P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work， the 6.07 kbp pcDNA3-FLAG-p53 plasmid， which encodes the p53 tumor suppressor， was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis， the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree， maximizing the recovery yield of the supercoiled plasmid DNA， the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05)， with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool， 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity， when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally， the purified sample was analysed to assess the content of endotoxins， proteins and genomic DNA， showing that all these impurity levels were below the recommendations of the regulatory agencies.