Production of plasmid DNA encoding human hepatocyte growth factor for gene therapy.
pUDK-HGF, recombinant plasmid DNA encoding human HGF (hepatocyte growth factor), is a potential agent for gene therapy of ischaemic disease. Production of pUDK-HGF is essential for its clinical application. In the present paper, a large-scale manufacturing process was developed, including fermentation, cell harvest, alkaline lysis, capturing plasmid DNA with Q-Sepharose XL chromatography, size-exclusion chromatography on a Sephacryl S1000 column and refining with Source 15Q anion-exchange chromatography. The quality criteria of pUDK-HGF such as purity, concentration, homogeneity, residual RNA, chromosomal DNA, contaminated protein, endotoxin and HGF expression efficacy all were analysed and met the requirements for pharmaceutical-grade plasmid DNA.
Zhang
Qinglin
Q
Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Taiping Road 27, Beijing 100850, People's Republic of China. qinglz@yahoo.com
Bi
Jianjing
J
Xiao
Fengjun
F
Duan
Haifeng
H
Wu
Bing
B
Wu
Zuze
Z
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant / Q000096:biosynthesis* / Q000627:therapeutic use*; D004926:Escherichia coli / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000096:biosynthesis / Q000627:therapeutic use; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis* / Q000235:genetics* / Q000627:therapeutic use; D006801:Humans; D010957:Plasmids / Q000096:biosynthesis
D004274:DNA, Recombinant; C066855:HGF protein, human; D017228:Hepatocyte Growth Factor