Preparation and Evaluation of Cellulose Adsorbents for Hydrophobic

Nov 12, 2008 - First, the cellulose beads were activated by allyl bromide (AB) or divinyl sulfone (DVS), and then they were coupled with three types o...
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9566

Ind. Eng. Chem. Res. 2008, 47, 9566–9572

Preparation and Evaluation of Cellulose Adsorbents for Hydrophobic Charge Induction Chromatography Hai-Feng Xia,† Dong-Qiang Lin,†,‡ Li-Ping Wang,† Zheng-Jie Chen,§ and Shan-Jing Yao*,†,§ Department of Chemical and Biochemical Engineering, Zhejiang UniVersity, Hangzhou 310027, People’s Republic of China, State Key Laboratory of Chemical Engineering, Zhejiang UniVersity, Hangzhou 310027, People’s Republic of China, and Zhejiang Key Laboratory of Antifungal Drugs, Hisun Pharmaceutical Co., Ltd., Taizhou 318000, People’s Republic of China

Hydrophobic charge induction chromatography (HCIC) has been proven to be an efficient technique for antibody purification. Several HCIC adsorbents were prepared with macroporous cellulose-tungsten carbide composite beads (Cell-TuC) as the matrix. First, the cellulose beads were activated by allyl bromide (AB) or divinyl sulfone (DVS), and then they were coupled with three types of mercaptoheterocyclic groupss4-mercaptoethyl-pyridine hydrochloride (MEP), 2-mercapto-1-methyl-imidazole (MMI), and 2-mercapto-benzimidazole (MBI)sas the HCIC ligands. Four types of HCIC adsorbents were obtained, labeled Cell-TuC-AB-MEP, Cell-TuC-DVS-MEP, Cell-TuC-DVS-MMI, and Cell-TuC-DVS-MBI. The activation and coupling conditions were optimized for high ligand density. The isotherm adsorption of immunoglobulin of egg yolk (IgY) on four HCIC adsorbents were investigated. High adsorption capacities of IgY could be obtained for all four adsorbents at pH 7, and low adsorption of IgY at pH 4 and of bovine serum albumin (BSA) at pH 7 was observed, which indicates that the HCIC adsorbents prepared have a potential application for antibody purification. 1. Introduction Nature or monoclonal antibodies have been widely researched and developed as therapeutical drug or in vitro diagnostics, especially after the hybridoma technology appeared in the 1980s. More-effective and more-efficient purification strategies were required to fulfill the needs of economical industrial-scale antibody production. Among varying separation methods, affinity-based chromatography was the method that was most widely used for antibody purification. A large number of biospecific molecules were used as antibody-binding ligands, including antigen, antiantibodies, lectins, or bacterial protein.1,2 One of the most valuable commercial adsorbents is the Protein A affinity adsorbent, which has been applied in many processes.3,4 Protein A binds specifically to the Fc domain, exhibiting excellent selectivity for antibody isolation. However, the high cost of Protein A affinity adsorbents limits its application for large-scale production. Moreover, Protein A adsorbents normally cannot tolerate sodium hydroxide (NaOH) at high concentration (>0.5 M) for the clean-in-place procedures and Protein A ligand might be degraded by the proteases in the feedstocks.5 Therefore, the pseudo-biospecific ligands have been developed to overcome these drawbacks. In 1985, Porath et al.6 reported on the concept of thiophilic interaction chromatography (TIC). The classical adsorbents, which are activated by divinyl sulfone and subsequently are coupled by 2-mercaptoethanol, are called T-gels. T-gels have been used for the successful separation of monoclonal antibodies.7 Hansen et al.8 reported the recovery close to 100%. Generally, antibody binding with TIC adsorbents was achieved * To whom correspondence should be addressed. Tel.: +86-57187951982. Fax: +86-571-87951015. E-mail address: [email protected]. † Department of Chemical and Biochemical Engineering, Zhejiang University. ‡ State Key Laboratory of Chemical Engineering, Zhejiang University. § Zhejiang Key Laboratory of Antifungal Drugs, Hisun Pharmaceutical Co., Ltd.

via the interactions of electron donor-acceptor complexes, because of the sulfone and thioether in the ligand structure.9,10 However, as observed with hydrophobic interaction chromatography, TIC is also salt-dependent, which means the adsorption should be conducted at relative high lyotropic salt concentrations while the desorption is achieved by reducing the salt concentration. In 1998, Burton and Harding11 described a new chromatographic technique: hydrophobic charge induction chromatography (HCIC). The ligands of HCIC have multiform functions, commonly combining thiophilic, hydrophobic, and electrostatic interactions. The target protein can be adsorbed on the HCIC ligand by thiophilic and hydrophobic effects and desorbed under the electrostatic repulsion between protein and the charged groups. The commercial HCIC adsorbent is MEP HyperCel, which was developed by the Pall Corporation. The dynamic binding capacities of MEP HyperCel for murine IgG could reach the range of 25-35 mg/mL.12 The ligand contains a pyridine ring with a pKa of 4.8 and a sulfur atom in the hydrophobic chain. The adsorption occurs at neutral pH, and the elution should be induced at pH