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　+/-　0.41g　of　pharmaceutical　pUDK-HGF　from　1.0kg　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.