## 1 Introduction  
Up to date more than 200 types of cancer have been described and characterized, and in all these cases, changes in the genome are involved, proving the complexity of this disease [1; 2]. When some mutations occur and are accumulated, the tumorigenesis process can start, leading the cell to gain different characteristics, namely: self-sufficiency in growth signals, insensitivity to antigrowth signals, sustained angiogenesis, capacity of invasion and metastasis, limitless replicative potential, and evasion of programmed cell death (apoptosis) [2]. The DNA mutations can occur not only by abiotic factors, such as environment exposure, but also could happen due to biotic factors, such as DNA replication errors or virus infection [3]. Cells have tumor suppressor genes that are responsible for the inhibition of cell growth and/or induction of cellular apoptosis, which therefore prevent cancer development. Amongst all the tumor suppressor genes, one of the most important/relevant is p53. Different research studies have demonstrated that it is mutated or deleted in more than 50% of all human cancers [4; 5]. In response to stress, p53 can induce cell senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis [6]. Concerning this, the p53 gene has attracted much attention as a candidate for cancer gene therapy, since the replacement of the abnormal gene by the normal counterpart could restore the protective function of the cell guardian. The manufacturing process of therapeutic molecules to be used in gene therapy encompasses an initial upstream stage foreseeing the production of this biomolecule by bacterial fermentation and a downstream stage comprising the its recovery and purification, usually based on chromatography, aiming for the removal of host's impurities, ensuring at the end of the process a high degree of purity of the purified supercoiled plasmid DNA (sc cDNA) according to some parameters established by regulatory agencies, such as the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) [7; 8]. Among these parameters are the product appearance (clear, colorless solution), the plasmid homogeneity (\(\geq\)90% supercoiled (sc) cDNA) and impurities levels (RNA and proteins should be undetectable, the amount of genomic DNA (gDNA) must be lower than 0.01 ug/ug of pDNA and the level of endotoxins should not be higher than 0.1 EU/ug of pDNA) [9]. All these impurities are derived from bacteria host cells used in the biosynthesis of plasmids and share some physicochemical characteristics with the target molecule, namely negative charge and molecular weight, which may hinder the pDNA purification [2]. Each impurity can promote some adverse effects on human cells, for example, gDNA can integrate in the cell genome, suppressing genes or even activating oncogenes [10], while the silencing of some cellular products can occur by bacterium RNA pairing with the messenger RNA of transfected cells. Moreover, endotoxins exhibit pyrogenic activities and may promote irreversible septic shock [11], as bacterial proteins can trigger several immunological responses, even if they are present in small amounts [12]. The need to obtain the sc pDNA topology is also related to its effectiveness in cell transfection and product expression, in comparison to other isoforms (open circular (oc) and linear (ln)). To purify the target molecule, following all requirements of regulatory agencies, affinity chromatography has shown to be a useful tool, mainly because of its ability to promote multiple reversible and specific interactions between biomolecules and ligands [13]. However, the chromatographic conditions used to bind and elute the target molecule also can irreversibly influence the plasmid stability, reducing its bioactivity or even making the therapeutic gene function unfeasible. In this way, the importance of the salt effect (type and ionic strength applied) once it can induce changes in DNA conformation should be remarked upon.