Fabrication Of Superporous Cellulose Matrices For Protein Chromatography

The thesis focuses on the fabrication of superporous cellulose bead for high-speed protein chromatography and its application in the purification of proteins. The details in this work are summarized as follows:A novel superporous cellulose (SC) matrix has been fabricated by water-in-oil emulsification-thermal regeneration using granules of calcium carbonate as porogenic agents. There were more irregular macropores and micropores scattering on the surface of the bead. The pore diffusivity of bovine serum albumin (BSA) in the diethylaminoehtyl-superporous cellulose (DEAE-SC) bead was two to three times larger than that in the diethylaminoehtyl-microporous cellulose (DEAE-MC) bead. In addition, the column packed with the DEAE-SC showed lower backpressure, higher column efficiency and dynamic binding capacity than the column packed with the DEAE-MC at a flow rate range of 150 to 900 cm/h.To enhance the adsorption capacity of BSA to DEAE-SC adsorbents, we fabiricated the grafted superporous cellulose beads using ceric ammonium nitrate as initiator. Infrared spectral analysis confirmed the grafting reaction. The adsorption capacity of BSA to the diethylaminoehtyl-grfted- superporous cellulose (DEAE-G-SC) was over two times increased as compared to the ungrafted DEAE-SC and no nonspecific adsorption of BSA was observed. The grafted polymer chains led to the decrease of flowthrough pores, so the convective mass transport in the particles was significantly reduced, leading to the decrease of column efficiency with flow velocity. Despite this unfavorable effect, however, due to the high adsorption capacity of the grafted beads, the DEAE-G-SC column could still be operated at a higher DBC than the normal DEAE-SC column in the range of 150 to 600 cm/h.The DEAE-SC beads and DEAE-MC beads were packed in a novel preparative electrochromatographic column to investigate the effect of elecric field on mass transfer. The preparative electrochromatographic column is constitutive with seven compartments which can provide two-dimensional (2D) electric field. The results showed that the two columns possessed higher dynamic binding capacity in the presence of 2D electric field, indicating which promote the mass transfer more efficiently. However, the effect of electric field on DEAE-MC column was more notable than DEAE-SC column. It is speculated that the mass transfer in DEAE-SC beads is superior compared with DEAE-MC beads.To determine the property of the DEAE-SC matrix, a purification of GroEL from the cell debris of recombinant E. coil was carried out by the column packed with DEAE-SC beads and DEAE-MC beads, respectively. The results of breakthrough experiments showed that the dynamic binding capacity of the DEAE-SC column was higer than that of DEAE-SC column. Moreover, the purity of GroEL by DEAE-SC column with stepwise gradient elution was higher than that by DEAE-MC column, and dese not affect by the mobile phase velocity.All the results exhibited the superior characteristics of the cellulose beads containing superpores as a potential medium for high-speed chromatography.