PhD Dissertation Defense: Vikash Kumar

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 facilitate 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. A completely non-invasive sensors for O2, CO2 and pH was 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 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 O2, CO2, 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.