Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.

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In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use.