Large-Scale Purification of Plasmid DNA using Membrane Ultrafiltration

使用膜超滤大规模纯化质粒 DNA

• 批准号: 0755816
• 负责人: Andrew Zydney
• 金额: $ 24万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0755816&HistoricalAwards=false
• 关键词: Large; Scale; Purification; Plasmid; DNA

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.

CBET-0755816Zydney 这项由化学和生物分离项目颁发的 NSF 奖项支持宾夕法尼亚州立大学化学工程系的 Andrew L. Zydney 教授研究膜超滤在大规模纯化质粒 DNA 中的应用。 目前正在进行许多关于使用质粒 DNA 作为基因治疗剂或基于 DNA 的疫苗的临床试验。 然而，该技术成功商业化的关键问题之一是与大规模纯化质粒 DNA 相关的成本和挑战。我们实验室获得的初步结果表明，由于质粒在膜上方流场内的伸长，质粒通过超滤膜的传输是滤液通量的重要函数。 我们的基本假设是，可以利用这种通量依赖性以及不同质粒亚型（线性、开环和超螺旋）的伸长灵活性的相关差异，以使用膜系统实现高分辨率质粒纯化。 实验研究将检查不同膜（包括中性和带电聚合物）的行为，以及溶液环境和操作条件对质粒 DNA 超滤行为的影响。 将使用适当的模型来分析数据，该模型考虑了流动引起的质粒伸长。这些结果将为开发用于去除宿主细胞蛋白质和分离不同 DNA 亚型的膜系统提供基本见解和实用指南。 此外，该研究项目将为研究生和本科生提供处理生物分子纯化膜工艺的实践经验，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。