsorption happened at neutral pH.  (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

Guerrier (Bioseparation 9: 211-221, 2000) discloses using MEP-HyperCell for the purificaiton of antibodies from ascite fluid, cell culture and cell culture supernatant. The column was initially equilibrated with a phosphate buffered slaid (25 mM phosphate containing 150 mM sodium chloride, pH 7.4), the colunm then loaded, washing tih 50 mM Tris-HCl buffer pH 8 to wash out protein impuriteis. 4-mecaptoethyl pyridine or 4-MEP has enhanced specificity for immunogloublins compared to a simple phenyl group. The pKa of the ligand is 4.8. At pH<4,7, the ligand takes on a predominant postivie charge. As a result the pH of the mobile phase needs only be reduced to 4.0-4.8 for desorption to ccur. 

HCIC in combination with other purification techniques: 

(As to other forms of chromatography followed by HCIC, see respective sections. For example, CEX-HCIC, see cation exchange for purification of antibodies)

HCIC – (Ion affinity or ion exchange or HIC or RPC): 

Rossi (WO/2005/049649) describes HIC used for the purification of IL-18 binding protein and further a step slected from immobilized metal ion affinity chromatography, ion exchange chromatrogaphy, HIC and RPC.

HCIC-AEX:

Boschetti (TRENDS in Biotechnology, 20(3) 2002) teaches that it is known to combine HCIC with AEX.

HCIC-CEX

Arunakumari (US13/410,740, now US8,697,847) teaches a method of purifying an antibody using CEX followed by HIC where there is no in -process tangential flow filtration step. 

Rossi (WO2005/049649) teaches purifcation of IL-18 binding protein using HCIC in combination with IEX such as CEX.

—HCIC-CEX-HIC-UF/DF: 

Boyle (US2004/0033535) discloses purification of alphaOPGL-1 antibody by HCIC with MEP HyperCel to remove a majority of host cell proteins and DNA. The MEP HypercerCel resin is washed with equilibration buffer (20 mM Tris pH 7.2) and the antibody eluted form the resin using a low pH buffer (20 mM Sodium Acetate, pH5). The MEP pool is titrated to pH 3.7 and help for about 60 minutes to inactivate virus (viral inactication), then filered and then the antibody is further purified by CEX using SP Sepharose HP which removed additional CHO cell proteins, DNA, lower weight MW proteins and aggregated forms of alphaOPGL-1. The column is washed and eluted with a linear gradient of increasing salt. The antibody is further purified by HIC and then concentration and fiafiltrated by TFF UF.

(HCIC -Hyrdroxyapatite (HT):  

Guerrier (J. Chromatogr. B. 755 2001, 37-46) teaches isolation of antibodies using HCIC in combination with hydroxyapatite as a second step. The optimal adsorption on the MEP HyperCel column was 7-9 at physiological ionic strengh.