Ruiz O.,Direccion de Desarrollo |
Limonta M.,Direccion de Desarrollo |
Valdes J.,Direccion de Desarrollo |
Marquez G.,Direccion de Desarrollo |
And 6 more authors.
Biotecnologia Aplicada | Year: 2012
A scalable high-cell-density Escherichia coli culture method was established to obtain pharmaceutical grade plasmid DNA (pDNA), together with an optimized purifi cation process. The effects of several components of the medium, such as carbon and nitrogen sources that ensure bacterial nutritional needs, were studied. The operation parameters, such as temperature, shaking and aeration, were set and the optimum values of cell growth and specifi c pDNA productivity in culture were determined. The subsequent purifi cation process for pharmaceutical grade pDNA was implemented, by combining RNA precipitation with ammonium sulfate and two successive chromatographic steps consisting of size exclusion chromatography and reverse phase-high performance liquid chromatography. This work comprised the fi rst report on the use of reverse phase to purify DNA for application in humans.