Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing.
Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.
Levy
M S
MS
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, UK. myriom.levy@ucl.ac.uk
Collins
I J
IJ
Tsai
J T
JT
Shamlou
P A
PA
Ward
J M
JM
Dunnill
P
P
D015139:Blotting, Southern; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000379:methods; D002876:Chromosomes, Bacterial / Q000737:chemistry; D003101:Collodion; D004345:Drug Industry / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005374:Filtration / Q000379:methods*; D008567:Membranes, Artificial; D009696:Nucleic Acids / Q000302:isolation & purification*; D010957:Plasmids / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000302:isolation & purification; D012965:Sodium Chloride / Q000737:chemistry
D008567:Membranes, Artificial; D009696:Nucleic Acids; D012329:RNA, Bacterial; D012965:Sodium Chloride; D003101:Collodion