Quality assessment of supercoiled minicircle DNA by cadaverine-modified analytical chromatographic monolith

Abstract

Minicircle DNA (mcDNA) is the ultimate non-viral DNA vector, presenting higher biosafety and therapeutic effect than conventional plasmid DNA (pDNA). However, given the similarity between mcDNA and its precursor, the parental plasmid (PP), analytical methodologies established for pDNA are unable to distinguish mcDNA from PP. Thus, a new need emerged for the implementation of suitable, rapid and non-expensive analytical methodologies for the characterization of mcDNA samples. Recently, our research group was able to develop a purification strategy for the isolation of supercoiled (sc) mcDNA resorting to cadaverine-modified monolith. Considering the promising results obtained with this strategy, a cadaverine-modified analytical monolith was prepared and explored for mcDNA quantification. Thus, a strategy of three-step increasing NaCl gradient was considered to first elute RNA/protein content, then isolate sc mcDNA and finally eliminate PP and other impurities still bounded to the matrix. A calibration curve was constructed with different sc mcDNA standards within a range of 1−25 μg/mL. Linearity, accuracy, precision and selectivity of this method were validated according to the international guidelines and the limit of detection and the lower limit of quantification were determined as 1 μg/mL. For the first time, to the best of our knowledge, an analytical method for mcDNA quantification is described. Besides ensuring the safety of mcDNA application by assessing the product purity, such methodology can be used in the future to control industrial mcDNA production and purification, perhaps aiding in the establishment of optimized and less expensive biotechnological operations.