Metabolic analysis of the asparagine and glutamine dynamics in an industrial Chinese hamster ovary fed‐batch process

Abstract

Chinese hamster ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13 C-glucose, 13 C-glutamine, and 13 C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition.Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-acetyl-glucosamine and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13 C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.

Metabolic Profiling of CHO Cells during the Production of Biotherapeutics

“…In low glutamine-containing medium, glycolytic fluxes were significantly increased while PPP was decreased 2-fold when compared to cultures growing in high glutamine medium. Pyruvate usage by the TCA cycle instead of lactate production was more important in low glutamine medium cultures, which was also observed by Kirsch et al [ 25 ]. An increase in TCA flux in such culture conditions was also reported in other studies, including in CHO-DHFR cells [ 50 ].…”

“…Similarly, another study concluded that glucose represents 65% of the entering carbon, with 10% coming from glutamine and 25% from other amino acids [ 23 ]. A lower contribution was also reported (40–50%) [ 24 , 25 ], although this can partly be explained by the fact that glucose consumption can vary simply due to different culture conditions, specifically, in the present case, culture volumes [ 26 ]. Besides these subtle differences, publications overall agree on the fact that glucose constitutes the main carbon source for growth.…”

“…The consumption and production of amino acids has been studied in CHO cells during the exponential growth phase ( Table 1 ), and amino acid-relevant metabolic fluxes were described to be significantly lower than glycolytic and TCA cycle fluxes [ 32 ]. MFA studies reported consistent percentages in terms of the contributions of amino acid to TCA cycle replenishment, with glutamine being the main carbon source, and it is known that its availability in the culture media, together with asparagine or serine, has a high impact on the metabolism of CHO cells [ 25 , 50 , 51 ]. Glutamine was found to contribute to about 40% of the total carbon supply of the TCA cycle.…”

Combined gene and environmental engineering offers a synergetic strategy to enhance r‐protein production in Chinese hamster ovary cells

“…Previous studies have associated glutamine depletion in cultures with the shift in lactate metabolism (Kirsch et al, 2021;Torres et al, 2018). Our data suggest that the decreased consumption of glutamine due to the interventions (observed in both cell lines) contributed to the delayed transition to lactate consumption in the CHO EPO-Fc cells.…”

Senescent cells develop PDK4-dependent hypercatabolism and form an acidic microenvironment to drive cancer resistance

“…Glucose is the primary carbon source to the tricarboxylic acid (TCA) cycle, followed by glutamate and aspartate (non-protonatable amino acids as glutamine or asparagine, respectively) as secondary sources (Fig. 3a) 30 . We first interrogated the metabolic pattern of glucose upon uptake by senescent cells, as glucose is supposed to act as a principal contributor to the TCA cycle when cells enter senescence, a stage that is considered metabolically active 31 .…”