Chávez-Peña C, Ayala-Mendivil NA, Ramírez OT, Palomares LA

Language: Spanish
References: 18
Page: 65-72
PDF size: 348.73 Kb.

ABSTRACT

The insect cell-baculovirus expression system is versatile and useful for the production of recombinant proteins. It consists in infecting insect cell cultures with a recombinant baculovirus that contains the gene of interest. An important limitation of the system is the limited availability of baculoviral stocks with high titers of infectious particles (plaque forming units, pfu). In this work, the effect of multiplicity of infection (MOI) and cell concentration at the time of infection (CCI) on the yield of baculovirus infectious particles was investigated. Combinations of two values for MOI (0.1 or 1 pfu/cell) and CCI (1 or 2 x10⁶ cel/mL) were tested. Kinetics of cell growth, total protein and gp64 concentrations in the culture supernatant, and the baculovirus titer at the time of harvest were determined. The highest viral titers, 3.8 ± 2 x10⁸ pfu/mL, were obtained in cultures infected at a MOI = 0.1 pfu/cell and a CCI = 1x10⁶ cell/mL. Such cultures had the lowest cell growth after infection. The results obtained in this work can be used for the efficient production of baculovirus stocks with high viral titers.

REFERENCES

1. Urabe, M., Ding, C. & Kotin, R.M. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy 13, 1935-1943 (2002).

2. Kost, T.A. & Condreay, J.P. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends in Biotechnology 20,173-180 (2002).

3. Palomares, L.A., Estrada-Mondaca, S. & Ramírez, O.T. “Principles and Applications of the Insect-Cell-Baculovirus Expression Vector System” en Cell Culture Technology for Pharmaceutical and Cellular Applications (ed. Ozturk, S. & W.S. Hu) 627-692 (Taylor and Francis, Nueva York, 2006).

4. Carinhas, N. et al. Baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange. Applied Microbiology and Biotechnology81, 1041-1049 (2009).

5. Radford, M.K. et al. The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non secreted products. Cytotechnology 24, 73-81 (1997).

6. Hu, Y.C. & Bentley, W.E. Effect of MOI ratio on the composition and yield of chimeric infectious bursal disease virus likeparticles by baculovirus co-infection: deterministic predictions and experimental results. Biotechnology and Bioengineering 75, 104-119 (2001).

7. Maranga, L., Cruz, P.E., Aunins, J.G. & Carrondo, M.J. Production of core and virus like particles with baculovirus infected cells. Advances in Biochemical Engineering and Biotechnology 74,183-206 (2002).

8. Mena, J.A., Ramírez, O.T. & Palomares, L.A. Population kinetics during simultaneous infection of insect cells with two recombinant baculoviruses for the production of virus-like particles. BMC Biotechnology 7, 39 (2007).

9. O’Reilly, D.R., Miller, L.K. & Luckow, V.A. Baculovirus expression vectors: a laboratory manual (Oxford University Press, New York, 1994).

10. Palomares, L.A., López, S. & Ramírez, O.T. Utilization of oxygen uptake rate to assess the role of glucose and glutamine in the metabolism of insect cell cultures. Biochemical Engineering Journal 19, 87-93 (2004).

11. Elias, C.B., Zeiser, A., Bédard, C. & Kamen, A.A. Enhanced growth of Sf-9 cells to a maximum density of 5.2 x107 cells per mL and production of b-galactosidase at high cell density by fed batch culture. Biotechnology and Bioengineering 68, 381-388 (2000).

12. Ross, O.H., MsCabe, D.D., Hollis, G.F. & Rosenfeld, S.A. Methods for baculovirus amplification- Application to large scale recombinant protein production in insect cells. Biotechnology Techniques 12, 463-465 (1998).

13. Tsai, C.T. et al. Factors influencing the production and storage of baculovirus for gene delivery: An alternative perspective from the transducing titer assay. Enzyme and Microbial Technology 40,1345-1351 (2007).

14. Mena, J.A., Ramírez, O.T. & Palomares, L.A. Titration of nonoccluded baculovirus using a cell viability assay. Biotechniques 34, 260-263 (2003).

15. Oomens, A.G.P., Monsma, S.A. & Blissard, G.W. The baculovirus gp64 envelope fusion protein: Synthesis, oligomerization and processing. Virology 209, 592-603 (1995).

16. Roldao, A., Oliveira, R., Carrondo, M.J.T. & Alves, P.M. Error assessment in recombinant baculovirus titration: Evaluation of different methods. Journal of Virological Methods 159, 69-80 (2009).

17. Wickham, T.J. et al. Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection. Biotechnology Letters 13(7), 483-488 (1991).

18. Palomares, L.A., González, M. & Ramírez, O.T. Evidence of Pluronic F-68 direct interaction with insect cells: Impact on shear protection, recombinant protein and baculovirus production. Enzyme and Microbial Technology 26, 324-331 (2000).