School of Life Science and Bio-Pharmaceutical, Guangdong Pharmaceutical University, 510006, Guangzhou, China.
Cytotechnology. 2011 Jan;63(1):7-12. doi: 10.1007/s10616-010-9322-9. Epub 2010 Dec 1.
To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.
为了建立一种经济有效的质粒 DNA 纯化工艺，用于基因治疗和 DNA 疫苗的大规模生产，本研究采用单一阴离子交换色谱（AEC）步骤，从澄清的裂解液中存在的其他形式和大肠杆菌杂质中纯化超螺旋质粒 DNA（sc pDNA）。两种不同大小和构象的质粒被用作模型靶标，并且在该色谱操作中表现出相似的洗脱行为，其中 sc pDNA 可在盐梯度中有效与开环质粒 DNA（oc pDNA）分离。该工艺可提供纯度高达 95 ± 1.1%、内毒素、基因组 DNA、RNA 和蛋白质含量几乎不可检测的高纯度 pDNA，产率为 65 ± 8%。此外，转染效率（29 ± 0.4%）显著高于 pDNA 对照（20 ± 0.1%）。本研究证实了在澄清的大肠杆菌裂解液中，使用单一 AEC 步骤从其他形式和宿主污染物中纯化 sc pDNA 的可能性。
