Abstract
A large-scale approach to the purification of plasmid DNA has been developed that overcomes many of the limitations of current chromatography-based processes. The process consists of a scaleable lysis using recombinant lysozyme and a rapid heating and cooling step followed by a selective precipitation with cetyltrimethylammonium bromide (CTAB). Calcium silicate batch adsorption is then utilized to remove residual genomic DNA, linear plasmid, open circular plasmid, endotoxin, detergents, and proteins. Finally, a concentration and diafiltration step utilizing ultrafiltration and a terminal sterile filtration complete the process. The final product exceeds the requirements for clinical-grade plasmid DNA, and the process has been scaled up to yield an average of 18 ± 4 g (over five lots) of pharmaceutically pure plasmid DNA per 140 L of lysate (from approx 1.3 kg Escherichia coli dry cell weight).
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Murphy, J.C., Winters, M.A., Sagar, S.L. (2006). Large-Scale, Nonchromatographic Purification of Plasmid DNA. In: Saltzman, W.M., Shen, H., Brandsma, J.L. (eds) DNA Vaccines. Methods in Molecular Medicine™, vol 127. Humana Press. https://doi.org/10.1385/1-59745-168-1:351
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DOI: https://doi.org/10.1385/1-59745-168-1:351
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