Advertisement

Cytotechnology

, 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
Article

Abstract

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 

Abbreviations

ONPG

ortho-phenyl 2-β-D-galactopyranoside

OUR

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

qglucose

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

qglutamine

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

qO2

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

MOI

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

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Altman SA, Randers L, Rao G (1993) Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol. Prog. 9: 671–674.Google Scholar
  2. Betenbaugh M, Balog L, Lee PS (1991) Production of recombinant proteins by baculovirus-infected gypsy moth cells. Biotechnol. Prog. 7: 462–467.Google Scholar
  3. Caron AW, Archambault J, Massie B (1990) High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system. Biotechnol. Bioeng. 36: 1133–1140.Google Scholar
  4. Hensler W, Singh V, Agathos SN (1994) Production of β-galactosidase in Sf9 insect cells in serum-containing and serum-free medium. Annals of N.Y.A.S. (in press).Google Scholar
  5. Jain D, Ramasumbramanyan K, Gould S, Lenny A, Candelore M, Tota M, Strader C, Cuca G, Tung JS, Hunt G, Junker B, Buckland BC, Silberklang M (1991) Large-scale recombinant protein production using the insect cell-baculovirus expression vector system: antistasin and β-adrenergic receptor. In: Spier RE, Griffiths JB and Meignier B (eds.) Production of Biologicals from Animal Cells in Culture (pp 345–350). Butterworth-Heinemann, Oxford.Google Scholar
  6. Kamen AA, Tom RL, Caron AW, Chavarie C, Massie B, Archambault J (1991) Culture of insect cells in a helical ribbon impeller bioreactor. Biotechnol. Bioeng. 38: 619–628.Google Scholar
  7. King GA, Daugulis AJ, Faulkner P, Goosen MFA (1992) Recombinant β-galactosidase production in serum-free medium by insect cells in a 14-L airlift bioreactor. Biotechnol. Prog. 8: 567–571.Google Scholar
  8. Licari P, Bailey JE (1992) Modelling the population dynamics of baculovirus-infected cells: optimizing infection strategies for enhanced recombinant protein yields. Biotechnol. Bioeng. 39: 432–441.Google Scholar
  9. Lin AA, Kimura R, Miller WM (1993) Production of tPA in recombinant CHO cells under oxygen-limited conditions. Biotechnol. Bioeng. 42: 339–3350.Google Scholar
  10. Lindsay DA, Betenbaugh MJ (1992) Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells. Biotechnol. Bioeng. 39: 614–618.Google Scholar
  11. Miller JH (1972) Assay of β-galactosidase. In: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press. pp. 352–355. Cold Spring Harbor, New York.Google Scholar
  12. Molecular Probes, Inc. (1992) ImaGene Green C2−C16 FDGlacZ Gene Expression Kit I-2896. Eugene Oregon, Bulletin 2896.Google Scholar
  13. Neutra R, Levi BZ, Shoham Y (1992) Optimization of protein production by the baculovirus expression vector system in shake flasks. Appl. Microbiol. Biotechnol. 37: 74–78.Google Scholar
  14. Ogonah O, Shuler M, Granados RR (1991) Protein production (β-galactosidase) from a baculovirus vector inSpodoptera frugiperda andTrichoplusia ni cells in suspension culture. Biotech. Lett. 13:265–270.Google Scholar
  15. Power J, Greenfield PF, Nielsen L, Reed S (1992) Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture. Cytotechnology 9:149–155.Google Scholar
  16. Reuveny S, Kim YJ, Kemp CW, Shiloach J (1993) Production of recombinant proteins in high-density insect cell cultures. Biotechnol. Bioeng. 42:235–239.Google Scholar
  17. Schopf B, Howaldt MW, Eailey JE (1990) DNA distribution and respiration activity ofSpodoptera frugiperda populations infected with wild-type and recombinantAutographa californica nuclear polyhedrosis virus. J. Biotechnology 15: 169–186.Google Scholar
  18. Scott RI, Blanchard JH, Ferguson C (1992) Effects of oxygen on recombinant protein production by suspension cultures ofSpodoptera frugiperda (Sf9) insect cells. Enzyme Microb. Technol. 14: 798–804.Google Scholar
  19. Singh V, Gunnarson A (1988) On-line method for measuring oxygen uptake rate. Annual ACS Meeting. Presentation 110. November Miami, FL.Google Scholar
  20. Streett DA, Hink WF (1978) Oxygen consumption ofTrichoplusia ni (TN-368) insect cell line infected withAutographa californica nuclear polyhedrosis virus. J. Invert. Pathol. 32: 112–113.Google Scholar
  21. Summers MD, Smith GE (1987) A manual of methods for baculovirus vectors and insect cell culture procedures. Bulletin No. 1555. Texas Agricultural Experimental Station.Google Scholar
  22. Wang MY, Kwong S, Bentley WE (1993a) Effects of oxygen/glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydrolase production. Biotechnol. Prog. 9: 355–361.Google Scholar
  23. Wang MY, Vakharia V, Bentley WE (1993b) Expression of epoxide hydrolase in insect cells; a focus on the infected cell. Biotechnol. Bioeng. 42: 240–246.Google Scholar
  24. Wu SC, Dale BE, Liao JC (1993) Kinetic characterization of baculovirus-induced cell death in insect cell cultures. Biotechnol. Bioeng. 41: 104–110.Google Scholar
  25. Zhang ZY, Naleway JJ, Larison KD, Huang Z, Haughland R (1991) DetectinglacZ gene expression in living cells with new lipophilic, fluorogenic β-galactosidase substrates. FASEB 5: 3108–3112.Google Scholar

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

Personalised recommendations