Large-scale, nonchromatographic purification of plasmid DNA.
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).
Murphy
Jason C
JC
Vaccines and Biologics Research, Merck Research Laboratories, West Point, PA, USA.
Winters
Michael A
MA
Sagar
Sangeetha L
SL
D017610:Calcium Compounds / Q000737:chemistry; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development*; D009113:Muramidase / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D017640:Silicates / Q000737:chemistry
D017610:Calcium Compounds; D002593:Cetrimonium Compounds; D017640:Silicates; D009113:Muramidase; C031293:calcium silicate; D000077286:Cetrimonium