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.

Project Narrative Biopharmaceutical production relies on complex expression systems to produce active pharmaceutical ingredients including natural products. Economically viable processes that produce high-quality drug substance are critical for product success and patient health. This R&D addresses a key limitation in biomanufacturing, accurately monitoring of cells within bioreactors in real time.