(669a) Challenges of Plasmid DNA Purification

In the biopharmaceutical industry, plasmid DNA (pDNA) is used to produce protein therapeutics and as gene therapy vectors. However, due to its large size, shear sensitivity, and similarity to impurities, pDNA poses several complications for purification by chromatography. Low resin binding capacity, plasmid damage, aggregation, and co-purification of impurities are among the challenges faced in pDNA purification, especially for large plasmids. In our work, we explore options to create a 2-column purification process that can be used for both large and small plasmids. We test anion exchange (AEX) and hydrophobic interaction (HIC) resins for efficacy at pDNA binding and yield, buffer composition as a mechanism for RNA removal, and optimizing ammonium sulfate concentration to minimize aggregation prior to the HIC column. Our results indicate that our process can be applied to pDNA ranging from 3-20 kb. Analytical analysis by agarose gel shows interesting differences in the behavior of small and large plasmids during chromatography and on the gel itself as well as identifies avenues for further development.