Metabolic responses of CHO cells to limitation of key amino acids

Abstract

Chinese hamster ovary (CHO) cells are the predominant host for production of therapeutic glycoproteins. In particular, the glutamine-synthetase (GS) expression system has been widely used in the biopharmaceutical industry for efficient selection of high-yielding clones. However, much remains unclear on how metabolic wiring affects culture performance. For instance, asparagine and serine have been observed to be the largest nitrogen sources taken up by GS-CHO cells, but their roles in biosynthesis and energy generation are poorly understood. In this work, a comprehensive profiling of extracellular metabolites coupled with an analysis of intracellular label distributions after 1-(13) C-pyruvate supplementation were used to trace metabolic rearrangements in different scenarios of asparagine and serine availability. The absence of asparagine in the medium caused growth arrest, and was associated with a dramatic increase in pyruvate uptake, a higher ratio of pyruvate carboxylation to dehydrogenation and an inability for de novo asparagine synthesis. The release of ammonia and amino acids such as aspartate, glutamate, and alanine were deeply impacted. This confirms asparagine to be essential for these GS-CHO cells as the main source of intracellular nitrogen as well as having an important anaplerotic role in TCA cycle activity. In turn, serine unavailability also negatively affected culture growth while triggering its de novo synthesis, confirmed by label incorporation coming from pyruvate, and reduced glycine and formate secretion congruent with its role as a precursor in the metabolism of one-carbon units. Overall, these results unfold important insights into GS-CHO cells metabolism that lay a clearer basis for fine-tuning bioprocess optimization.

A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

“…The CHO-specific auxotrophies include cysteine, proline, and at least one report of an asparagine auxotrophy (Duarte et al, 2014). While cysteine, proline, asparagine, and arginine are essential based on experimental evidence, homologs for all genes required for their biosynthesis are in the C. griseus genome.…”

Responses of CHO-DHFR cells to ratio of asparagine to glutamine in feed media: cell growth, antibody production, metabolic waste, glutamate, and energy metabolism

“…The respiratory quotient of cells was constant in metabolic flux analysis, and the value was one (Sengupta et al 2011;Ferreira et al 2005;Duarte et al 2014;Ahn and Antoniewicz 2012). So, the specific rate of CO 2 production was equal to that of O 2 consumption.…”

“…2a, b). Both glutamine and asparagine were firstly metabolized by deamination (Additional file 1: Table S1) which produced ammonia and then aspartate tended to occur transamination reaction (Huang et al 2007;Duarte et al 2014;Ahn and Antoniewicz 2012;Ozturk et al 1992;Ahn and Antoniewicz 2012;Rajendra et al 2012). Amino acid, such as aspartate, could use α-ketoglutarate to initiate transamination and generate glutamate simultaneously.…”

Scale-down of CHO cell fed-batch cultures

“…The formic acid concentration in this experiment reached 4 mM on day 5 (Figure 2b), which concentration was shown to inhibit CHO cell growth 97 . Formic acid is considered a breakdown product of serine 71,98 . In the present study, formic acid is mainly produced by the breakdown of serine through folate metabolism (ESM_1 of online published data), which is also reported by Carinhas et al 71 and Duarte et al 98 and a small fraction is generated in the breakdown of tryptophan and during synthesis of cholesterol (ESM_1 of online published data).…”

“…Formic acid is considered a breakdown product of serine 71,98 . In the present study, formic acid is mainly produced by the breakdown of serine through folate metabolism (ESM_1 of online published data), which is also reported by Carinhas et al 71 and Duarte et al 98 and a small fraction is generated in the breakdown of tryptophan and during synthesis of cholesterol (ESM_1 of online published data). The high breakdown rates for some amino acids are likely caused by the overfeeding of these nutrients after the NI phase as can be seen in Figure 2.…”