## 3 Results and Discussion
### Preparative Chromatography
Plasmid purification by chromatography deals with some concerns associated with the diversity of biomolecules present in the lysate extracts and their characteristics such as: size, shape, conformation, and rheological properties. Moreover, most impurities like RNA, gDNA, or endotoxins share with pDNAsome properties of molecular mass, charge, or hydrophobicity [30]. Thus, the applied chromatographic methods must guarantee the presence of a final sc pDNA sample with the purity degree in accordance with the regulatory agency requirements (Table 1). Nonetheless, the residual amounts of proteins, gDNA, and endotoxins also need to be quantified to ensure that these values are in accordance with these specifications of the regulatory agencies. To accomplish that, several specific analyses were performed on the purified sc pDNA fractions and the observed results are presented in Table 1.  
Regarding the overall quality of purified sc pDNA fractions, it should be remarked that all samples were colorless and both proteins and RNA were undetectable. The endotoxin content was also quantified by the LAL method that revealed the reduction of this impurity to acceptable levels when the sc pDNA was purified by histidine-agarose and arginine-macroporous supports. However, the sc pDNA purified through the histidine-monolithic support presented a slightly higher content of endotoxins (Table 1), resulting in a value above the limit established by the regulatory agencies. Endotoxins are part of the cell wall constitution of the host and as mentioned above, must be removed from the lysate, being acceptable only at minimal amounts (<0.1 EU/ug pDNA), because they exhibit pyrogenic activity when they are present in high doses [11]. The amphipatic feature of endotoxins makes the process of eliminating this component more difficult [31]. Actually, some authors have described the high affinity of histidine for endotoxins [32]. In our work, despite the use of two different supports functionalized with histidine, the histidine-sepharose support was more effective at eliminating the endotoxins, while histidine-monolith was not so effective. This difference can be justified by the different pH conditions, where at pH 8.0 the major interactions that are promoted are hydrophobic, while at pH 5.0, the imidazole ring becomes protonated, and both electrostatic and hydrophobic interactions are explored. Therefore, it is reasonable to hypothesize that this combined effect can favor the endotoxin adsorption onto the histidine-monolith. The endotoxin levels present in sc pDNA purified by histidine-monolith were above the maximum limit established by FDA, a previous study indicates that the use of acidic pH can promote irreversible changes which may deactivate its biological activity [33]. In what concerns the gDNA, it was also verified the reduction of this impurity to residual levels in the three samples purified through the different chromatographic strategies, and the final pDNA samples were characterized with a gDNA content below the limit established by the regulatory agencies, thus evidencing the efficiency of the three chromatographic matrices in removing this impurity (Table 1). This assessment is extremely important because some studies demonstrated that the existence of gDNA in a purified plasmid sample for clinical application was directly related with necrosis of the muscle cells [34]. After endogenous biosynthesis of therapeutic plasmid in the _E. coli_ host, the alkaline lysis used to extract this molecule can induce some structural damage in the plasmid, converting the sc pDNA in other isoforms such as linear (ln) or oc pDNA. For this reason, the chromatographic supports explored in this work must be able to purify the most biologically active conformation, namely sc pDNA, and homogeneity higher than 90% of this topology must be obtained. Therefore, the global sc pDNA