Studies On The Process Intensification Of Protein Chromatography

This thesis is focused on the process intensification of protein chromatography towards the objective of high performance preparation. The main approaches proposed here are the preparative electrochromatography and the superporous agarose gel. The development and application of preparative two-dimensional electrochromatography (2DEC) as well as the fabrication, grafting modification and adsorption performance of superporous agarose gel were all investigated in this work. The details are summarized as follows:A novel preparative 2DEC column with seven compartments was developed. The design gave promise to provide transverse and longitudinal electric field on the column in order to induce two-dimensional electrokinetic transports in the central gel compartment. The dye-ligand affinity chromatography and immobilized metal affinity chromatography (IMAC) were performed in the 2DEC respectively. It was confirmed that the dynamic binding capacity (DBC) of protein in 2DEC was promoted more efficiently than that in one-dimensional electrochromatography. Moreover, the results indicated that the electroosmotic flow on the charged adsorbent surface was the predominant electrokinetic transport in the affinity electrochromatography. The effect of ionic strength proved that low-salt concentration was more suitable for the electrochromatography. Then, alcohol dehydrogenase was purified by the 2DEC packed with Zn-Sepharose FF at loading volume which was 2-fold higher than that by the traditional IMAC. The results have proved that the 2DEC is an efficient technique for the process intensification of protein chromatography and promising for the high capacity protein separation in the downstream processing.The entropic interaction chromatographic (EIC) resins were fabricated by the grafting of glycidyl methacrylate to TOYOPEARL HW-65. The resins gave a high resolution of solutes during the separation range of 0.67 kD to 158 kD. The EIC resins of different grafting densities were used to investigate the transport mechanism in EIC with an oscillatory electric field perpendicular to mobile phase flow driven by pressure. The results demonstrated that the electrophoresis was the dominante electrokinetic transport in the EIC resin with a low grafting density for all the solutes employed in this research and in the resin with a high grafting density for the solutes of low molecular weights. However, the concentration polarization played a key role in the retention behaviors for the solutes of high molecular weights. The concentration polarization parameters and the electrophoretic mobilities in the station phase of solutes were calculated by a mathematical model developed. The concentration polarization parameters would increase with the increasing molecular weight and free electrophoretic mobilities but decrease with the increasing partition coefficient and free solution diffusivity.The scaling-up of the fabrication of superporous agarose gel (SA) was accomplished in a reactor of 10 L with Brumagin agitator and four baffles in it. Dextran chains of 40 kD were grafted into the pores of SA in order to improve the adsorption properties. Besides the increase of adsorption capacity led by the grafting of dextran chains, the protein uptake rate was also greatly increased. So, the grafted SA resin showed the high DBC and adsorption efficiency even at a high flow rate. Hence, it is considered that grafting modification was a prominent approach to improve the properties of superporous agarose gel.