Optical Redox Probe for Continuous Metabolic Monitoring during Natural Products Bioprocessing

项目摘要/摘要 天然产物包括多酮、脂肪酸、氨基酸、萜类和类固醇，正在发挥着重要的作用。 在制药业中扮演着越来越重要的角色。尽管许多天然产品是 这些分子的大规模生产是在植物中发现的，依赖于在生物反应器中培养微生物。 然而，开发和保持较高的生物制造生产率和产量往往是一个挑战 实现低成本、高质量的规模化生产。一个尚未得到满足的需求是能够迅速和 准确测量生物反应器内微生物在细胞水平上的生理状态。量测 细胞的代谢状态，特别是能量和氧化还原参数是指导决策的关键 饲喂策略和其他操作动作。由于生物反应器细胞培养的复杂性 目前，直接测量细胞代谢参数是不切实际的，而且依赖于 定期取出培养样本进行离线分析。然而，频繁采样会带来时间 延误、污染风险和制造成本增加。 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将开发一种新型双光子激发(TPE)荧光氧化还原传感器 用于在线、实时测量天然产物发酵生物反应器中的细胞代谢。一个 将开发出坚固的TPE纤维探头，这种探头可以消毒并插入生物反应器培养物中 连续测量重要细胞内代谢物的动态变化。细胞内氧化还原 将使用内源性荧光团的自体荧光来监测比率，从而消除了 外源荧光标记。这种基于光纤的多功能氧化还原探头的开发利用了 创新技术，包括通过专业设备高通量传输飞秒激光脉冲 使用独特的光纤束设计的光纤线和高效的信号采集。 在这个拟议的第一阶段计划中，PSI领导的团队将制造一个原型氧化还原传感器，并 评价其在酵母生物反应器中的性能。将进行概念验证实验，以 演示用于在线、连续代谢测量的原型的有效性 天然产物发酵生物反应器中的微生物。基于实时氧化还原的新陈代谢 测量将与标准的离线新陈代谢测量进行比较。两者之间的关联性 将调查细胞比率氧化还原评估和生物处理产率，以展示其价值 这项传感技术在指导有效的操作行动以提高生产率方面的作用 天然产物的生物加工。在随后的第二阶段计划中，PSI团队将开发 技术走向成熟，并进行大规模生物和工艺开发研究以证明 它在生物制造过程中的广泛应用。

Project Summary/Abstract Natural products including polyketides, fatty acids, amino acids, terpenoids and steroids, are playing an increasingly important role in the pharmaceutical industry. 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 guiding decisions on feeding strategies and other operation actions. Due to the complex nature of bioreactor cell culture systems, direct measurements of cellular metabolic parameters is current impractical, and relies on periodic removal of culture samples for off-line analysis. However, frequent sampling introduces time delays, contamination risks, and increased manufacturing costs. During this R&D program, Physical Sciences Inc. (PSI) in collaboration with the University of Massachusetts Lowell (UML) 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 robust TPE fiber probe will be developed that can be sterilized and inserted into bioreactor cultures for continuous measurement of dynamic changes of important intracellular metabolites. The intracellular redox ratio will be monitored using the autofluorescence of endogenous fluorophores, eliminating the need for exogenous fluorescence labeling. Development of this versatile, fiber-based redox probe draws on innovative techniques including high-throughput delivery of femtosecond laser pulses through a specialty fiber cord and efficient signal collection using a unique fiber bundle design. During this proposed Phase I program, the PSI led team will fabricate a prototype redox sensor and evaluate its performance in yeast bioreactors. Proof of concept experiments will be performed to demonstrate the effectiveness of the prototype for on-line, continuous metabolic measurement of microorganisms in natural products fermentation bioreactors. The real-time redox based metabolic measurement will be compared with standard off-line metabolism measurements. The correlation between cell ratiometric redox assessment and the bioprocessing yield will be investigated, demonstrating the value of this sensing technology in guiding effective operation actions for improved productivity during bioprocessing of natural products. During a subsequent Phase II program the PSI team will develop the technology to maturity and conduct large-scale biological and process development studies to demonstrate its broad application in biomanufacturing processes.