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 requi res the development of reproducible and scaleable downstream processes. She aring of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Reg ulatory standards will probably require that the level of chromosomal DNA c ontamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports th e use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-1 bioreactor. Clarified lys ates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA wa s selectively retained by the membrane while most supercoiled plasmid DNA w as 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 r etained by the nitrocellulose membrane. (C) 2000 Elsevier Science B.V. All rights reserved.