PMID:23175141
| Citation |
Gomes, AG, Azevedo, AM, Aires-Barros, MR and Prazeres, DM (2012) Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography. J Sep Sci 35:3190-6 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1002/jssc.201200225 |
| Keywords |
Adsorption; Boronic Acids/chemistry; Chromatography, Affinity/instrumentation; Chromatography, Affinity/methods; DNA/chemistry; DNA/isolation & purification; DNA, Bacterial/chemistry; DNA, Bacterial/isolation & purification; Escherichia coli/chemistry; Plasmids/chemistry; Plasmids/isolation & purification |
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