Location: JHE 326H
There is growing interest in using plasmid DNA for gene therapy and DNA-based vaccines. Existing methods for the purification of plasmid DNA are inadequate for large-scale commercial production; thus, there is a critical need for the development of new separations technology specifically targeted for plasmid purification. This talk examines the possibility of using membrane ultrafiltration for the purification of supercoiled plasmid DNA, including removal of host cell-related impurities and product-related isoforms. Ultrafiltration experiments were performed with Ultracel composite regenerated cellulose membranes and a 3000 base pair plasmid. Open-circular and linear isoforms were prepared using appropriate restriction enzymes. Plasmid transmission during ultrafiltration was a strong function of filtrate flux and ionic strength. This flux-dependence was described using a model that accounts for the elongation of the plasmid associated with the converging flow into the membrane pores. Plasmid sieving coefficients were in good agreement with model calculations over a wide range of conditions, providing an appropriate framework for analysis of plasmid ultrafiltration data. Transmission of the open circular DNA was significantly less than that of the supercoiled plasmid, while transmission of the linear DNA was considerably enhanced due to differences in the conformational flexibility of these DNA isoforms. These data were used to identify optimal conditions for purification of the supercoiled plasmid, which is the desired therapeutic isoform. The results clearly demonstrate the potential application of ultrafiltration for the commercial-scale purification of plasmid DNA.
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