Optical Redox Probe for Continuous Metabolic Monitoring during Natural Products Bioprocessing

项目总结/摘要 市场上几乎一半的药物是天然产物，包括聚酮化合物、脂肪酸、氨基甲酸酯、 酸类、萜类和类固醇。虽然许多天然产物是在植物中发现的， 这些分子的生产依赖于在生物反应器中培养微生物。然而，它往往是一个 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 大规模的优质生产。一个未满足的需求是能够快速准确地测量 生物反应器内细胞水平的微生物生理状态。测量细胞代谢状态， 特别是能量和氧化还原参数，是开发改进的生物合成过程的关键， 指导喂养战略和其他业务行动。由于生物反应器细胞培养的复杂性， 系统，目前无法获得真实的时间测量细胞代谢参数的能力，以及 代谢评估依赖于耗时的、周期性的取出培养物样品用于离线分析。 在这个研发项目中，物理科学公司。(PSI)，与大学合作， 马萨诸塞州洛厄尔（UML）和东北大学（NEU），将开发一种新的双光子激发 (TPE)荧光氧化还原传感器，用于在线实时测量生物反应器中的细胞代谢， 天然产物发酵将开发一种小型化的光学探头， 插入生物反应器培养物中，用于连续测量重要的细胞内 代谢物。该技术相当于生物反应器内的在线细胞计数器，提供关键的 细胞水平的生理数据，否则只能从离线测量中获得。的 将使用内源性荧光团的自发荧光监测细胞内氧化还原比，而不 需要（但不排除选择）外源荧光标记。 在第一阶段计划中，PSI领导的团队成功地证明了拟议的 技术.第一代原型氧化还原传感器的制造和测试过程中的连续代谢 在运行的生物反应器中进行测量，减轻拟议的第二阶段的关键技术风险 research.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， 用于商业化。特别是，第二代原型具有显著改善的信噪比， 将制造系统鲁棒性。第二代仪器将在大量的 生物反应器操作，以证明新的实时细胞代谢数据将如何用于指导 开发和操作新的和更有效的生物反应器工艺，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 理解和控制过程。

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.