Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption.
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.
Gomes
A Gabriela
AG
IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisboa, Portugal. miguelprazeres@ist.utl.pt
Azevedo
Ana M
AM
Aires-Barros
M Raquel
MR
Prazeres
D Miguel F
DM
D000327:Adsorption; D001897:Boronic Acids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology*; D046911:Macromolecular Substances / Q000302:isolation & purification*; D009696:Nucleic Acids / Q000302:isolation & purification; D010957:Plasmids / Q000737:chemistry*; D012996:Solutions
D001897:Boronic Acids; D046911:Macromolecular Substances; D009696:Nucleic Acids; D012996:Solutions; C010686:benzeneboronic acid