PLASMID TRANSMISSION THROUGH NUCLEPORE TRACK ETCHED MEMBRANES

Open Access
Author:
Harter, Allison Beth
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Andrew Zydney, Thesis Supervisor
  • Ali Borhan, Honors Advisor
Keywords:
  • ultrafiltration
  • track etched membranes
  • plasmid DNA
  • sieving coefficient
Abstract:
Recent results have demonstrated that ultrafiltration can be effectively used for the large-scale purification of plasmid DNA for the production of gene therapy agents and DNA-based vaccines. The objectives of this thesis were to obtain experimental data for: (1) plasmid transmission through a series of track-etched (Nuclepore) membranes with very uniform pores to examine the role of the pore size distribution, and (2) the effect of membrane porosity on plasmid ultrafiltration. Experiments were performed with Nuclepore track-etched membranes with pore sizes of 15, 30, and 50 nm using a linearized 3 kilobase pair plasmid at both low and high salt concentrations. A low porosity version of the 50 nm pore size membrane was produced by blocking a percentage of the pores with polystyrene microspheres that were subsequently held in place by melting the polystyrene. Plasmid transmission was negligible below a critical value of the filtrate flux but then increased to nearly 100% transmission at high filtrate flux. The increase in plasmid transmission occurred over an approximately 9-fold range in filtrate flux, which is very similar to the transition seen previously with composite regenerated cellulose membranes that have a broad pore size distribution. These results strongly suggest that the gradual transition in sieving is unrelated to the breadth of the pore size distribution. The critical flux for plasmid transmission for the low porosity membrane was significantly smaller than that for the unmodified Nuclepore membrane, with the observed dependence on membrane porosity in good agreement with theoretical predictions of an elongational flow model previously developed to describe transmission of a flexible polymer through a cylindrical pore. These data provide the first direct verification of the predicted dependence on membrane porosity, while also giving additional insights into the physical phenomena controlling plasmid transmission through small pore size ultrafiltration membranes.