Analysis And Quality Control Of Genetherapeutic PUDKH

pUDKH, a new plasmid DNA comprising human hepatocyte growth factor(HGF) was developed as a potential genetherapeutic product for peripheral arterial disease. In this paper an anion-exchange HPLC method was developed to determine the proportion of plasmid DNA opened and supercoiled forms. Tskgel DNA-NPR column was equilibrated with 50% buffer A (20mmol/L Tris-HCl, pH8.8) and 50% buffer B (buffer A with additional Imol/L NaCl), pUDKH was analyzed with a gradient from 50% to 80% B. The flow rate was 0.5 ml/min, UV detected at 260nm. Also this chromatographic method was used to monitor the production procedures.In the process of plasmid pUDKH production, Triton X-100 was used as a virus inactivated solvent. The residual Triton X-100 concentrations must be determined in final products. A reversed-phase high performance liquid chromatography was used. The system included a Hypersil silica column, eluted with acetonitrile -H2O(70:30,V/V). The flow rate was l.Oml/min. The detection limit was low (1.25 u g/ml) with detection at 223nm. The calibration linear range was between 1.25 u g/ml and 20 u g/ml. The method was rapid and did not need sample pretreatment. The residual Triton X-100 in our final pUDKH products were determined at 2.27-3.00 u g/ml level.Host protein in plasmid pUDKH is monitored by ELISA, through Elx-800 reading the production in the 96 microplate at 570nm.We half-quantify the percent of host DNA in production by Northernblot and tachypleus amebocyte lysate for endotoxin detection. It is resulted that the percentage of supercoiled pUDKH in plasmid is more than 95%,and RNA undetected. The content of host protein remained is 0.0166 u g/mg and chDNA is less than 2ng/mg,endotoxin is0.00178EU/mg.