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

Authors

  • A. Gabriela Gomes,

    1. IBB, Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal
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  • Ana M. Azevedo,

    1. IBB, Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal
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  • M. Raquel Aires-Barros,

    1. IBB, Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal
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  • D. Miguel F. Prazeres

    Corresponding author
    1. IBB, Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal
    • Correspondence: Professor D. Miguel F. Prazeres, IBB, Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

      E-mail: miguelprazeres@ist.utl.pt

      Fax:351-218419062

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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 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 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 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.

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