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    Analysis of selective metal-salt-induced endotoxin precipitation in plasmid DNA purification using improved limulus amoebocyte lysate assay and central composite design

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    Authors
    Ongkudon, C.
    Danquah, Michael
    Date
    2011
    Type
    Journal Article
    
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    Citation
    Ongkudon, C. and Danquah, M. 2011. Analysis of selective metal-salt-induced endotoxin precipitation in plasmid DNA purification using improved limulus amoebocyte lysate assay and central composite design. Analytical Chemistry. 83 (1): pp. 391-397.
    Source Title
    Analytical Chemistry
    DOI
    10.1021/ac1026379
    ISSN
    0003-2700
    School
    Curtin Sarawak
    URI
    http://hdl.handle.net/20.500.11937/36784
    Collection
    • Curtin Research Publications
    Abstract

    Recent advancements in plasmid DNA (pDNA) production involve the development of innovative and cost-effective methods as well as reduced number of unit operations. This study investigates the feasibilities of using a metal salt to selectively remove endotoxins from clarified cell lysates containing plasmid DNA. Screening of endotoxin precipitation in various metal salt solutions and optimization of process conditions (pH, ion concentration, temperature, and incubation time) using central composite design experiments have been carried out successfully. Results show that selective endotoxin precipitation (<0.05 EU/µg) can economically be carried out during the alkaline cell lysis procedure (neutralization step) at a pH condition similar to that of alkaline-lysed cell lysate, a low ZnSO 4 concentration (0.5 M), a minimum incubation time (30 min), and a temperature of 15 °C. In summary, this method provides ease of subsequent plasmid DNA purification due to reduced bulk impurities and cost-effective and most importantly high endotoxin removal (>80%) and plasmid recovery (>90%). © 2010 American Chemical Society.

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