Two-stage baculovirus production in insect-cell bioreactors

Baculoviruses are insect-pathogenic viruses with a narrow host range. The viruses can be an alternative to chemical insecticides. From research aimed at improving the efficacy of the viruses in insect control another application evolved: the use of the baculovirus to express foreign proteins in insect cells. To produce large amounts of baculovirus or baculovirus- expressed proteins, large-scale culture and subsequent infection of insect cells is necessary. Chapter 1 of this thesis reviews the research on factors influencing insect-cell culture and infection and the status of production with insect-cell cultures.One of the production strategies reviewed in chapter 1 is a continuous two-stage bioreactor configuration. In the first reactor cells are cultured and the effluent is led to second reactor where the cells are infected with the baculovirus. The first results reported with this system showed two disadvantages. The number of cells infected was relatively small and production in the infection reactor was limited to about one month.To increase the number of infected cells the residence time distribution in the infection reactor was altered (chapter 2). The infection reactor was replaced by two infection reactors in series, each containing half the volume of the original infection reactor. Therefore, the mixing characteristics of the infection part of the production system became more plug-flow like. This resulted in a higher number of cells which produced polyhedra (encapsulated virus). However, the time during which the cells in the infection reactors produced polyhedra was diminished to about two weeks.To gain more insight in the decrease of production a recombinant virus was used in a study described in chapter 3. In this recombinant virus the polyhedrin gene (coding for the major protein in the virus matrix) was replaced by the lacZ gene of Escherichia coli. Production of β- galactosidase in a continuous two-reactor configuration gave analogous results to production with the wild-type virus. Production was maintained for about four weeks then it rapidly decreased. Upon restriction enzyme analysis it was shown that the decrease of β-galactosidase resulted from the disappearance of the gene from the virus population rather than inactivation of the gene.Research at the department of Virology of the Agricultural University of Wageningen revealed that the reduction of production resulted from the occurrence of defective mutants of the baculovirus. These defective virus lack the polyhedrin-promoter driven gene and became predominant in the bioreactor due to interference with the replication of non-mutated virus.On the defective viruses another highly expressed gene, the p10 gene, was found to be still present. A recombinant virus containing the lacZ from E. coli under control of the p10 promoter was used in the study described in chapter 4. The recombinant virus produced both polyhedra and β-galactosidase in a continuous bioreactor system. Again production lasted for about four weeks both for polyhedra and β-galactosidase This indicates that the presence of the p10-promoter driven gene per se is not enough for recombinant protein production.In chapter 5 a model is presented which describes the kinetics of virus infection. Besides the infectious virus particles and the defective mutants, a third virus type is taken into account. This so-called abortive virus is capable of blocking an entry site of a cell but is not leading to production of new virus particles. By assuming a limited amount of entry sites on an insect cell it was possible to calculate when production of infectious virus decreased. The model indicates that the build-up of defective viruses can be postponed by keeping the number of infectious viruses per cell low. A way of accomplishing this is the use of repeated batch infections.In chapter 6 experiments with repeated (fed-)batch infections are presented. These experiments confirmed calculations with the model. A production involving a series of batch infections resulted in prolongation of production time to 60 days.The application of the baculovirus expression vector in the light of the findings described in the thesis is discussed in chapter 7.