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    Removal of endotoxins from plasmid DNA: Analysis of aggregative interaction of mobile divalent metal cations with endotoxins and plasmid DNA

    Access Status
    Fulltext not available
    Authors
    Ongkudon, C.
    Hodges, E.
    Murphy, K.
    Danquah, Michael
    Date
    2012
    Type
    Journal Article
    
    Metadata
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    Citation
    Ongkudon, C. and Hodges, E. and Murphy, K. and Danquah, M. 2012. Removal of endotoxins from plasmid DNA: Analysis of aggregative interaction of mobile divalent metal cations with endotoxins and plasmid DNA. Journal of Separation Science. 35 (22): pp. 3208-3216.
    Source Title
    Journal of Separation Science
    DOI
    10.1002/jssc.201200237
    ISSN
    1615-9306
    School
    Curtin Sarawak
    URI
    http://hdl.handle.net/20.500.11937/47699
    Collection
    • Curtin Research Publications
    Abstract

    Endotoxin lipopolysaccharide removal from plasmid DNA-based vaccine remains a very challenging task for bioprocess engineers. This paper examined the potential use and advantages of divalent cation (Zn2+, Ca2+, Mg2+) induced aggregation as a plasmid DNA purification method for lipopolysaccharide removal. Analysis of zeta potential, hydrodynamic size, percentage of aggregation; UV-Vis spectroscopy and electron microscopy were performed to determine the optimal cation for preferential aggregation of lipopolysaccharide over plasmid DNA. The results from the hydrodynamic size analysis showed that the addition of Zn2+ resulted in the maximum theoretical number of lipopolysaccharide molecules per aggregate particle. Dynamic light scattering analysis showed that plasmid DNA aggregates formed a larger maximum hydrodynamic size when it was treated with Ca 2+ than the other two cations. The Km value for lipopolysaccharide-Zn2+ was substantially low (0.28 M) and considerably large (>2 M) for plasmid DNA-Zn2+. Scatchard plots for plasmid DNA cations showed positive slopes indicating that there was a minimum concentration of plasmid DNA or cations before a significant aggregation occurred. This work concluded that Zn2+ had the most preferential aggregative interaction with lipopolysaccharide compared to Mg2+ and Ca2+.

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