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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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Mass transfer coefficient and culture apparent viscosity assessment. Batch 1 was scaled-up and run at DO 12.5% at the large (10,000 L) scale bioreactor. Run 5 and 7 were consistency runs at DO 12.5% at the 10 L scale bioreactor. (A) Mass transfer coefficient (kLa) comparison between two scales. (B) Apparent viscosity comparison. The apparent viscosity for the 10,000 L scale Batch 2 and one parallel at 10 L scale run at DO 12.5% was measured at 750 s−1 shear rate as described in “Methods”.
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Figure 3: Mass transfer coefficient and culture apparent viscosity assessment. Batch 1 was scaled-up and run at DO 12.5% at the large (10,000 L) scale bioreactor. Run 5 and 7 were consistency runs at DO 12.5% at the 10 L scale bioreactor. (A) Mass transfer coefficient (kLa) comparison between two scales. (B) Apparent viscosity comparison. The apparent viscosity for the 10,000 L scale Batch 2 and one parallel at 10 L scale run at DO 12.5% was measured at 750 s−1 shear rate as described in “Methods”.

Mentions: To evaluate the causes of higher pure oxygen sparge demand in the 10,000 L scale bioreactor, we compared the mass transfer coefficient (kLa) from both scales. As seen in Figure 3A, kLa initially increased as the agitation speed was ramped up from approximately EFT 10 to 34 hr, but then decreased during the constant maximum agitation speed irrespective of increasing the supply of oxygen-enriched air. The kLa temporal profiles had significant changes between approximately EFT 34 and 54 hr for both scales, but more pronounced at the 10,000 L scale (Figure 3A). After EFT 54 hr, the kLa appeared to level off at the 10 L scale, whereas kLa continued to decrease at lower pace at the 10,000 L scale. By the EOR, kLa dropped to ~2.5 min−1 and 6.5 min -1 at the 10,000 L and 10 L scale, respectively. The net decrease in mass transfer coefficient was significantly higher at 10,000 L than at 10 L.


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)

Mass transfer coefficient and culture apparent viscosity assessment. Batch 1 was scaled-up and run at DO 12.5% at the large (10,000 L) scale bioreactor. Run 5 and 7 were consistency runs at DO 12.5% at the 10 L scale bioreactor. (A) Mass transfer coefficient (kLa) comparison between two scales. (B) Apparent viscosity comparison. The apparent viscosity for the 10,000 L scale Batch 2 and one parallel at 10 L scale run at DO 12.5% was measured at 750 s−1 shear rate as described in “Methods”.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Mass transfer coefficient and culture apparent viscosity assessment. Batch 1 was scaled-up and run at DO 12.5% at the large (10,000 L) scale bioreactor. Run 5 and 7 were consistency runs at DO 12.5% at the 10 L scale bioreactor. (A) Mass transfer coefficient (kLa) comparison between two scales. (B) Apparent viscosity comparison. The apparent viscosity for the 10,000 L scale Batch 2 and one parallel at 10 L scale run at DO 12.5% was measured at 750 s−1 shear rate as described in “Methods”.
Mentions: To evaluate the causes of higher pure oxygen sparge demand in the 10,000 L scale bioreactor, we compared the mass transfer coefficient (kLa) from both scales. As seen in Figure 3A, kLa initially increased as the agitation speed was ramped up from approximately EFT 10 to 34 hr, but then decreased during the constant maximum agitation speed irrespective of increasing the supply of oxygen-enriched air. The kLa temporal profiles had significant changes between approximately EFT 34 and 54 hr for both scales, but more pronounced at the 10,000 L scale (Figure 3A). After EFT 54 hr, the kLa appeared to level off at the 10 L scale, whereas kLa continued to decrease at lower pace at the 10,000 L scale. By the EOR, kLa dropped to ~2.5 min−1 and 6.5 min -1 at the 10,000 L and 10 L scale, respectively. The net decrease in mass transfer coefficient was significantly higher at 10,000 L than at 10 L.

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