Purification of plasmid DNA from clarified and non-clarified lysates

26 27 Several small molecules, like some therapeutic agents, are able to bind DNA with high 28 specificity. These may represent a relevant alternative as ligands in affinity and pseudo-29 affinity chromatographic processes for plasmid DNA (pDNA) purification. In the 30 present study, berenil (a DNA intercalator used an anti-trypanosomal agent in veterinary 31 applications) was tested as a ligand to specifically purify plasmids with different sizes, 32 pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from the impurities 33 present in Escherichia coli alkaline lysates. For this purpose, chromatographic 34 experiments were set using Sepharose derivatized with berenil. The results showed that 35 both pDNA molecules are completely purified using smaller amounts of salt in the 36 eluent than those reported before for other pseudo-affinity and hydrophobic 37 chromatography based processes. Total retention of all lysate components was achieved 38 with 1.3 M ammonium sulphate in the eluent buffer and pDNA elution was obtained by 39 decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing 40 the concentration to 0 M. The recovery yield for the larger pDNA molecule pCAMBIA-41 1303 (45%) is lower than that obtained for pVAX1-LacZ (85%), presenting however a 42 higher final purity. Furthermore, pVAX1-LacZ purification studies were also performed 43 using non-clarified E. coli process streams, replacing the clarification step with a second 44 chromatographic run on the berenil-Sepharose support. Using the same binding and 45 elution conditions as before, a pure plasmid sample was obtained with a 33% yield. The 46 pDNA fractions were analysed for E. coli host impurities, and all levels were in 47 accordance to the requirements established by the regulatory agencies (FDA). These 48 results suggest that this chromatographic support is a promising alternative to purify 49 pDNA for therapeutic use. 50 3