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目的提取、纯化靶向治疗肺癌的质粒TV-BIKDD,并进行质量检定。方法采用碱裂解法对工程菌DH5α/TVBIKDD菌体进行质粒初提,通过两步整体柱层析(离子柱层析和疏水柱层析)进行质粒纯化,并检测质粒含量、DNA超螺旋比例;用该方法连续纯化3批以超螺旋质粒DNA为样品的溶液制剂,对纯化后的质粒进行质量检定。结果质粒初提液经离子柱层析纯化后,去除了大量的RNA和蛋白质,再经过疏水柱层析纯化,最终获得的质粒DNA超螺旋比例为95.21%以上,质粒总回收率为78%。纯化后的3批溶液制剂全面质量检定结果均符合美国FDA标准以及我国人基因治疗用产品的技术指导原则规定。结论建立了稳定的TV-BIKDD质粒的纯化工艺,为进一步制备临床药用级样品的规模化工艺研究奠定了基础。
Objective To extract and purify the plasmid TV-BIKDD targeted for lung cancer and conduct quality control. Methods The plasmid of DH5α / TVBIKDD was digested with alkaline lysis method. The plasmid was purified by two steps of monolithic column chromatography (ion and column chromatography), and the plasmid content and DNA supercoiling ratio were detected. Using this method, three batches of solution preparation containing supercoiled plasmid DNA as samples were continuously purified and the quality of purified plasmids was checked. Results After purified by ion-exchange column chromatography, a large amount of RNA and protein were removed and purified by hydrophobic column chromatography. The ratio of supercoiled plasmid DNA was over 95.21%, and the total recovery of plasmid was 78%. The purified batches of three batches of solutions were tested in accordance with the US FDA standards and the technical guidelines for human gene therapy products in China. Conclusion A stable purification process of TV-BIKDD plasmid was established, which laid the foundation for the further study of large-scale production of clinical medicinal grade samples.