Precipitation of RNA impurities with high salt in a plasmid DNA purification process: use of experimental design to determine reaction conditions.
The use of high salt solution to precipitate RNA in a pharmaceutical-grade plasmid DNA purification process was investigated. Five antichaotropic salts were tested for their potential to precipitate RNA. Calcium chloride was by far the best precipitant with high RNA removal in a very short incubation time. Calcium chloride precipitation conditions were investigated at two stages of a plasmid purification process using experimental design techniques. The effect of up to five factors on RNA precipitation and plasmid recovery was assessed by statistical modeling. Optimized conditions for calcium chloride precipitation were then introduced to the plasmid purification process resulting in the efficient removal of most impurities (RNA, chromosomal DNA, proteins, and endotoxins).
Eon-Duval
Alex
A
GlaxoSmithKline Research and Development Ltd, BioPharmaceutical Development, South Eden Park Road, Beckenham, Kent, BR3 3BS, United Kingdom. aed98800@gsk.com
Gumbs
Karlene
K
Ellett
Christopher
C
D002122:Calcium Chloride / Q000737:chemistry* / Q000302:isolation & purification; D011232:Chemical Precipitation; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D015233:Models, Statistical; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification*; D011786:Quality Control; D012329:RNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification*; D012492:Salts / Q000737:chemistry / Q000302:isolation & purification; D012996:Solutions; D014462:Ultrafiltration / Q000379:methods*
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012492:Salts; D012996:Solutions; D002122:Calcium Chloride