Indexed on: 12 Mar '10Published on: 12 Mar '10Published in: Journal of Chromatography A
The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (approximately pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6+/-3.1% of RNA, 94.6+/-0.8% of proteins and 96.7+/-11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2+/-4.0%), even though the RNA (65.5+/-2.8%), protein (84.4+/-1.3%) and gDNA clearance (44.7+/-14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.