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Validation and scale‐up of plasmid DNA purification by phenyl‐boronic acid chromatography
Author(s) -
Gomes A. Gabriela,
Azevedo Ana M.,
AiresBarros M. Raquel,
Prazeres D. Miguel F.
Publication year - 2012
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201200225
Subject(s) - chromatography , chemistry , elution , lysis , adsorption , filtration (mathematics) , boronic acid , yield (engineering) , extraction (chemistry) , agarose , solid phase extraction , monolithic hplc column , high performance liquid chromatography , biochemistry , materials science , organic chemistry , statistics , mathematics , metallurgy
This study addresses the feasibility of scaling‐up the removal of host cell impurities from plasmid DNA (p DNA )‐containing E scherichia 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 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 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.