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Cytotechnology

, Volume 15, Issue 1–3, pp 129–138 | Cite as

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

  • C. Bédard
  • A. Kamen
  • R. Tom
  • B. Massie
Article

Abstract

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing β-galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific β-galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (≤3×106 cells mL−1). In cultures infected at later growth stages, β-galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific β-galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.

Key Words

Baculovirus expression vector system insect cell culture multiplicity of infection nutrients metabolic byproducts culture medium 

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Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • C. Bédard
    • 1
  • A. Kamen
    • 1
  • R. Tom
    • 1
  • B. Massie
    • 1
  1. 1.Animal Cell Engineering Group, Biotechnology Research InstituteNational Research Council CanadaMontréalCanada

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