Several recent studies have demonstrated that small pore size ultrafiltration membranes can be used for purification of supercoiled plasmid DNA for therapeutic applications, but the performance of these membrane systems is severely limited by membrane fouling and low selectivity. The objective of this work was to examine the potential of using “pre-conditioning”, in this case accomplished by pre-elongating the DNA by passage through a region with large pore size, to minimize fouling and enhance DNA separations.
Data were obtained using both asymmetric hollow fiber ultrafiltration membranes, with flow in either the normal or reverse orientation, and with composite membrane structures made by placing a larger pore size flat sheet microfiltration membrane in series with an ultrafiltration membrane. In all cases, flow through the larger pore size region pre-stretched the plasmid, leading to an increase in plasmid transmission and a significant reduction in fouling. This pre-conditioning also provided a significant increase in selectivity for separation of the linear and supercoiled isoforms. The effectiveness of the pre-conditioning was a function of the pore size and morphology of the top layer in the composite membrane structure, with the best performance obtained using a pre-conditioning layer that had a pore size similar to the radius of gyration of the plasmid. These results clearly demonstrate the potential for dramatically increasing the performance of membrane systems for plasmid DNA separations by controlling the pore morphology to effectively pre-stretch the DNA before passing through the narrow pores of the selective ultrafiltration membrane.