Preparation Of Cellulose Matrices With N-methylmorpholine Oxide Dissolution And Application For Hydrophobic Charge-Induction Chromatography

Protein purification is a series of processes intended to isolate target protein from a complex mixture. A comprehensive improvement of chromatography method includes matrix, operation mode and separation conditions. The traditional cellulose-based matrices are produced by cellulose xanthate viscose method, which requires relatively harsh conditions and the use of expensive and uncommon solvents and some environmentally hazardous byproducts are generated. Therefore, this research used N-methyl morpholine oxide (NMMO) as the non-derivatizing "green" solvent to dissolve cellulose and prepare the cellulose-tungsten carbide composite beads. Then, the composite matrix was functionalized as hydrophobic charge-induction chromatography (HCIC) adsorbent with 2-mercaptoimidazole (MI) as the functional ligand. The adsorption properties of new adsorbent as well as the application for the separation of immunoglobulin G (IgG) were investigated. The main points of this thesis were listed as follows.Firstly, with the NMMO as the non-derivatizing dissolution solvent, cellulose can be dissolved directly. Addition of tungsten carbide powder as the densifier, the cellulose-tungsten carbide composite beads could be formed through the method of water-in-oil suspension. In general, the composite beads had the spherical appearance, suitable size distribution, appropriate wet density of 1.6～1.7 g/ml, water content of 48～53%, porosity of 80～90%, pore volume of 1.05～1.13 ml/g for large fraction (Cell-TuC-N-L) and 0.92～1.08 ml/g of small fraction (Cell-TuC-N-S), pore radius of 72～91 nm, and specific surface area of 17.64～23.25 m2/ml. Compared to the traditional technology with cellulose xanthate viscose, new method based on the NMMO dissolution is more environment-friendly, reduces the preparation time and shows a potential application for large scale.Secondly, the Cell-TuC-N-S composite beads prepared with NMMO dissolution method were tested with the expansion characteristic and the axial mixing in an expanded bed for a potential EBA application. The results indicated that the cellulose composite beads prepared have a good expansion property and could be used at high operation flow velocity (1100～1800 cm/h).Thirdly, the Cell-TuC-N-S matrix was used to couple a novel HCIC ligand,2-mercaptoimidazole (MI), to prepare new Cell-TuC-N-S-DVS-MI resin after the activation with divinyl sulfone. The density of vinylsulfone group on the Cell-TuC-N-S-DVS could reach 100μmol/ml gel, and the ligand density was about 60μmol/ml gel. Using porcine IgG as a model protein, the adsorption behaviors were investigated. It was found that the adsorption capacity of IgG could reach a high level at pH 7 and pH 8 with the Qm of 77.68 and 78.02 mg/ml gel, respectively. When the pH was below the pI of protein and pKa of ligand the protein could be desorbed by protein-ligand electrostatics repulsion. It was found that the adsorption capacity decreased significantly at pH 4.Finally, new HCIC resin, Cell-TuC-N-S-DVS-MI, was used to purify IgG from crude porcine plasma using packed bed chromatography. The crude sample was loaded at pH 7 and the elution was induced at acidic pH. The purity of IgG could reach more than 80% with the maximum of 89.4% for the elution at pH 3.6 with the purification factor of 3.35. The HCIC process with new Cell-TuC-N-S-DVS-MI resin could provide a powerful technique with high selectivity for the IgG purification from complex feedstock.The thesis focused on the preparation, functionalization and application of novel cellulose-based composite matrix for protein separation chromatography. Some information obtained would certainly be useful for the developments of new adsorbent and the application of antibody purification.