Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells.
The baculovirus infection process of Spodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinant Autographa californica (AcNPV) virus expressing beta-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 beta-galactosidase production. Specific beta-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 beta-galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.
Hensler
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Schering Plough Research Institute, Union, NJ 07083.
Agathos
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D000818:Animals; D016367:Baculoviridae; D001709:Biotechnology / Q000379:methods*; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D046508:Culture Techniques / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D010101:Oxygen Consumption; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis*
D011994:Recombinant Proteins; D005973:Glutamine; D001616:beta-Galactosidase; D005947:Glucose