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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, line plot graphs and corresponding heat map table showing the changes in branch chain amino acid levels over time between scales. (A) Line plot graphs and pathway diagram comparison between two scales. (B) 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 10: Pathway diagram, line plot graphs and corresponding heat map table showing the changes in branch chain amino acid levels over time between scales. (A) Line plot graphs and pathway diagram comparison between two scales. (B) 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: Further examination of mitochondrial function at both scales was performed through analysis of biochemical markers. An increase in branched chain amino acids, valine, isoleucine and leucine, and their alpha-keto acid intermediates are known metabolic indicators of mitochondrial dysfunction or respiratory-deficient cells[18,19]. As shown in Figure 10, branched chain amino acids (valine, isoleucine and leucine) and their alpha-keto acid intermediates (alpha-keto acids 3-methyl-2-oxobutyrate, 3-methyl-2-oxovalerate and 4-methyl-2oxopentanoate) either stayed constant or decreased over time at the 10 L scale. In contrast, branched chain amino acids were notably elevated in media at the EFT 54 hr at the 10,000 L scale. These results suggested mitochondrial dysfunction or impairment that occurred in parallel with accumulation of glycerol-3-phosphate and intermediates of TCA cycle and mevalonate pathways. Mitochondrial impairment could also reflect cell hypoxia at the 10,000 L scale culture resulting in changes in ergosterol synthesis and glucose degradation. Importantly, associated with the hypoxia phenomenon the marked differences in lanosterol and ergosterol levels found in the 10 L and 10,000 L scale media at the earliest time point (EFT 46 hr) (Figure 6A), may indicate the divergence in their metabolic behavior thus initiating the scale-specific differences unveiled by exometabolomics.


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, line plot graphs and corresponding heat map table showing the changes in branch chain amino acid levels over time between scales. (A) Line plot graphs and pathway diagram comparison between two scales. (B) 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 10: Pathway diagram, line plot graphs and corresponding heat map table showing the changes in branch chain amino acid levels over time between scales. (A) Line plot graphs and pathway diagram comparison between two scales. (B) 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: Further examination of mitochondrial function at both scales was performed through analysis of biochemical markers. An increase in branched chain amino acids, valine, isoleucine and leucine, and their alpha-keto acid intermediates are known metabolic indicators of mitochondrial dysfunction or respiratory-deficient cells[18,19]. As shown in Figure 10, branched chain amino acids (valine, isoleucine and leucine) and their alpha-keto acid intermediates (alpha-keto acids 3-methyl-2-oxobutyrate, 3-methyl-2-oxovalerate and 4-methyl-2oxopentanoate) either stayed constant or decreased over time at the 10 L scale. In contrast, branched chain amino acids were notably elevated in media at the EFT 54 hr at the 10,000 L scale. These results suggested mitochondrial dysfunction or impairment that occurred in parallel with accumulation of glycerol-3-phosphate and intermediates of TCA cycle and mevalonate pathways. Mitochondrial impairment could also reflect cell hypoxia at the 10,000 L scale culture resulting in changes in ergosterol synthesis and glucose degradation. Importantly, associated with the hypoxia phenomenon the marked differences in lanosterol and ergosterol levels found in the 10 L and 10,000 L scale media at the earliest time point (EFT 46 hr) (Figure 6A), may indicate the divergence in their metabolic behavior thus initiating the scale-specific differences unveiled by exometabolomics.

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