Zhang Qinglin, Bi Jianjing, Xiao Fengjun, Duan Haifeng, Wu Bing, Wu Zuze
Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Taiping Road 27, Beijing 100850, People's Republic of China.
Biotechnol Appl Biochem. 2008 Jan;49(Pt 1):11-6. doi: 10.1042/BA20060251.
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.
pUDK-HGF，即编码人肝细胞生长因子（hepatocyte growth factor，HGF）的重组质粒DNA，是一种用于缺血性疾病基因治疗的潜在药物。pUDK-HGF的生产对于其临床应用至关重要。在本文中，开发了一种大规模生产工艺，包括发酵、细胞收获、碱裂解、用Q-Sepharose XL色谱法捕获质粒DNA、在Sephacryl S1000柱上进行尺寸排阻色谱以及用Source 15Q阴离子交换色谱法精制。分析了pUDK-HGF的质量标准，如纯度、浓度、均一性、残留RNA、染色体DNA、污染蛋白、内毒素和HGF表达效率，均符合药用级质粒DNA的要求。
