Project Summary Abstract Continuous Bioproduction of Viral Vectors via High Cell Density, Membrane-less Perfusion Culture In this project, we propose to intensify the viral vector production process by the membrane-less perfusion bioreactor system, which was developed by our team at MIT. Based on the inertial microfluidics and fully validated in CHO cell cultivation, this system has many critical advantages over existing perfusion bioreactors, including high product recovery and avoiding membrane fouling, continuous removal of dead cells, cell debris, and cell aggregates, and easy maintenance and automation. As a result, our bioreactors can maintain high cell density reliably even when the cell viability and culture conditions change significantly, ideally suited for controlling viral vector production processes. Teaming with experts in HEK293 and Sf9 based production systems, we will implement long-term, high cell density cultures of host cells and explore and validate several innovative vector production platforms that provide more accurate control over fresh cell growth, transfection, incubation/production, and recovery of the products. Inertial microfluidic cell manipulation processes allow many novel controls over host cells in bioreactors, including selective separation of transfected from untransfected host cells. Finally, a continuous bioprocessing system will be optimized to maximize the quantity and quality of the finished viral vector products. For this, we will employ novel microfluidic electrokinetic separation of empty from filled viral capsid, which can provide near real-time determination of the quality of the product. In contrast to fed-batch or batch cultures, continuous monitoring of vector quality and quantity enables rapid adjustment of the culture protocols to restore/maintain optimal conditions for vector production over an extended period. This project is expected to have a broader impact on the bioproduction of viral vectors, but especially on rare and ultra-rare diseases where low disease prevalence requires relatively small production volumes. Such diseases do not justify the biopharmaceutical industry’s large investment needed for vector manufacturing.

Project Narrative Continuous Bioproduction of Viral Vectors via High Cell Density, Membrane-less Perfusion Culture This project will develop novel bioreactor technology for generating critical viral vectors used for gene and cell therapy applications, potentially reducing the cost and time necessary for manufacturing vaccines, gene and cell therapy products for various diseases. The developed technology will be flexible and cost-effective, applicable to small- and medium-scale production ideal for rare diseases.