Capillary zone electrophoresis of a recombinant adenovirus

Adenovirus preparations are used as vectors in a number of gene therapy clinical development programs. The success of commercial production of adenovirus will strongly depend on the development of methods to define the recombinant virus product by analysis as opposed to being defined by the manufacturing process. While most analytical techniques examine portions of the virus, e.g. proteins or DNA, ion-exchange chromatography has been used to separate intact virus at low efficiency. A free zone capillary electrophoretic method was developed for high-efficiency separations of adenovirus 5. Experimental conditions such as buffer pH and concentration were explored which produced a high-efficiency separation in less than 20 min. The virus band was identified by collection of CE fractions and examination using a cell based assay. Initially, a single virus peak is found in fresh virus samples. After as little as one freeze-thaw in 1 x phosphate-buffered saline with 2% sucrose, the active virus migrates as a regular series of peaks. The nature of the virus modification leading to the differing electrophoretic mobilities is presently under investigation.