BME Seminar Series: David Latulippe
Separation of Plasmid DNA Isoforms Using Ultrafiltration
Ph.D. Candidate, Department of Chemical Engineering
The Pennsylvania State University
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. 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 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 transmission 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. 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.