Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process

• Published in: Analytical biochemistry 2003-05, Vol.316 (1), p.66-73
• Main Authors: Eon-Duval, Alex, MacDuff, Robert H, Fisher, Carol A, Harris, Mark J, Brook, Chris
• Format: Article
• Language: English
• Subjects: Calcium Chloride - chemistry; Chromatography, High Pressure Liquid; DNA, Bacterial - isolation & purification; Escherichia coli - genetics; Filtration - methods; Plasmid purification; Plasmids - isolation & purification; Ribonucleases - metabolism; RNA removal; RNA, Bacterial - isolation & purification; RNA, Bacterial - metabolism; Tangential flow filtration; Time Factors
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(03)00050-2

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.