Cytotechnology

, Volume 22, Issue 1, pp 239–250

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Authors

  • Weichang Zhou
    • Merck Research Laboratories
  • Theodora Bibila
    • Merck Research Laboratories
  • Konstantin Glazomitsky
    • Merck Research Laboratories
  • Javier Montalyo
    • Merck Research Laboratories
  • Christine Chan
    • Merck Research Laboratories
  • Daniel DiStefano
    • Merck Research Laboratories
  • Sonal Munshi
    • Merck Research Laboratories
  • David Robinson
    • Merck Research Laboratories
  • Barry Buckland
    • Merck Research Laboratories
  • John Aunins
    • Merck Research Laboratories
Special Issue

DOI: 10.1007/BF00353944

Cite this article as:
Zhou, W., Bibila, T., Glazomitsky, K. et al. Cytotechnology (1996) 22: 239. doi:10.1007/BF00353944

Abstract

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×109 cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.

Key words

GS-NSO cellsfed-batch culturesscale upcellular metabolismlactate consumptionrecombinant mouse growth hormonerecombinant rat growth hormone

Copyright information

© Kluwer Academic Publishers 1996