Optical Redox Probe for Continuous Metabolic Monitoring during Natural Products Bioprocessing

项目摘要/摘要 市场上几乎一半的药品是天然产品，包括多酮、脂肪酸、氨基酸 酸、萜类和类固醇。尽管许多天然产物是在植物中发现的，但马萨诸塞州 这些分子的生产依赖于在生物反应器中培养微生物。然而，它通常是一种 开发和保持高生物制造生产率和产量以实现低成本和高产量的挑战 大规模的高质量生产。一个未得到满足的需求是快速而准确地测量 生物反应器内微生物在细胞水平上的生理状态。测量细胞代谢状态， 尤其是能量和氧化还原参数，是开发改进的生物合成工艺和 指导喂养策略和其他操作行动。由于生物反应器细胞培养的复杂性 系统，目前无法实时测量细胞代谢参数，以及 新陈代谢评估依赖于耗时的、定期取出培养样本进行离线分析。 在这一研发计划期间，物理科学公司(PSI)与加州大学 马萨诸塞州洛厄尔大学和东北大学将开发一种新的双光子激发 用于在线、实时测量生物反应器中细胞代谢的(TPE)荧光氧化还原传感器 天然产物发酵。将开发一种微型光学探头，它可以消毒和 插入到生物反应器培养物中，用于连续测量重要细胞内的动态变化 代谢物。这项技术相当于生物反应器内的在线细胞仪，提供了关键的 细胞水平的生理学数据，否则只能从离线测量中获得。这个 细胞内的氧化还原比率将使用内源性荧光团的自体荧光进行监测，而不是 外源荧光标记的必要性(但不排除该选项)。 在第一阶段计划期间，PSI领导的团队成功地证明了拟议的 技术制作了第一代氧化还原传感器样机，并在连续代谢过程中进行了测试 在运行中的生物反应器中进行测量，减轻拟议第二阶段的关键技术风险 研究。在第二阶段计划中，研究工作将集中在优化技术上， 展示其在推进生物制造工艺开发和运营方面的价值，并准备 为了商业化。特别是，第二代原型具有显著提高的信噪比和 系统的健壮性将被制造出来。第二代仪器随后将在大量的 生物反应器操作，演示如何利用新的实时细胞代谢数据来指导 新的和更有效的生物反应器工艺的开发和运行，以提高产品产量。 实验数据也将被输入到理论生物过程模型中，以促进改进 对过程的理解和控制。

Project Summary/Abstract Almost half of the medicines on the market are natural products including polyketides, fatty acids, amino acids, terpenoids and steroids. Though many of the natural products are discovered in plants, mass production of these molecules relies on culturing microorganisms in bioreactors. However, it is often a challenge to develop and maintain high biomanufacturing productivity and yield to enable low-cost and high- quality production at large scales. One unmet need is an ability to rapidly and accurately measure the physiological status of microbes at the cellular level within bioreactors. Measuring the cellular metabolic state, especially energetic and redox parameters, is key to developing improved biosynthesis processes and to guiding feeding strategies and other operational actions. Due to the complex nature of bioreactor cell culture systems, currently the capability of measuring cellular metabolic parameters in real time is unavailable, and metabolic assessment relies on time-consuming, periodic removal of culture samples for off-line analysis. During this R&D program, Physical Sciences Inc. (PSI), in collaboration with the University of Massachusetts Lowell (UML) and Northeastern University (NEU), will develop a novel two-photon excitation (TPE) fluorescence redox sensor for on-line, real-time measurement of cell metabolism in bioreactors for natural products fermentation. A miniaturized optical probe will be developed that can be sterilized and inserted into bioreactor cultures for continuous measurement of dynamic changes of important intracellular metabolites. The technology is equivalent to an online cytometer within the bioreactor, providing critical cellular-level physiology data that would otherwise be only available from off-line measurements. The intracellular redox ratio will be monitored using the autofluorescence of endogenous fluorophores, without the need (but not excluding the option) for exogenous fluorescence labeling. During the Phase I program, the PSI led team successfully demonstrated the feasibility of the proposed technology. A Gen-I prototype redox sensor was fabricated and tested during continuous metabolism measurements in operational bioreactors, mitigating the critical technical risks of the proposed Phase II research. During the Phase II program, the research effort will be focused on optimizing the technology, demonstrating its value in advancing biomanufacturing process development and operations, and preparing for commercialization. In particular, a Gen-II prototype with significantly improved signal to noise ratio and system robustness will be fabricated. The Gen-II instrument will then be tested in a large-number of bioreactor operations to demonstrate how the new real-time cellular metabolism data will be utilized to guide the development and operation of novel and more effective bioreactor processes for improved product yield. The experimental data will also be input into theoretical bioprocess models to promote improved understanding and control of the processes.