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Large-scale, nonchromatographic purification of plasmid DNA.

Authors
  • Murphy, Jason C
  • Winters, Michael A
  • Sagar, Sangeetha L
Type
Published Article
Journal
Methods in molecular medicine
Publication Date
Jan 01, 2006
Volume
127
Pages
351–362
Identifiers
PMID: 16988465
Source
Medline
License
Unknown

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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