(240d) Ultrafiltration Characteristics of Plasmid DNA

Plasmids are extra-chromosomal circular double-stranded DNA that can be used for development of recombinant cell lines and as vectors for delivery of therapeutic genes. These applications require the development of improved methods for the purification of plasmid DNA. Membrane ultrafiltration (UF) is of particular interest since the large size of plasmids creates significant challenges for traditional chromatographic resins. The objective of this study was to obtain quantitative data on the effects of solution environment and filtration conditions on plasmid transmission through different UF membranes.

Experiments were conducted with a 3000 base pair pBluescript plasmid using Ultracel composite regenerated cellulose membranes. Plasmid concentrations were evaluated using PicoGreen DNA fluorescent reagent in combination with a microplate reader allowing accurate detection of concentrations as low as 250 ng/L. Stirred-cell ultrafiltration experiments were conducted over a range of filtrate flux rates and solution ionic strength. The plasmid transmission was a very strong function of the filtrate flux with the observed sieving coefficient increasing by over 100 fold, from <0.01 to 0.85, as the flux increased from 7 to 40 µm/s at a NaCl concentration of 150 mM. This increase in transmission was greater than that predicted using available concentration polarization models, suggesting that there may have been a flow-induced conformational change in the plasmid DNA. The plasmid sieving coefficient was also a strong function of the salt concentration; the sieving coefficient increased by more than 80 fold as the NaCl concentration increased from 1 to 150 mM at a constant flux of 40 µm/s. These results clearly demonstrate the importance of both solution conditions and operating parameters on the ultrafiltration behavior of plasmid DNA.