Metabolic responses of CHO cells to limitation of key amino acids

Tiago M. Duarte, Nuno Carinhas, Laura C. Barreiro, Manuel J T Carrondo, Paula M. Alves, Ana P. Teixeira

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 1-13C-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.

Original languageEnglish
Pages (from-to)2095-2106
Number of pages12
JournalBiotechnology and Bioengineering
Volume111
Issue number10
DOIs
Publication statusPublished - Oct 2014

Fingerprint

Asparagine
Cricetulus
Glutamate-Ammonia Ligase
Metabolism
Amino acids
Labels
Ovary
Pyruvic Acid
Carboxylation
Nitrogen
Amino Acids
Glycoproteins
Biosynthesis
Serine
Electric wiring
Dehydrogenation
Metabolites
formic acid
Ammonia
Tuning

Keywords

  • Asparagine modulation
  • GC-MS analysis
  • GS-CHO cells
  • Serine modulation

Cite this

Duarte, Tiago M. ; Carinhas, Nuno ; Barreiro, Laura C. ; Carrondo, Manuel J T ; Alves, Paula M. ; Teixeira, Ana P. / Metabolic responses of CHO cells to limitation of key amino acids. In: Biotechnology and Bioengineering. 2014 ; Vol. 111, No. 10. pp. 2095-2106.
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Metabolic responses of CHO cells to limitation of key amino acids. / Duarte, Tiago M.; Carinhas, Nuno; Barreiro, Laura C.; Carrondo, Manuel J T; Alves, Paula M.; Teixeira, Ana P.

In: Biotechnology and Bioengineering, Vol. 111, No. 10, 10.2014, p. 2095-2106.

Research output: Contribution to journalArticle

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AU - Carinhas, Nuno

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