Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography.

This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (p DNA)-containing Escherichia coli lysates by phenyl-boronic ( PB) acid chromatography using columns packed with 7.6 and 15.2 cm³ of controlled porous glass beads ( CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm³/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 p DNA 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 cm³) 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 dm³ of alkaline lysate per hour per dm³ 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. [ABSTRACT FROM AUTHOR]