Recombinant plasmid DNA can be used as a novel therapeutic molecule for gene therapy and DNA vaccination. One of the challenges in plasmid DNA production is the separation of the desired supercoiled isoform from host-cell related impurities including genomic DNA, RNA, proteins, as well as the undesirable linear and open-circular plasmid DNA isoforms formed by irreversible breaks in the DNA backbone. Current FDA regulations specify that at least 90% of the plasmid DNA in the final product must be in the supercoiled form. Due to the very large size of plasmid DNA, traditional chromatographic strategies suffer from a number of limitations including limited resolution, very low binding capacity, mass transfer limitations, and high processing costs. Membrane-based processes have great potential for large-scale plasmid DNA purification. The objective of this work was to obtain quantitative data on the effects of plasmid isoform structure on the transmission of plasmid DNA during ultrafiltration.

Experiments were conducted with Ultracel composite regenerated cellulose membranes and a 3000 base pair pBluescript plasmid. The plasmid as supplied was predominantly in the supercoiled form (>90%). Open-circular and linear isoforms were prepared by incubating the plasmid with appropriate restriction enzymes. Plasmid concentrations were determined using PicoGreen DNA fluorescent reagent allowing accurate detection of concentrations as low as 250 ng/L. Stirred-cell ultrafiltration experiments were conducted over a range of filtrate flux and solution ionic strength using different molecular weight cut-off membranes. The different DNA isoforms were analyzed using agarose gel electrophoresis.

Experiments with the individual isoforms showed that transmission was a strong function of both the filtrate flux and solution ionic strength. The strong flux-dependence was due to the elongation of the DNA in the converging flow field into the membrane pores. For all conditions tested, the linear DNA had a higher sieving coefficient than the supercoiled and open-circular forms due to differences in conformation and flexibility of the DNA isoforms. Experiments with a mixture of plasmid isoforms confirmed that it was possible to purify the different isoforms by proper selection of the membrane pore size, filtrate flux, and solution ionic strength. For example, very good separation of the linear form from the supercoiled and open-circular forms was achieved with the largest pore size membrane by operating at a filtrate flux slightly above the critical flux for transmission of the linear DNA. This work demonstrates that high-resolution purification of supercoiled DNA can be achieved using membrane ultrafiltration by exploiting differences in the conformational flexibility of the different plasmid isoforms.