Project Summary/Abstract Gene therapy is an emerging therapeutic technology that uses genes in targeted cells to prevent and/or treat acquired disorders and inherited genetic diseases. The rapid advancement of human gene and disease research has increased the demand for gene therapy and investment in the production of large-scale clinical applications. However, the clinical potential of gene therapy is limited by the current manufacturing capability of gene delivery products. Gene therapy biologics are mostly produced by harvesting viral vectors in mammalian cell cultures, which is a delicate and intricate process that can be affected by many environmental parameters. Inefficient monitoring and control of the viral production processes can lead to various manufacturing defects such as cell instability, high impurities, difficult scalability, and low productivity. Therefore, in order to develop efficient and robust biomanufacturing processes, comprehensive knowledge and understanding of bioprocess dynamics are needed. This in turn necessitates the need to develop capable analytical technologies to provide accurate and timely information on key bioprocess parameters and quality attributes. Physical Sciences Inc. (PSI), in collaboration with the University of Massachusetts Lowell (UML), proposes to develop a novel in-line biomanufacturing process analytical technology tool. It enables real-time monitoring of the biomolecules of host cells and their environment in large-scale bioreactors. The technology is based on deep ultraviolet excited optical spectroscopic techniques that combine Raman scattering and autofluorescence to differentiate cellular biomolecules including DNA and protein produced during cell culture processes. An innovative spatially and temporally confined spectroscopy cytometry design will also be adopted to efficiently distinguish intra-cellular biomolecules within the host cells from extra-cellular biomolecules in surrounding media. A miniaturized optical probe will be designed that will inserted into an agitated bioreactor for in-line cytometry measurements. In this Phase I program, a prototype cytometry probe system will be designed, constructed, and evaluated by measuring and analyzing cellular biomolecules in real-time during viral vector production bioreactor operations. During the Phase II program, the technology will be improved, and its performance will be validated in different bioreactors using different culture organisms. Successful development of this technology will support biotech R&D to improve the understanding and monitoring of bioprocesses, which in turn will promote the development of new processes for large-scale production of viral vector products for gene therapy applications.