Institute of Genetic Engineering, Southern Medical University, Guangzhou, PR China.
Pharm Biol. 2013 Jan;51(1):42-8. doi: 10.3109/13880209.2012.703678. Epub 2012 Sep 27.
The recent developments in non-viral gene therapy and DNA vaccine have fostered the development of efficient plasmid DNA (pDNA) purification processes.
This work aimed to establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccine.
E. coli DH5α harboring pCDNA3.1-GFP (7200 base pairs) was used as a model plasmid. Hydrophobic-interaction chromatography (HIC) was employed to purify supercoiled plasmid DNA (sc pDNA).
With this method, not only host contaminants, but also open circular plasmid DNA (oc pDNA) could be removed from sc pDNA. Anion-exchange HPLC analysis proved that the recovery of HIC could reach 75%. The plasmid DNA exhibited high purity with supercoiled percentage of 98 ± 1.2% and undetectable residual endotoxins, genomic DNA, RNA and protein. The purity of pDNA had nothing to do with the flow rate in the range at least up to 400 cm/h. Liposomes transfection experiment prove that the purified pDNA in this article had higher transfection efficiency than the control pDNA.
In the present work, we confirmed the possibility of separation of sc pDNA from oc pDNA and other host contaminants using a single HIC chromatography.
最近非病毒基因治疗和 DNA 疫苗的发展促进了高效质粒 DNA（pDNA）纯化工艺的发展。
本研究旨在建立一种用于基因治疗和 DNA 疫苗大规模生产质粒 DNA 的经济高效的纯化工艺。
以含有 pCDNA3.1-GFP（7200 个碱基对）的大肠杆菌 DH5α 为模型质粒。采用疏水性相互作用色谱（HIC）纯化超螺旋质粒 DNA（sc pDNA）。
该方法不仅可以去除宿主污染物，还可以去除开环质粒 DNA（oc pDNA）。阴离子交换 HPLC 分析证明 HIC 的回收率可达 75%。质粒 DNA 具有高纯度，超螺旋百分比为 98±1.2%，且检测不到残留内毒素、基因组 DNA、RNA 和蛋白质。质粒 DNA 的纯度与流速无关，至少在 400cm/h 的范围内没有关系。脂质体转染实验证明，本文纯化的 pDNA 比对照 pDNA 具有更高的转染效率。
在本研究中，我们证实了使用单一 HIC 色谱分离 sc pDNA 与 oc pDNA 和其他宿主污染物的可能性。
