Current technologies for the purification of supercoiled plasmids are limited. The use of cesium chloride gradients in the presence of ethidium bromide is time consuming, labor intensive, requires the use of known mutagens and is not conducive to large scale. As a result, first-generation high-performance liquid chromatography (HPLC) methods based on anion-exchange and size exclusion have been developed but are difficult to accommodate production at large scale and still result in compromised purity (1,2). The success of DNA vaccines in animal models and the initiation of human trials (3,4) has led to a need to increase the level of supercoiled plasmid purity as well as the methodology utilized to produce these plasmids at large scale. Several parameters of the purification process need to be addressed:

–	• The ability to prepare supercoiled plasmid at purity levels acceptable for clinical material.
–	• The ability to prepare clinical grade supercoiled plasmid that will be scalable in order to produce gram quantities of product.
–	• The ability to prepare clinical grade supercoiled plasmid in accordance with cGMP principles.
–	• The ability to develop validated assays to assess purity, yield, and contamination levels.