A novel superporous agarose medium for high-speed protein chromatography

Abstract

A novel superporous agarose (SA) bead characterized by the presence of wide pores has been fabricated by water-in-oil emulsification using solid granules of calcium carbonate as porogenic agent. After cross-linking, the solid granules were removed by dissolving them in hydrochloric acid. Then, the gel was modified with diethylaminoethyl groups to create an anion exchanger, SA-DEAE, for protein adsorption. A homogeneous agarose (HA) bead was also produced and modified with DEAE for comparison. It was found that the porosity of SA-DEAE was about 6% larger than that of HA-DEAE. Moreover, both optical micrographs and confocal laser scanning microscopy (CLSM) of the ion exchangers with adsorbed fluorescein isothiocyanate (FITC) labeled IgG revealed the superporous structure of the SA medium. In addition, the SA-DEAE column had lower backpressure than the HA-DEAE column, confirming the convective flow of mobile phase through the wide pores. Due to the presence of the wide pores, more channels were available for protein transport and, furthermore, more diffusive pores in the agarose network were accessible for the protein approach from different directions. This led to 40% higher protein capacity and two times higher effective pore diffusivity in the SA-DEAE than in HA-DEAE. Moreover, an increase of the efficiency of the SA-DEAE column until a flow rate of 5 cm/min and the independency of the column efficiency at flow rates from 5 to 17.8 cm/min was found, indicating that intraparticle mass transfer was intensified by convective flow at elevated flow rates. Therefore, the chromatographic resolution of IgG and BSA was little affected up to a flow rate of 17.8 cm/min. The results indicate that the SA medium is favorable for high-speed protein chromatography.