Study On The Production Technology Of Recombinant Human Hepatocyte Growth Factor Naked Plasmid

Arterial ischemia, a kind of vascular disease, threat human’s health seriously, however, at present there exists no effective treatment. Recombinant hepatocyte growth factor (HGF) naked plasmid injection (hereinafter referred to as: NL003) is the recombinant naked plasmid gene therapy medicine, which can be used for ischemic diseases. After injected into the ischemic muscle tissue, the naked plasmid is transfected into the striated muscle cells, then it synthesizes and secretes HGF protein factor by protein synthesis system, which will promote the formation of collateral circulation in ischemic part and achieve the purpose of treatment of lower limb arterial ischemiaIn order to promote the industrialization of the NL003 project, this study focuses on the establishment of large-scale production technology, the key quality control standards and the treatment effects of the final product on ischemic animal models. The main contents are as follows:Firstly, the culture process of engineering bacteria (NL003) was investigated. The appropriate conditions for NL003 growth and plasmid amplification in shake flask were determined. And under the optimum condition, the influence of culture medium, incubation time and inoculum on growth and plasmid amplification was also studied. The suitable fermentation process in the 30 liter fermentor was built for the first time, that is, organic nitrogen and trace element-containing substances as culture medium, 1:18 to 1:9 of inoculum concentration, 37oC, 20 l/min air flow, over 20% dissolved oxygen and pH 7.0. In the process of fermentation, 50% (w/v) glucose solution was added into the culture. And the bacteria were collected after 10h fermentation.Secondly, the purification process of plasmid was studied. The small scale purification process of NL003 programme was determined as follows: alkaline lysis breaking strain - ultrafiltration - first chromatography (complete removal of RNA) - second chromatography (purification of supercoiled plasmid DNA) - Third chromatography (removal of residual endotoxin.). And then, according to the principle of the same column height and flow rate, just increasing the column diameter, the 10 times scale-up of chromatograph coumn was developed.Three continuous batches of production was performed, and the as-prepared plasmid met the quality requirements, which indicated the stability of purification process Thirdly, the quality control standards were explored. According to 2010 version of "Chinese Pharmacopoeia" Part III and the new drug technical requirements, the assay method of plasmid contents and biological activity was set up for NL003. The assay method of plasmid contents was as follows: it was calculated according to OD. The assay method of biological activity was as follows: the plasmid was transfected into target cell and the quantity of secreted HGF was determined firstly, and then the biological activity of HGF was measured.Finally, the as-prepared NL003 samples were used for the lower limb ischemic disease treatment. Additionally, the effect of the re-formation of collateral circulation was evaluated. Compared with the experimental control group, NL003 can promote collateral angiogenesis in ischemic area, promote angiogenesis in muscle tissue, and increase the flux of arterial flow in internal iliac.