(498e) Simplified Lab Scale and Pilot Scale Bioreactor Processes for Recombinant Butyrylcholinesterase Production in Transgenic Rice Cell Suspension Cultures

Human butyrylcholinesterase (BChE), a complex tetrameric hydrolase enzyme composed of four ~85 kDa identical monomers circulating in human blood plasma, is a potent bio-scavenger enzyme against organophosphorus (OP) nerve agents and pesticides due to its tight binding to these compounds. Conventionally, human BChE is purified from expired blood plasma, an approach which is exceptionally expensive. This makes recombinant butyrylcholinesterase (rBChE) a promising source of BChE for prophylactic and therapeutic purposes. Among various rBChE expression systems, metabolically regulated transgenic rice cell suspension culture controlled by the rice alpha-amylase promoter that is turned on under sugar starvation conditions leading to the production of rBChE, is one potentially cost-effective platform primarily due to its simple culture medium. Our group has produced and reported an active and tetrameric form of rBChE produced in transgenic rice cell suspension cultures via a cyclical semicontinuous operation in which media exchanges are performed to provide sugar-rich medium for the growth phase and sugar-free medium for the production phase. However, further process development is needed to enhance rBChE yield and simplify the process to facilitate scale-up in conventional bioreactors.

The maximum active cell-associated rBChE level is around 50 μg rBChE/g fresh weight, 2-fold higher than previous reports using the same cell-line, in a two-stage (growth and induction phases) batch operation and did not change significantly over a range (10% to 40%) of controlled dissolved oxygen (DO) concentration. When DO is uncontrolled with a fixed aeration rate (a constant flowrate of compressed air is sparged to the system) during the production phase, the maximum expression level is also equivalent to above experiments in which DO is controlled using a feedback control system with the composition of the sparging gas as the manipulated input. We also found that a single-stage batch operation with no medium exchange (the production of rBChE is triggered simply through sugar depletion due to uptake by the cells) yields maximum active cell-associated rBChE similar to a two-stage batch operation with a medium exchange. Furthermore, simplified small scale bioreactor operations with uncontrolled DO and no medium exchange were performed, and we found that they produce comparable rBChE levels to the others. Applying the simplified process from small scale bioreactors (uncontrolled DO and no media exchange) to a larger scale, we successfully scaled up the production of rBChE in transgenic rice cell suspension culture to a 40-L pilot scale bioreactor. Altogether, these studies demonstrate a simpler and potentially more efficient, economical, and scalable bioreactor process for rBChE production from metabolically regulated transgenic rice cell cultures.