Purification of a Type 5 Recombinant Adenovirus Encoding Human p53 by Column Chromatography

We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications. We have devised a chromatographic protocol for the purification of recombinant adenoviruses intended for use in human gene therapies that allows for production on an industrial scale. This method is intended to replace the current methodology of density-gradient ultracentrifugation. A comparison of the purity and potency of a recombinant adenovirus type 5 bearing the human p53 gene (ACN53) derived from chromatographic and ultracentrifugation methods is presented, and the advantages of virus purification by column chromatography are discussed.