Studies Of Novel Liquid Chromatography Matrices For High-Speed Biopolymer Purification

Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and the mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly (glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N, N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores that are, micropores (10-90 nm) and macropores (180-4000 nm). Because of the presence of the macro pores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was high and the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.Then the epoxy-contained biporous micro sphere matrix which was modified by iminodiacetic acid (IDA) was prepared for metal chelate affinity chromatography (IMAC). The retention behaviors of four model proteins on IDA coupling column and metal chelate adsorbents packed column loading with Cu2+and Ni2+ was studied at a wide pH range 4.0-9.0. The high performance separation of four proteins within 300cm/h-2400cm/h by the biporous Ni2+-IDA beads column was demonstrated effectively. The recombinant fusion protein His6-IL-11 was purified by Ni2+-IDA biporous affinity chromatography. The collected peak was analyzed by SDS-PAGE. Result show that the purity of recombinant IL-11 was 94%.A rigid spherical giant porous matrix has been prepared by radical suspension-polymerization on the basis of the novel porogenic mode only using superfine granules of calcium carbonate as solid porogen. Scanning electron microscopy reveals that the bead has giant pore as large as 8μm. The hydrodynamic properties show that the superporous column has good strength and low backpressure when flow velocity reached to 3000cm/h. After being modified to anion-exchanger, above 1000 (μg plasmid/mL bed) high dynamic binding capacity of plasmid DNA by the matrix packed column could be obtained. Furthermore, 2mL and 20mL scale preparative chromatography of plasmid separations from cell lysate were investigated. 75% purification yield and 94.9% purity of plasmid DNA were obtained by 20 mL superporous matrix chromatography separation.