Fundamental Research On The Functionalization And Application Of Cellulose-Based Composite Matrix For Chromatography

The chromatography has been developed with new methods and integrated technologies for bioseparation due to varying separation mechanisms and convenient operation modes. Expanded bed adsorption (EBA) and hydrophobic charge induction chromatography (HCIC) are two of important chromatographic techniques. Based on our previous work, a novel macroporous cellulose-tungsten carbide composite matrix was designed and prepared. Then, the composite matrix was functionalized as anion-exchanger for EBA and HCIC adsorbents. The adsorption properties of new adsorbents as well as the application for antibody separation were investigated. The main points of this thesis were listed as follows.Firstly, with the method of water-in-oil suspension thermal regeneration and starch addition, a series of cellulose-tungsten carbide composite matrix with different densities and porosities were developed. With cellulose as the skeleton, gelatinized cassava starch solution and tungsten carbide were used as porogenic agent and inert densifier, respectively. The matrix prepared showed good sphericity and a logarithmic symmetrical distribution of particle size. The mean pore diameter could reach 129 nm, while particle wet density could be increased with the addition of tungsten carbide. The results demonstrated that the macroporous matrix could be used in the packed bed and expanded bed with perfect properties, such as fast diffusion, low column pressure and good liquid mixing in the bed. In addition, the density of composite matrix could be controlled by altering the densifier addition to match the requirement of varying fluid velocity and expansion factor in expanded bed.Secondly, a new anion-exchanger Cell-TuC-DEAE for EBA application was prepared by coupling diethylaminoethyl (DEAE) ligand on the macroporous cellulose-tungsten carbide composite.matrix. With bovine serum albumin (BSA) as model protein, the static and dynamic adsorption properties of Cell-TuC-DEAE were studied. The saturated adsorption capacity could reach 97.1mg/ml, and the dynamic adsorption capacity was 66.6mg/ml at 500cm/h and 54.5 mg/ml at 900cm/h in expanded bed. The results of adsorption kinetic indicated that the protein diffusion was enhances due to large pore in the matrix, which could be used for high fluid velocity in expanded bed.Thirdly, four HCIC adsorbents were prepared. The macroporous cellulose-tungsten carbide composite matrix was activated with allyl bromide (AB) and divinyl sulfone (DVS), then coupled with three kinds of mercapto heterocyclic groups, 4-mercapto-ethyl-pyridine hydrochloride (MEP), 2-mercapto-1-methyl-imidazole (MMI) and 2-mercapto-benzimidazole (MBI), to form four HCIC adsorbents, Cell-TuC-AB-MEP, Cell-TuC-DVS-MEP, Cell-TuC-DVS-MMI andCell-TuC-DVS-MBI. The preparation conditions were optimized. The content of allyl group could reach 137μmol/ml matrix and the MEP density was 90μmol/ml adsorbents; while the content of vinyl sulfone could reach 93μmol/ml matrix and the ligands density was about 60μmol/ml adsorbents.Fourthly, with model protein, Immunoglobulin of egg yolk (IgY) and BSA, the adsorption mechanism and performance of HCIC adsorbents was discussed. The static adsorption equilibrium, adsorption kinetics, retention factor and protein breakthrough in the column were investigated. The results indicate that the interactions between HCIC adsorbents and protein were determined mainly by pH and salt concentration. At pH 5, the adsorption reached the maximal value and the adsorption capacity was above 100mg/ml. With the increase of pH, the adsorption capacity decreased. When the pH was below the pI of protein and pK_a of ligand, the protein could be desorbed by the protein-ligand electrostatics repulsion. It was also found that the addition of lyotropic salt could enhance the protein adsorption. For all HCIC adsorbents tested, Cell-TuC-DVS-MBI showed highest adsorption capacity, while Cell-TuC-AB-MEP was the least. Cell-TuC-DVS-MMI and Cell-TuC-AB-MEP showed the excellent adsorption selectivity for immunoglobulin. Based on the study of retention factor, the protein could be easily eluted on Cell-TuC-AB-MEP, while harsh elution terms should be used for Cell-TuC-DVS-MBI. The results of adsorption kinetics indicated that the experimental data could be fitted with simplified diffusion model. The total effective diffusion coefficients of HCIC adsorbent were smaller than that of ion exchanger. The protein breakthrough behaviors demonstrated that the adsorbents prepared could be used in packed bed and expanded bed.Finally, Cell-TuC-AB-MEP was used to separate IgY from egg yolk. For packed bed chromatography, the yield was 58.4%, and the purification factor was 2.9 with the purity of 92%. For expanded bed adsorption, the yield was 48.4% with the purification factor of 2.9 and the purity of 89% for the fluid velocity of 500cm/h. These results indicate that the HCIC adsorbents prepared was suitable for the purification of immunoglobulin both in packed bed and expanded bed.The thesis focused on the preparation, functionalization and application of novel cellulose-based composite matrix for chromatography, especially for expanded bed adsorption and HCIC. Some important information on the adsorbent preparation, adsorption mechanism and antibody separation were obtained, which would certainly be useful for the developments of new adsorbent and chromatographic methods.