Large-Scale Purification of Plasmid DNA using Membrane Ultrafiltration

CBET-0755816Zydney这项由化学和生物分离计划授予的NSF奖项支持宾夕法尼亚州立大学化工系安德鲁·L·齐德尼教授研究使用膜超滤大规模纯化质粒DNA的工作。目前正在进行一些临床试验，将质粒DNA用作基因治疗剂或基于DNA的疫苗。然而，这项技术成功商业化的关键问题之一是大规模纯化质粒DNA所带来的成本和挑战。我们实验室获得的初步结果表明，由于质粒在膜上方的流场中的伸长，通过超滤膜的质粒传输是滤液通量的强烈函数。我们的基本假设是，这种通量依赖性，以及不同质粒异构体(线形、开环和超螺旋)在伸长灵活性方面的相关差异，可以利用膜系统实现高分辨率的质粒纯化。实验研究将考察不同膜的行为，包括中性和带电聚合物，以及溶液环境和操作条件对质粒DNA超滤行为的影响。数据将使用适当的模型进行分析，该模型解释了由于流动而导致的质粒的伸长。这些结果将为开发用于去除宿主细胞蛋白和分离不同DNA亚型的膜系统提供基本的见解和实用的指导方针。此外，该研究项目将为研究生和本科生提供处理生物分子提纯的膜过程的实践经验，为他们在生物技术和制药行业追求职业生涯提供理想的背景。

CBET-0755816ZydneyThis NSF award by the Chemical and Biological Separations program supports work by Professor Andrew L. Zydney in the Department of Chemical Engineering at The Pennsylvania State University to examine the use of membrane ultrafiltration for the large-scale purification of plasmid DNA. A number of clinical trials are currently underway on the use of plasmid DNA as a gene therapy agent or a DNA-based vaccine. However, one of the critical issues for the successful commercialization of this technology is the cost and challenge associated with the large-scale purification of plasmid DNA. Preliminary results obtained in our laboratory have demonstrated that plasmid transmission through ultrafiltration membranes is a strong function of the filtrate flux due to elongation of the plasmid within the flow field above the membrane. Our underlying hypothesis is that this flux dependence, and the associated differences in elongational flexibility of the different plasmid isoforms (linear, open-circular, and supercoiled), can be exploited to achieve high-resolution plasmid purification using membrane systems. Experimental studies will examine the behavior of different membranes, including both neutral and charged polymers, as well as the effects of solution environment and operating conditions on the ultrafiltration behavior of plasmid DNA. Data will be analyzed using appropriate models that account for the elongation of the plasmid due to flow. These results will provide fundamental insights and practical guidelines for the development of membrane systems for the removal of host cell proteins and the separation of different DNA isoforms. In addition, this research program will provide graduate and undergraduate students hands-on experience dealing with membrane processes for biomolecule purification, giving them an ideal background to pursue careers in the biotechnology and pharmaceutical industries.