## 4 Conclusions  
Three methods involving histidine-agarose, arginine-macroporous, and histidine-monolith supports were applied to isolate sc cDNA encoding p53, by manipulating the chromatographic conditions: pH, temperature, ionic strength, and kind of salt in the binding/elution buffers. These main parameters of the mobile phases influenced not only the physical structure of the plasmid but also the bioactivity of this therapeutic molecule. With the topology studies it was verified that, despite small non-significant differences detected by dichroism signals, the CD spectra clearly show that the B topology of pDNA was maintained in the samples purified through the three chromatographic supports. In zeta potential assay, the results suggested that the colloidal stability of purified samples inside formulations are preserved and the more stable formulation follow the order of the purification with histidine-monolith \(>\) arginine-macroporous \(>\) histidine-agarose. Overall, the bioactivity assays indicate that the arginine-macroporous seems to be the most efficient matrix used in this work, despite the 92% of purity degree and the 43% of recovery yield. In addition, cells transfected with this sample have a tendency to induce the expression of higher amounts of the therapeutic p53 protein and also induce higher levels of apoptosis in cancer cells. This behavior can be related to the chromatographic conditions, namely the use of a milder salt (NaCl), the low ionic strength, neutral pH, and the low temperatures used in arginine-macroporous when compared to the high ionic strength of ammonium sulfate and room temperature or acidic pH used in histidine-based columns. The data obtained in this work show the features of each purification condition and this information allows the elaboration of good chromatographic strategies based on arginine which, in addition to purifying pDNA with a high pharmaceutical grade, was efficient in preserving the bioactivity of the plasmid.  
[PERSON] and [PERSON] designed the concept and developed the practical work. [PERSON] helped on the measurability of the DNA bioactivity and [PERSON] and [PERSON] helped on the structural analysis of the DNA. [PERSON] and [PERSON] provided scientific and technical advice as well as critical revision of the manuscript. All authors provided critical feedback in the revision of the manuscript.  
Figure 7: p53-mediated apoptosis in HeLa cells after transfection with a sc p53 plasmid recovered from different chromatographic supports.  
**Funding:** 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. [PERSON] acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq/203482/2014-0). [PERSON] acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.  
**Acknowledgments:** The authors would like to thank [PERSON] for providing the pcDNA3-FLAG-p53 construct through Addgene, ref: 10838.  
**Conflicts of Interest:** The authors declare no conflicts of interest.