Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity


The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical's activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine agarose, arginine-macroporous, and histidine monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine-macroporous support, followed by histidine monolith and histidine agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine monolithic support (26%) and histidine-agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.



subject category

Chemistry; Engineering; Materials Science; Physics


Azevedo, GM; Valente, JFA; Sousa, A; Pedro, AQ; Pereira, P; Sousa, E; Queiroz, JA

our authors


This work was supported by FEDER funds through the POCI-COMPETE 2020 Operational Programme Competitiveness and Internationalization in Axis I-Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491), and National Funds by FCT Foundation for Science and Technology (Project UID/Multi/00709/2013). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. G.M. Azevedo acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq/203482/2014-0). J.F.A. Valente acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.

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