Optical Redox Probe for Continuous Metabolic Monitoring during Natural Products Bioprocessing

用于天然产品生物加工过程中连续代谢监测的光学氧化还原探针

• 批准号: 9907720
• 负责人: Youbo Zhao
• 金额: $ 23.23万
• 依托单位: PHYSICAL SCIENCES, INC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-20 至 2020-12-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907720
• 关键词: Academia; Address; Aerobic; Amino Acids; Biological Process; Biological Products; Biomanufacturing; Bioreactors; Calibration; Carotenoids; Cell Culture System; Cell Proliferation; Cell physiology; Cells; Cellular Metabolic Process; Collaborations; Collection; Complex; Data; Detection; Development; Drug Industry; Effectiveness; Engineering; Equilibrium; Escherichia coli; Excision; Failure; Fatty Acids; Fermentation; Fiber; Flavin-Adenine Dinucleotide; Fluorescence; Glucose; Goals; Health; Industry; Label; Lasers; Lead; Massachusetts; Measurement; Measures; Metabolic; Metabolism; Methodology; Microbe; Monitor; Natural Products; Nature; Nicotinamide adenine dinucleotide; Noise; Omega-3 Fatty Acids; Optics; Organism; Oxidation-Reduction; Oxygen; Pathway interactions; Patients; Performance; Periodicity; Pharmacologic Substance; Phase; Physiologic pulse; Physiological; Plants; Play; Process; Production; Productivity; Respiration; Risk; Role; Sales; Sampling; Signal Transduction; Small Business Innovation Research Grant; Steroids; System; Techniques; Technology; Terpenes; Testing; Time; Universities; Validation; Yarrowia lipolytica; Yeasts; base; bioprocess; cost; cost effectiveness; design; drug quality; experimental study; extracellular; feeding; fluorophore; improved; inhibitor/antagonist; innovation; instrument; lycopene; medical specialties; metabolic engineering; metabolomics; microbial; microorganism; microorganism culture; miniaturize; novel; operation; physical science; process optimization; programs; promoter; prototype; ratiometric; research and development; sensor; sensor technology; success; temporal measurement; two-photon

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.

项目总结/摘要 天然产物包括聚酮化合物、脂肪酸、氨基酸、萜类化合物和甾体化合物， 在医药行业中的作用越来越大。虽然许多天然产品 这些分子在植物中发现，大规模生产这些分子依赖于在生物反应器中培养微生物。 然而，开发和保持高的生物制造生产率和产量， 实现低成本和高质量的大规模生产。一个未满足的需求是能够快速和 在生物反应器内的细胞水平上准确测量微生物的生理状态。测量 细胞代谢状态，特别是能量和氧化还原参数是指导决策的关键， 喂养策略和其他操作动作。由于生物反应器细胞培养的复杂性， 系统中，细胞代谢参数的直接测量目前是不切实际的，并且依赖于 定期取出培养物样品用于离线分析。然而，频繁采样会引入时间 延迟、污染风险和增加的制造成本。 在这个研发项目中，物理科学公司。(PSI)在与大学合作， 马萨诸塞州洛厄尔（UML）将开发一种新型的双光子激发（TPE）荧光氧化还原传感器 用于天然产物发酵生物反应器中细胞代谢的在线实时测量。一 将开发一种坚固的TPE纤维探针，可以灭菌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发荧光来监测比率，从而消除了对 外源荧光标记这种多功能的基于纤维的氧化还原探针的开发利用了 创新技术，包括飞秒激光脉冲通过专业 采用独特的光纤束设计，实现了高效率的信号采集。 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， 评价其在酵母生物反应器中的性能。将进行概念验证实验， 证明了原型的有效性，用于在线，连续的代谢测量， 天然产物发酵生物反应器中的微生物。实时氧化还原代谢 将测量结果与标准离线代谢测量结果进行比较。的相关性 将研究细胞比率氧化还原评估和生物加工产率，证明其价值 这种传感技术在指导有效的操作行动，以提高生产率， 天然产物的生物处理。在随后的第二阶段计划中，PSI团队将开发 技术成熟并进行大规模生物和工艺开发研究以证明 其在生物制造过程中的广泛应用。