Glutamine replenishment and ammonia removal in hybridoma cell cultures via immobilized glutamine synthetase

Carl D. Okeson, Mark Riley

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Batch in vitro hybridoma cell culture efficiency frequently is hindered by the depletion of the nutrient glutamine and the accumulation of toxic ammonia within the culture medium. This paper presents a novel approach for ammonia removal and glutamine replenishment in hybridoma cell culture through use of ex vivo immobilized glutamine synthetase (GS). Experiments involving soluble GS revealed that the ammonia conversion was sensitive to the concentration of reaction substrates. Glutamate and Mg2+ concentrations had the largest positive effects on initial ammonia conversion rate, whereas glutamate and ATP had the largest positive effects on total ammonia conversion. Ca2+ was found to be highly inhibitory to GS activity; 1.8 mM Ca2+ (as present in a standard culture medium) reduced GS conversion activity by 66%. Of three enzyme immobilization methods tested, covalent binding to Sepharose yielded the largest increase in GS thermostability at 37°C, but encapsulation in dialysis tubing provided the best overall conversion activity (100% of soluble GS activity). A hybridoma culture system incorporating encapsulated GS maintained the culture medium ammonia concentration below 4 mM and substantially prolonged hybridoma metabolic activity. Overall, immobilized GS shows promise as a means of extending the "useful life" of hybridoma cell cultures without the product diluting effect of medium perfusion.

Original languageEnglish (US)
Pages (from-to)125-133
Number of pages9
JournalBiochemical Engineering Journal
Volume9
Issue number2
DOIs
StatePublished - Nov 28 2001

Fingerprint

Glutamate-Ammonia Ligase
Hybridomas
Glutamine
Cell culture
Ammonia
Cell Culture Techniques
Culture Media
Enzyme immobilization
Glutamic Acid
Dialysis
Adenosinetriphosphate
Tubing
Encapsulation
Nutrients
Poisons
Immobilization
Sepharose
Perfusion
Adenosine Triphosphate
Substrates

Keywords

  • Ammonia removal
  • Bioreactors
  • Enzyme activity
  • Glutamine synthetase
  • Hybridoma cultures
  • Immobilized enzymes

ASJC Scopus subject areas

  • Biotechnology
  • Environmental Engineering
  • Bioengineering
  • Biomedical Engineering

Cite this

Glutamine replenishment and ammonia removal in hybridoma cell cultures via immobilized glutamine synthetase. / Okeson, Carl D.; Riley, Mark.

In: Biochemical Engineering Journal, Vol. 9, No. 2, 28.11.2001, p. 125-133.

Research output: Contribution to journalArticle

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