Department of Experimental Hematology, Beijing Institute of Radiation Medicine, People's Republic of China.
Biotechnol Appl Biochem. 2011 May;58(3):162-5. doi: 10.1002/bab.23. Epub 2011 May 26.
Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.
用于生物制药应用的质粒 DNA 很容易在大肠杆菌中产生。细胞裂解是质粒 DNA 纯化的最关键步骤。本文描述了一种连续的细胞碱性裂解、中和和澄清组合工艺，使用中空纤维超滤柱作为裂解室，生产质粒 pUDK-HGF，并分别与 T 型连接器和手动工艺进行比较。结果表明，组合工艺的质粒 pUDK-HGF 产量比手动裂解高 13%，比使用 T 型连接器高 2 倍。当裂解细胞和中和溶液的比例为 3:1 时，质粒 pUDK-HGF 的产量可提高 70%。该工艺可轻松放大，以满足细胞裂解的工业规模需求。
