克级生产的质粒pUDK-HGF与目前良好的生产规范,用于基因治疗严重肢体缺血

Chunsheng Hu, Xiaochen Cheng, Yuxin Lu, Zu-ze Wu, Qingling Zhang
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引用次数: 0

摘要

在临床试验中,对大量、高纯度、高浓度编码人肝细胞生长因子基因(pUDK-HGF)的质粒的需求日益增加,用于危重肢体缺血(CLI)的基因治疗。在本文中,我们按照当前的克级良好生产规范生产了pUDK-HGF。该工艺包括50 l批量发酵,连续碱性裂解,在Sepharose 6 Fast Flow, PlasmidSelect Xtra和Source 15Q上集成三步色谱。经扩大生产工艺,1.0 kg细菌胞浆可制得4.24±0.41 g药用pUDK-HGF,总产率为58.37% ~ 66.70%。最终产物纯度高,超卷绕率> 95.8%,检测不到残留RNA、污染蛋白和细菌内毒素。I期临床研究表明,肌内注射pUDK-HGF是安全的,耐受性良好,可以缓解CLI患者的症状。这些结果表明,我们的制备工艺可以有效地生产药用级质粒DNA,并且可以安全用于临床应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.
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