, Volume 15, Issue 1–3, pp 177–186 | Cite as

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

  • William T. Hensler
  • Spiros N. Agathos


The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing β-galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG) — was used to monitor β-galactosidase production. Specific β-galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units β-galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.

Key words

Baculovirus infection β-galactosidase insect cell aggregation fluorescent microscopy EXCELL 401 serum-free medium Spodoptera frugiperda (Sf9) insect cells 



ortho-phenyl 2-β-D-galactopyranoside


oxygen uptake rate (μ-mol O2/liter/hour)


specific glucose uptake rate (mg glucose/106cell/hour)


specific glutamine uptake rate (mg glutamine/106cell/hour)


specific oxygen uptake rate (μ-mol O2/106cell/hour)


virus multiplicity of infection (viral plaque forming units/cell)


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Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • William T. Hensler
    • 1
  • Spiros N. Agathos
    • 2
  1. 1.Schering Plough Research InstituteUnionUSA
  2. 2.Unit of BioengineeringCatholic University of LouvainLouvain-la-NeuveBelgium

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