Large-scale capture and partial purification of plasmid DNA using anion-exchange membrane capsules.
The demand for larger quantities of high-purity plasmids continues to increase. Substantial quantities of plasmid DNA are needed to support viral and plasmid-based gene-therapy programmes, including drug delivery, preclinical and clinical studies and production of DNA vaccines. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of plasmid DNA is becoming more widespread. Development of an efficient capture-unit operation for very large plasmid DNA molecules from these large lysate volumes can present a considerable challenge. Use of conventional chromatographic beaded media for plasmid capture is problematic, owing to their restrictive pores, which limits access of the large DNA molecules to only those binding sites on the beads' outer surface. Anion-exchange membranes, on the other hand, have large convective pores that allow the plasmid DNA to readily access all of the membrane's anionic binding sites, even at very high flow rates. The convenience of anion-exchange membranes, configured in ready-to-use capsules, can greatly simplify large-scale plasmid purification strategies. The effectiveness of membrane-based technology for the capture of a pCAT reporter plasmid from large volumes of clarified Escherichia coli lysate was demonstrated. The captured and eluted plasmid pool was found to have been concentrated 10-fold with a reduction in endotoxin of four orders of magnitude.
Zhang
Shiying
S
Pall Corporation, 8780 Ely Road, Pensacola, FL 32514, USA.
Krivosheyeva
Alla
A
Nochumson
Samuel
S
D000327:Adsorption; D000837:Anion Exchange Resins / Q000737:chemistry*; D001433:Bacteriolysis; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008567:Membranes, Artificial; D010957:Plasmids / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*
D000837:Anion Exchange Resins; D004269:DNA, Bacterial; D008567:Membranes, Artificial