High-cell-density cultivation and recombinant protein production with Komagataella pastoris in stirred-tank bioreactors from milliliter to cubic meter scale

High-cell-density cultivations (HCDC) with methylotrophic yeasts need continuous methanol feeding at controlled process conditions. However, none of the miniaturized bioreactor systems in use for screening studies facilitates continuous feeding. Hence, continuous feeding of methanol in pH-controlled parallel stirred-tank bioreactors on a milliliter scale was ensured by supplying air with varying methanol concentrations in the gas phase. The gas-liquid methanol transfer rates were characterized as a function of the operating conditions. Methanol feeding rates of up to 5.3 g L-1 h(-1) were possible without oxygen limitation at the chosen process conditions. HCDC processes with 50-60 g L-1 cell dry weight within 49-69 h were established on the milliliter scale with extracellular production of Candida antarctica lipase B2 variant (CaL-B2) as an example (16,604 U L-1). The scalability was demonstrated by transferring the Komagataella pastoris HCDC processes to the liter and cubic meter scale with the maximum oxygen transfer rate as the scale-up criterion. Biomass concentrations and lipase activities were the same at all scales within the estimation error. Thus, parallel-operated stirred-tank bioreactors on a milliliter scale with continuous methanol feeding via the gas phase will reduce the time and cost of screening methylotrophic yeast strains and cultivation conditions. (C) 2015 Elsevier Ltd. All rights reserved.