Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption.
There is an increasing interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The use of an integrated unit operation, comprising tangential flow microfiltration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was examined in this work. Experiments were performed using a custom-built tangential flow microfiltration rig (membrane area=12.5 cm(2)). Tangential flow filtration-adsorption of E. coli lysates containing a plasmid product removed most solids (>75%) and decreased chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed, indicating a recovery yield of 100% (transmission equal to 1). Results were similar for E. coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin, and protein levels were also observed. The reproducibility and potential for scale up of this integrated filtration-adsorption operation makes it at attractive option for intermediate- to large-scale pharmaceutical-grade plasmid processing.
Kendall
D
D
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, United Kingdom.
Lye
G J
GJ
Levy
M S
MS
D000327:Adsorption; D000818:Animals; D002417:Cattle; D003101:Collodion / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification*; D004731:Endotoxins / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D008567:Membranes, Artificial; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000235:genetics / Q000302:isolation & purification; D013950:Thymus Gland / Q000502:physiology; D014462:Ultrafiltration / Q000295:instrumentation* / Q000379:methods*
D004731:Endotoxins; D008567:Membranes, Artificial; D012710:Serum Albumin, Bovine; D003101:Collodion; D004247:DNA