Real-time monitoring of glycerol and methanol to enhance antibody production in industrial Pichia pastoris bioprocesses

Monoclonal antibody production in glycoengineered Pichia pastoris was optimized for high cell density fed-batch fermentations. Results show that antibody productivity was 1.72-fold higher when maintaining oxygen-limited conditions compared to methanol-limited conditions during the protein production phase of the bioprocess. Under oxygen-limited conditions with a 30 mmol L-1 h(-1) of oxygen uptake rate, an optimum methanol dosage level of 10 gL(-1) was established by comparing antibody titers under different methanol dosages during protein production. Real-time on-line NIR monitoring of glycerol concentration, methanol concentration, and relative cell density provided valuable data for process characterization and optimization. Real-time profiles of glycerol concentration and relative cell density documented run-to-run consistency during the batch and fed-batch phases by analyses of the glycerol consumption and cell growth kinetics. The volumetric methanol consumption rate was characterized for the induction process following the end of the glycerol fed-batch phase. Under oxygen-limited conditions, the volumetric methanol consumption rate increased over a 60-h period and this increase was independent of the methanol concentration over a range of 2.5-30 gL(-1) when the oxygen utilization rate was maintained at 30 mmol L-1 h(-1). Utility of the on-line NIR monitor is demonstrated over the one-year period of this investigation by accurately tracking biomass, glycerol concentration, and methanol concentration without user required recalibration as well as by documenting abnormal events during high cell density bioprocesses. (C) 2014 Elsevier B.V. All rights reserved.