, Volume 30, Issue 1–3, pp 159–168 | Cite as

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

  • Ruth Freitag
  • Sabine Vogt


The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities.

continuous bed column displacement chromatography lipopolysaccharide plasmid DNA SMA model 


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  1. Antia F and Horvath C (1989) Operational modes of chromatographic separation processes. Ber Bunsenges Phys Chem 93: 961.Google Scholar
  2. Bernardi G (1971) Chromatography of nucleic acids on hydroxyapatite columns. Methods Enzymol 21: 95.CrossRefGoogle Scholar
  3. Brooks CA and Cramer SM (1992) Steric mass action ion exchange displacement profiles and induced salt gradients. AIChE J 38: 1969-1978.CrossRefGoogle Scholar
  4. Freitag R and Breier J (1995) Displacement chromatography in biotechnological downstream processing. J Chromatogr 691: 101.CrossRefGoogle Scholar
  5. Freitag R, Fix M and Brüggemann O (1997) Analysis of endotoxins by capillary electrophoresis. Electrophoresis 18(10): 1899-1905.PubMedCrossRefGoogle Scholar
  6. Kasper C, Vogt S, Breier J and Freitag R (1996) Protein displacement chromatography in hydroxy-and fluoroapatite columns. Bioseparation 6: 247-262.Google Scholar
  7. Kundu A, Barnthouse KA and Cramer S (1997) Selective displacement chromatography of proteins. Biotech Bioeng 56: 119-129.CrossRefGoogle Scholar
  8. Sten RG, MacDonald CG, Weaver AL and Pitt AM (1993) Rapid fluorescence quantitation of plasmid miniprep DNA, BioTechniques 15(5): 932-933.Google Scholar
  9. Vogt S and Freitag R (1997) Comparison of Anion Exchange and Hydroxyapatite Displacement Chromatography for the Isolation of Whey Proteins. J Chromatogr 760: 125-137.CrossRefGoogle Scholar
  10. Vogt S and Freitag R (1998) Displacement chromatography using the UNOTM continuous bed column as stationary phase. Biotechnol Progr (submitted).Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Ruth Freitag
    • 1
  • Sabine Vogt
    • 1
  1. 1.Laboratoire de Biotechnologie Cellulaire (CBUE)Ecole Polytechnique Fédérale de LausanneEcublensSwitzerland

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