Exometabolome analysis reveals hypoxia at the up-scaling of a Saccharomyces cerevisiae high-cell density fed-batch biopharmaceutical process

Scale-up of industrial fermentation process occurs after the process is optimized in the small scale and is critical for a successful technology transfer and subsequent commercialization of the product of interest. Here we characterize process performance changes that were observed during the scaling-up of a Saccharomyces cerevisiae high-cell density fed-batch culture process to commercial scale (10,000L) bioreactor. The fermentation process was optimized and demonstrated to be robust and reproducible in small scale (10L) bioreactors. However, differences were observed at large scale that affected process controllability including increases in wet cell weight; culture viscosity and pure oxygen sparge supply. Mitigation of these differences to ensure process performance at large scale required changes in the dissolved oxygen and pressure set points as well as addition of diluents. To further understand the physiological changes between two scales, we performed a comprehensive quantitative and time-dependent analysis of the exometabolome of the culture at both scales. A metabolome profiling approach was used to identify groups of biochemicals associated to known metabolic pathways in S. cerevisiae. At both scales and different time points a diversity of intermediates was found from central carbon metabolism, mevalonate/ergosterol synthesis pathway, and precursor synthesis devoted to glycosylation. At 10,000L the profiling revealed exacerbated hypoxia affecting cell structures, membranes and cell wall; and cell functions, ergosterol biosynthesis and Krebs cycle. The contribution of this work is the knowledge of how cells respond to such an environment at the large scale and how those responses are related to the process performance.