September 11-16, 2016
Several recent studies have demonstrated that small pore size ultrafiltration membranes can be used for purification of supercoiled plasmid DNA for use in both gene therapy applications and as DNA-based vaccines. Plasmid transmission during ultrafiltration is governed by the elongation of the large DNA molecules in the converging flow-field entering the membrane pores. However, the performance of these membrane systems is severely limited by membrane fouling, and the selectivity for the separation of the DNA isoforms is often low. 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 larger pore size, to minimize fouling and enhance DNA separations.
Stock solutions of 3.0 and 16.9 kbp (kilo-base pair) supercoiled plasmids were prepared by Aldevron; solutions of the linear and open-circular isoforms were generated by enzymatic digestion of the supercoiled DNA. Ultrafiltration experiments were performed using both asymmetric hollow fiber polyethersulfone ultrafiltration membranes, with flow in either the normal or reverse orientation, and with composite (layered) 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 caused an increase in plasmid transmission and a significant reduction in fouling by pre-stretching (or pre-conditioning) the DNA. This facilitated transmission of the large DNA through the small pores of the ultrafiltration membrane while simultaneously minimizing DNA trapping at the pore entrance. Results for the critical filtrate flux for DNA transmission were in good qualitative agreement with available theories for the flow-induced elongation of flexible polymer chains.
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Andrew L. Zydney and Ying Li, "Development of novel membrane structures for enhanced purification of plasmid DNA using small pore size ultrafiltration membranes" in "Advanced Membrane Technology VII", Isabel C. Escobar, Professor, University of Kentucky, USA Jamie Hestekin, Associate Professor, University of Arkansas, USA Eds, ECI Symposium Series, (2016). http://dc.engconfintl.org/membrane_technology_vii/11