(5bb) DNA Therapeutics – New Challenges for Separation of Plasmid Isoforms | AIChE

(5bb) DNA Therapeutics – New Challenges for Separation of Plasmid Isoforms

Authors 

Latulippe, D. R. - Presenter, Pennsylvania State University
Zydney, A. L. - Presenter, The Pennsylvania State University


Recent progress in the development of gene therapies and DNA-based vaccines has created a need for new separations technologies suitable for the large scale production and purification of plasmid DNA. Plasmids exist in one of three topological morphologies or isoforms: supercoiled, open-circular, and linear. These double-stranded DNA molecules are all naturally produced in typical plasmid manufacturing processes. The supercoiled isoform has the greatest transfection efficiency and is thus the FDA recommended morphology for therapeutic applications; the other isoforms are considered process impurities. The objective of our research has been two-fold: to develop a quantitative understanding of the behavior of the different plasmid isoforms in membrane ultrafiltration (UF) and size exclusion chromatography (SEC), and to use appropriate theoretical frameworks to analyze the experimental results in terms of the underlying bio-physical properties of the different isoforms.

UF experiments were conducted over a range of filtrate flux, membrane pore size, and solution properties. Plasmid transmission was a strong function of the filtrate flux with very high transmission obtained at the higher filtration rates even for plasmids that were more than an order of magnitude larger than the effective membrane pore size. Plasmid transmission was nearly independent of the plasmid size (number of base pairs), but varied with solution ionic strength and membrane pore size. The data were analyzed using a model for flexible polymers that accounts for the elongation of the plasmid in the converging flow-field above the membrane pores. At a given filtrate flux, transmission of the linear isoform was greater than that for the supercoiled plasmid, with the open-circular plasmid being almost completely retained out to very high filtrate flux. These differences were directly related to the underlying differences in the flexibility of the three isoforms and were successfully exploited to develop a novel ?flexibility-based' UF process that was able to achieve high-resolution purification of the different plasmid isoforms.

In contrast to the behavior seen in UF, plasmid separation in SEC was governed primarily by the plasmid size. Thus, the linear isoform showed the least retention while the supercoiled isoform had the greatest retention (for plasmids with the same number of base pairs). These trends were confirmed by static light scattering measurements of the radii of gyration. The resolution between the different isoforms was dramatically reduced for the larger plasmids, resulting in low product yields and quality under these conditions. The experimental data were analyzed using theoretical models for partitioning behavior of flexible linear and cyclic polymers. The results provide a framework for analysis and design of SEC systems for plasmid separations.

The results obtained in these studies clearly demonstrate the critical relationship between the underlying physical properties and the separation characteristics of the plasmid DNA isoforms. Ongoing advances in our understanding of the biophysics of plasmid DNA could provide exciting opportunities for the development of new / improved separations technologies that specifically exploit the unique physical properties of plasmid isoforms.