title: Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process.
abstract: Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.
authors.affiliation: BioPharmaceutical Development, GlaxoSmithKline Research and Development Ltd, South Eden Park Road, Kent BR3 3BS, Beckenham, UK. aed98800@gsk.com
authors.forename: Alex
authors.initials: A
authors.lastname: Eon-Duval
authors.forename: Robert H
authors.initials: RH
authors.lastname: MacDuff
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fisher
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Harris
authors.forename: Chris
authors.initials: C
authors.lastname: Brook
mesh_terms: D002122:Calcium Chloride / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005374:Filtration / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012260:Ribonucleases; D002122:Calcium Chloride