Preparation Of Thiophilic Mixed-Mode Adsorbents And Its Application For Immunoglobulin Yolk Separation

Combining the thiophilic chromatography and mixed mode adsorption,new adsorbent with thiophilic mixed-mode ligands were developed and used for antibody separation.Thiophilic chromatography is a novel technology to separate and purify antibody.The special thio-ethylsulfone structures have shown a good selective binding for antibodies in the presence of high concentration of salts.Considering the mixed-mode adsorption,heterocyclic compounds would be introduced as the ligands and new adsorbents called thiophilic mixed-mode chromatography will be designed. The heterocyclic compound residues in the ligands enhance the adsorption of antibody and show a salt-independent adsorption property for the feedstock with medium salt concentration.With the cellulose-tungsten carbide composite beads as the matrix,new kinds of thiophilic mixed-mode adsorbents were prepared.Then the adsorption and separation behavior was measured with immunoglobulin of egg yolk(IgY)and bovine serum albumin(BSA)as model proteins.Finally,new adsorbent prepared was used to separate IgY from egg yolk.With the cellulose-tungsten carbide composite beads as the matrix and divinylsulphone and aUyl bromide as the activation reagent,a series of thiophilic mixed-mode adsorbents were prepared with the heterocyclic compounds as the ligands,including mercaptomethyl-imidazole(MMIM),2-mercapto-benzimidazole (MBIM)and 4-mercaptoethylpyridine(MEP).New adsorbents were named as Cell-WC-DVS-MEP,Cell-WC-DVS-MMIM,Cell-WC-DVS-MBIM and Cell-WC-AB-MEP,respectively.The activation and coupling conditions were optimized for high ligand density.When allyl bromide was used as the activation reagent,the ligand density could reach about 90μmol/ml.The ligand density reached 60μmol/ml with divinyl-sulphone as the activation reagent.With IgY and BSA as model proteins,the isotherm adsorption equilibria for four adsorbents prepared were investigated.The results indicated thiophilic mixed-mode adsorbents showed good adsorption capacity for IgY and have the salt-independent property.The most important factor on the adsorption was pH.High adsorption capacity could be obtained as pH 5,and the conditions of pH lower than 4 could be used for desorption of IgY.Addition of(NH₄)₂SO₄ would enhance the adsorption of IgY.Different adsorbents with varying ligands showed different adsorption properties. Cell-WC-DVS-MMIM was suitable of purifying IgY with high adsorption capacity and good selectivity.Cell-WC-DVS-MMIM was used to separate IgY from egg yolk.IgY could be separated directly from diluted egg yolk with Cell-WC-DVS-MMIM,but the purity was low.Then diluted egg yolk was precipitated at isoelectric point and IgY was salted out.The precipitate was redissolved and loaded to the column with Cell-WC-DVS-MMIM.The purity of IgY in the elution was as high as 98%,and the purify could be kept for high loading concentration and fluid velocity.The results indicated that Cell-WC-DVS-MMIM could be used for purifying IgY,and the application of expanded bed adsorption would be hoped.