title: Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography.
abstract: This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm(3) of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm(3) /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of pDNA was recovered in the flow through while 66-71% of impurities remained bound (~2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm(3) ) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm(3) of alkaline lysate per hour per dm(3) of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration.
authors.affiliation: IBB, Institute for Biotechnology and Bioengineering, Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal.
authors.forename: A Gabriela
authors.initials: AG
authors.lastname: Gomes
authors.forename: Ana M
authors.initials: AM
authors.lastname: Azevedo
authors.forename: M Raquel
authors.initials: MR
authors.lastname: Aires-Barros
authors.forename: D Miguel F
authors.initials: DM
authors.lastname: Prazeres
mesh_terms: D000327:Adsorption; D001897:Boronic Acids / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D001897:Boronic Acids; D004269:DNA, Bacterial; D004247:DNA; C010686:benzeneboronic acid