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

Fu Z, Verderame TD, Leighton JM, Sampey BP, Appelbaum ER, Patel PS, Aon JC - Microb. Cell Fact. (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA. juan.c.aon@gsk.com.

ABSTRACT

Background: Scale-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.

Results: Heterogeneity 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.

Conclusions: An 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.

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Pathway diagram and corresponding heat map table and line plot graphs showing biochemical differences in the intermediates of the mevalonate/ergosterol pathway between scales. (A) Line plot graphs comparison between two scales. (B) Pathway diagram comparison between two scales. (C) Heat map table comparison between two scales. Red and green arrows show changes in the 10,000 L scale with respect to that at the 10 L scale. The intermediates measurement and data analysis were determined as described in “Methods”. Error bars represent “mean +/− one standard deviation”. “M” and “F” in the time axis for every line plot stand for Medium and Feed samples, respectively.
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Figure 6: Pathway diagram and corresponding heat map table and line plot graphs showing biochemical differences in the intermediates of the mevalonate/ergosterol pathway between scales. (A) Line plot graphs comparison between two scales. (B) Pathway diagram comparison between two scales. (C) Heat map table comparison between two scales. Red and green arrows show changes in the 10,000 L scale with respect to that at the 10 L scale. The intermediates measurement and data analysis were determined as described in “Methods”. Error bars represent “mean +/− one standard deviation”. “M” and “F” in the time axis for every line plot stand for Medium and Feed samples, respectively.

Mentions: Alterations in the mevalonate/ergosterol pathway have been shown to affect membrane composition and permeability. These effects could potentially influence the volume occupied by cells as shown in Figure 5A, or trafficking of metabolites through membranes and cell wall. Figure 6 depicts the metabolite profiles from the mevalonate/ergosterol pathway obtained at 10 L and 10,000 L production scales. Concentrations from both ergosterol and its precursor lanosterol were lower at 10,000 L than at 10 L scale, but the kinetics of increase was similar (Figure 6A). However, 3-hydroxy-3-methylglutarate and acetoacetate increased over time at 10,000 L scale while they decreased at 10 L scale (Figure 6A). The consistent pattern of elevated concentrations from upstream metabolites, 3-hydroxy-3-methylglutarate and acetoacetate, and decreased levels from downstream metabolites, lanosterol and ergosterol, lend notable credence to a diminished or impaired ergosterol synthesis at the 10,000 L scale culture (Figure 6B).


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

Fu Z, Verderame TD, Leighton JM, Sampey BP, Appelbaum ER, Patel PS, Aon JC - Microb. Cell Fact. (2014)

Pathway diagram and corresponding heat map table and line plot graphs showing biochemical differences in the intermediates of the mevalonate/ergosterol pathway between scales. (A) Line plot graphs comparison between two scales. (B) Pathway diagram comparison between two scales. (C) Heat map table comparison between two scales. Red and green arrows show changes in the 10,000 L scale with respect to that at the 10 L scale. The intermediates measurement and data analysis were determined as described in “Methods”. Error bars represent “mean +/− one standard deviation”. “M” and “F” in the time axis for every line plot stand for Medium and Feed samples, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016033&req=5

Figure 6: Pathway diagram and corresponding heat map table and line plot graphs showing biochemical differences in the intermediates of the mevalonate/ergosterol pathway between scales. (A) Line plot graphs comparison between two scales. (B) Pathway diagram comparison between two scales. (C) Heat map table comparison between two scales. Red and green arrows show changes in the 10,000 L scale with respect to that at the 10 L scale. The intermediates measurement and data analysis were determined as described in “Methods”. Error bars represent “mean +/− one standard deviation”. “M” and “F” in the time axis for every line plot stand for Medium and Feed samples, respectively.
Mentions: Alterations in the mevalonate/ergosterol pathway have been shown to affect membrane composition and permeability. These effects could potentially influence the volume occupied by cells as shown in Figure 5A, or trafficking of metabolites through membranes and cell wall. Figure 6 depicts the metabolite profiles from the mevalonate/ergosterol pathway obtained at 10 L and 10,000 L production scales. Concentrations from both ergosterol and its precursor lanosterol were lower at 10,000 L than at 10 L scale, but the kinetics of increase was similar (Figure 6A). However, 3-hydroxy-3-methylglutarate and acetoacetate increased over time at 10,000 L scale while they decreased at 10 L scale (Figure 6A). The consistent pattern of elevated concentrations from upstream metabolites, 3-hydroxy-3-methylglutarate and acetoacetate, and decreased levels from downstream metabolites, lanosterol and ergosterol, lend notable credence to a diminished or impaired ergosterol synthesis at the 10,000 L scale culture (Figure 6B).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA. juan.c.aon@gsk.com.

ABSTRACT

Background: Scale-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.

Results: Heterogeneity 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.

Conclusions: An 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.

Show MeSH
Related in: MedlinePlus