title: Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia.
abstract: The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.
authors: a College of Life Science and Bio-Engineering , Beijing University of Technology , Beijing , China.|b Department of Experimental Hematology , Beijing Institute of Radiation Medicine , Beijing , China.
authors: ChunSheng
authors: C
authors: Hu
authors: b Department of Experimental Hematology , Beijing Institute of Radiation Medicine , Beijing , China.
authors: XiaoChen
authors: X
authors: Cheng
authors: b Department of Experimental Hematology , Beijing Institute of Radiation Medicine , Beijing , China.
authors: YuXin
authors: Y
authors: Lu
authors: a College of Life Science and Bio-Engineering , Beijing University of Technology , Beijing , China.|b Department of Experimental Hematology , Beijing Institute of Radiation Medicine , Beijing , China.
authors: ZuZe
authors: Z
authors: Wu
authors: b Department of Experimental Hematology , Beijing Institute of Radiation Medicine , Beijing , China.
authors: QingLin
authors: Q
authors: Zhang
mesh_terms: D004247:DNA / Q000235:genetics / Q000627:therapeutic use; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D005121:Extremities / Q000098:blood supply; D015316:Genetic Therapy; D017228:Hepatocyte Growth Factor / Q000235:genetics*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007218:Industrial Microbiology / Q000295:instrumentation / Q000379:methods; D007511:Ischemia / Q000235:genetics / Q000628:therapy*; D010957:Plasmids / Q000235:genetics / Q000627:therapeutic use*
chemical_list: D017228:Hepatocyte Growth Factor; D004247:DNA
keywords: Continuous alkaline lysis; critical limb ischemia; hepatocyte growth factor; plasmid DNA; purification