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

Microbial Cell Factories

Research

Edward R Appelbaum¹  Juan C Aon¹  Zhibiao Fu¹  Julie M Leighton¹  Pramatesh S Patel¹  Thomas D Verderame²  Brante P Sampey³ 

[1] Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, 19406, King of Prussia, PA, USA;Department of Process Engineering Manufacturing, Global Manufacturing and Supply, GlaxoSmithKline, 893 River Road, 19428, Conshohocken, PA, USA;Metabolon, Inc, 617 Davis Drive, Suite 400, 27713, Durham, NC, USA;

关键词: Saccharomyces cerevisiae;    Fermentation process;    Scale-up;    Mass transfer coefficient;    Oxygen uptake rate;    Exometabolomics;    Metabolome profiling;    Hypoxia;   

DOI  :  10.1186/1475-2859-13-32

 received in 2013-09-30, accepted in 2014-02-19,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundScale-up to industrial production level of a fermentation process occurs after optimization at small scale, a critical transition for successful technology transfer and commercialization of a product of interest. At the large scale a number of important bioprocess engineering problems arise that should be taken into account to match the values obtained at the small scale and achieve the highest productivity and quality possible. However, the changes of the host strain’s physiological and metabolic behavior in response to the scale transition are still not clear.ResultsHeterogeneity in substrate and oxygen distribution is an inherent factor at industrial scale (10,000 L) which affects the success of process up-scaling. To counteract these detrimental effects, changes in dissolved oxygen and pressure set points and addition of diluents were applied to 10,000 L scale to enable a successful process scale-up. A comprehensive semi-quantitative and time-dependent analysis of the exometabolome was performed to understand the impact of the scale-up on the metabolic/physiological behavior of the host microorganism. Intermediates from central carbon catabolism and mevalonate/ergosterol synthesis pathways were found to accumulate in both the 10 L and 10,000 L scale cultures in a time-dependent manner. Moreover, excreted metabolites analysis revealed that hypoxic conditions prevailed at the 10,000 L scale. The specific product yield increased at the 10,000 L scale, in spite of metabolic stress and catabolic-anabolic uncoupling unveiled by the decrease in biomass yield on consumed oxygen.ConclusionsAn optimized S. cerevisiae fermentation process was successfully scaled-up to an industrial scale bioreactor. The oxygen uptake rate (OUR) and overall growth profiles were matched between scales. The major remaining differences between scales were wet cell weight and culture apparent viscosity. The metabolic and physiological behavior of the host microorganism at the 10,000 L scale was investigated with exometabolomics, indicating that reduced oxygen availability affected oxidative phosphorylation cascading into down- and up-stream pathways producing overflow metabolism. Our study revealed striking metabolic and physiological changes in response to hypoxia exerted by industrial bioprocess up-scaling.

【 授权许可】

Unknown   
© Fu et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.