Abstract
Baculovirus has been proved to be able to infect mammalian cells at 1995 and have the potential as a gene delivery vehicle. As a gene therapy vector, generation of high viral purity and concentration is prerequisite. Traditional virus purification by several ultra centrifugations is not suitable for virus purification on large scale, because it is time consuming and hard to scale up. Comparatively, chromatography has been widely used on protein purification but has not been widely used on mass purification of gene therapy vehicle. In precious research of our laboratory, we combine immobilized metal affinity chromatography (IMAC) and ultrafiltration system to purify baculovirus, and the recovery of virus titer is 8 % in IMAC process. To apply this result for purification on large scale, we should improve virus recovery. In this research, we try to modify parameters of IMAC and ultrafiltration, such as feed flow rate of IMAC (to improve virus combining rate and shorten operating time), salt concentration of IMAC buffers (to improve virus combining rate and shorten operating time), pH of IMAC buffers (to improve virus combining rate and shorten operating time), gradient of IMAC elution buffer (to improve purification efficiency), and pore size of ultrafiltration membrane (improve ultrafiltration efficiency) to improve recovery and purity. We try to find applicable purification process by using End Point Dilution Assay (EPDA) and flow cytometry to detect virus recovery in each step of purification process.