DEVELOPMENT AND VALIDATION OF A BREATHABLE SHAKE FLASK WITH INTEGRATED NON-INVASIVE SENSORS FOR REAL-TIME CRITICAL PROCESS PARAMETERS MONITORING AND CONTROL

Abstract

Shake flasks are one of the most widely used bioreactors in industrial biotechnology research and process development. For over a century of usage in microbiology, shake flasks have remained unchanged. The only innovations have been the introduction of baffles and the move towards disposable plastic material to fabricate the flask and closure plugs. Shake flask suffers from a significant problem. The opening on top of the shake flask is the only route for O2 to diffuse in and CO2 to diffuse out. However, this opening is often sealed with a closure plug to prevent spill-over and maintain sterility. These seal closures provide undesired mass transfer resistance to the gaseous exchange between the shake flask and the external environment, which creates a severe paucity of O2 and accumulation of CO2, leading to poor growth conditions in the flask. There is no monitoring or control over culture conditions like pH, dissolved O2, or dissolved CO2. These often result in inconsistencies in process development studies, culminating in inefficient recombinant protein production, and constitute a major source of heterogeneities as the process moves from flasks to stirred tank bioreactors. Breathable flask has been invented to address the limitations of a shake flask. It is made of a biocompatible silicone membrane that is highly permeable to O2 and CO2. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of O2 and CO2 between the flask and the external environment, leading to a 28-66% growth in biomass and 41-56% improvement in recombinant protein titer. Completely non-invasive sensors for O2, CO2 and pH were developed and integrated with the breathable flask for real time process monitoring. Further, a jacket was developed that encapsulates a breathable flask. This jacket facilitated control of O2 and CO2 concentrations inside the breathable flask by flushing the jacket with desired gas mixes. By actively monitoring and controlling the O2 in the jacketed breathable flask by controlling the gas mix and flow rate in the jacket, O2 limitations were completely eliminated in the microbial cultures for the first time. Growth was optimized in the jacketed flask, with an improvement of 81-156% in biomass and 76-140% in recombinant yield. A hydrodynamic model has been developed to make informed decisions on selecting the agitation rate and fill volume by modeling the fluid behavior in the shake flask. The last part of the work focused on developing a commercial manufacturing capacity by following DoD’s technology and manufacturing readiness level guidelines to commercialize the flask technology. The work delivered a breathable flask system equipped with integrated real-time sensors for monitoring and controlling dissolved O?, CO?, and pH. This innovation transforms ubiquitous laboratory shake flasks into a smart, high-throughput platform capable of generating invaluable process data for optimizing cellular expression and scaling new biomanufacturing technologies affordably at the lab bench.