Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy， since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However， the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work， two monolithic supports were modified with lysine and its decarboxylated derivative， cadaverine， to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general， cadaverine mo... More
Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy， since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However， the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work， two monolithic supports were modified with lysine and its decarboxylated derivative， cadaverine， to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general， cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith， at acidic pH. For instance， at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59， respectively， while for cadaverine modified monolith was 20.32 and 27.12， respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However， recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith， achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample， obtained by the column that showed the best performance， was characterized by real-time PCR， presenting 98.4% purity and 78.6% recovery yield. The impurities content， namely genomic DNA， proteins and endotoxins， was found within the criteria established by regulatory agencies. Overall， a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column， with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.
