Kendall, Diane; (2003) Novel approaches to the purification of plasmid DNA for therapeutic application. Doctoral thesis (Ph.D), UCL (University College London).

Text 10013935.pdf Download (12MB)

Abstract

At the present time there is considerable interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The work presented in this thesis addresses two of the most significant purification challenges namely, the initial purification of the plasmid DNA from related contaminants in high yield and the subsequent high-resolution separation of different plasmid DNA forms. The ability of nitrocellulose to selectively bind single stranded DNA (ssDNA), both in powder and sheet membrane form, was initially investigated. In order to maximise the volume of lysate that could be processed per m2 membrane area, the use of an integrated unit operation, comprising tangential-flow filtration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was then examined, and indicated the utility of the operation to adsorb ssDNA and proteins from model solutions. Tangential-flow filtration-adsorption of E.coli lysates containing a plasmid product was shown to decrease the levels of chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed indicating close to 100% yield. Results were similar for E.coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin and protein levels were also observed. The use of liquid-liquid countercurrent chromatography (CCC) for the preparative scale fractionation of different forms of plasmid DNA was subsequently examined. Fractionation of supercoiled and open circular plasmid DNA (6.9 kb) could be achieved using a phase system comprising 12.5% w/w PEG 600 and 18% w/w K2HPO4. Addition of isopropyl alcohol (2% w/w) was found to be beneficial to the separation by alteration of the phase physical properties. Residual protein, RNA and chromosomal DNA did not co-purify with the plasmid DNA fractions further increasing the purity of the final product. Preparation of lysate prior to loading onto the CCC column by aqueous two-phase partitioning was found to decrease chromosomal contamination by 90% with 25% w/w yield loss of plasmid DNA.

Download activity - last month

Export as XMLJSONCSV

Download activity - last 12 months

Export as XMLJSONCSV

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item