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PURIFICATION OF PLASMID DNA USING ULTRAFILTRATION MEMBRANES
Restricted (Penn State Only)
Doctor of Philosophy
Date of Defense:
May 31, 2017
Andrew Zydney, Dissertation Advisor
Andrew Zydney, Committee Chair
Manish Kumar, Committee Member
Janna Kay Maranas, Committee Member
William O Hancock, Outside Member
Previous studies have demonstrated that plasmid transmission through ultrafiltration membranes can be controlled by adjusting the filtrate flux thereby controlling the extent of plasmid elongation. This phenomenon can be used for the separation of different plasmid isoforms by exploiting the differences in flexibility of the supercoiled, linear, and open-circular isoforms. However, there are a number of critical challenges that still need to be overcome in order to apply these novel membrane-based processes for commercial scale manufacture of DNA for gene therapy applications and for use as DNA-based vaccines. The overall objectives of this dissertation are to develop novel strategies to enhance the separation resolution during ultrafiltration of different plasmid isoforms and control membrane fouling during ultrafiltration of concentrated DNA solutions. This work first focused on developing the strategy of pre-conditioning, 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 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 performance of composite membrane system can be optimized by controlling the pore size and morphology of the microfiltration membranes. This work also examined the effects of ionic conditions (including solution ionic strength and ion type) on separation of the different plasmid isoforms. The transmission of the linear and open-circular isoforms slightly increased with increasing solution ionic strength (NaCl or MgCl2 concentration) due to shielding of intramolecular electrostatic interactions. The effect of ionic strength was greatest for the supercoiled plasmid due to changes in its plectonemic structure, providing opportunities for enhanced purification of this therapeutically active isoform. Polycation spermine was found to induce DNA condensation at a threshold concentration, above which transmission of the plasmid DNA dropped rapidly with the membranes becoming nearly completely retentive to the plasmid. DNA condensation was reduced in the presence of high concentrations of monovalent salts, potentially providing an opportunity to “tune” the transmission of the DNA isoforms by proper of addition of spermine and NaCl to the solutions. Solution conditions also have a significant effect on the fouling characteristics of supercoiled plasmid DNA isoforms with different numbers of base pairs. Sieving coefficient and filtrate flux data were analyzed using a model based on the partial blockage of the membrane pores by trapped plasmids. Fouling increased dramatically at low ionic strength, with the flux decline parameter for the 3.0 kbp plasmid in a 1 mM NaCl solution being an order of magnitude greater than that in a 10 mM solution. Fouling was also most pronounced for the larger 16.8 kbp plasmid, consistent with the greater probability of plasmid trapping at the pore entrance. Ultrafiltration membranes also have the potential to separate supercoiled plasmids based on differences in their size (i.e., number of base pairs). An up to 30-fold selectivity between 3.0 and 16.8 kbp plasmids was achieved using commercial ultrafiltration membranes. The reduction in transmission of the supercoiled plasmids with increasing chain length was a direct result of the morphology of the supercoiled isoform; no significant affect of plasmid size was seen during ultrafiltration of linear versions of the same plasmids. The supercoiled isoforms adopt a branched structure due to the under-twisting of the DNA, with the number of branches increasing, and the DNA transmission decreasing, with increasing chain length. It is anticipated that the results from this study will provide important information needed for successfully implementing UF processes into commercial systems for the large-scale manufacture of therapeutic DNA products.
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