(550a) Size-Based Separation Processes for Plasmid DNA – Comparison Between Size Exclusion Chromatography, Agarose Gel Electrophoresis, and Membrane Ultrafiltration

Plasmids are circular pieces of double-stranded DNA that exist in different topological morphologies known as isoforms. The supercoiled isoform is characterized by a ?self-coiling' of the DNA induced by enzyme-generated helical stresses. A nick or break in one strand of the DNA double helix releases the helical stresses and creates the open-circular isoform which lacks any supercoiling. A break in both strands of the DNA double helix creates the linear isoform. The different plasmid isoforms have identical base pair sequences and net charge, but they differ in effective size as determined by static light scattering measurements. Thus, a number of size-based separation techniques have been proposed for both the analysis and purification of plasmid isoforms for gene therapy and DNA-based vaccines. However, there is currently no fundamental understanding of how the morphology of the different plasmid isoforms controls the thermodynamic and transport phenomena involved in these separation processes. The objective of this study was to evaluate the behavior of the supercoiled, open-circular, and linear plasmid isoforms in size exclusion chromatography (SEC), agarose gel electrophoresis (AGE), and membrane ultrafiltration (UF).

Experiments were performed with a 5.76 kbp plasmid supplied in the supercoiled form. Linear and open-circular isoforms were prepared by enzymatic digestion of the supercoiled plasmid. The radius of gyration of the plasmid was evaluated from static light scattering measurements obtained over a range of scattering angles and concentrations. SEC was performed using Sephacryl S-1000 SF resin, with the elution volume evaluated over a range of flow rates and buffer conditions. AGE was performed using 0.7 % horizontal agarose gels, which were stained and then imaged using a UV trans-illuminator. UF was conducted with Ultracel composite regenerated cellulose membranes, data were obtained over a range of filtrate flux using a small laboratory stirred cell.

The radius of gyration of the different plasmid isoforms ranged from 104 nm for the supercoiled isoform to 136 nm for the open-circular and 160 nm for the linear plasmid. The same general behavior was seen in SEC, with the supercoiled isoform having the largest elution volume followed by the open-circular and then the linear plasmid. In contrast, the open-circular isoform migrated the shortest distance in AGE, corresponding to the largest effective size, while the supercoiled plasmid showed the greatest penetration through the gel. This anomalous behavior is likely due to the greater hindrance to electrophoretic motion experienced by the more rigid open-circular plasmid; the linear and supercoiled isoforms have greater flexibility and are thus more easily transported through the agarose gel. The open-circular isoform also had the greatest retention in UF, but in this case the smallest retention was obtained with the linear isoform instead of the supercoiled. The UF data were analyzed using a scaling model based on elongation of the plasmid in the converging flow-field into the membrane pore. Plasmid transmission is predicted to be independent of the plasmid size but to depend critically on the plasmid flexibility, with the greatest transmission of the linear plasmid associated with the increased flexibility of this isoform. The very different behavior observed in SEC, AGE, and UF are directly related to differences in the underlying separation mechanisms and the physical properties of the different isoforms.