DNA therapeutics – New challenges for the separation of plasmid isoforms

David Latulippe, Pennsylvania State University, Sustainability Engineering Faculty Candidate

04 May 2010 at 11:00

Location: JHE 342

Recent progress in the development of DNA therapeutics 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. The different plasmid isoforms have identical base pair sequences, but they differ in effective size. The supercoiled isoform has the greatest transfection efficiency and thus is the FDA recommended morphology for therapeutic applications; the other isoforms are considered process impurities. A number of size-based separation techniques have been proposed for both the analysis and purification of plasmid isoforms as DNA therapeutics. However, there is currently no fundamental understanding of how the morphology of the different plasmid isoforms controls their thermodynamic behaviour and transport phenomena in these separation processes.
The objective of this research has been two-fold: to develop a quantitative understanding of the behaviour of the different plasmid isoforms in ‘large-scale’ separation processes, and to use appropriate theoretical frameworks to analyze the experimental results in terms of the bio-physical properties of the DNA molecules. This combination of experimental and modeling work has demonstrated the critical relationship between the underlying physical properties and the separation characteristics of the plasmid DNA isoforms. A novel membrane-based separation strategy that exploits the differences in isoform flexibility was developed and shown to perform significantly better than conventional processes.
Ongoing advances in our understanding of the conformational, dynamic, and entropic properties of DNA will provide exciting opportunities for the development of new separation technologies that specifically exploit the unique physical properties of plasmid isoforms. This rational design approach will achieve unprecedented levels of performance and thus address the practical problems associated with the production and purification of both existing high-value biopolymers and ‘next-generation’ bio-molecules developed by the biotechnology industry.

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