Full text
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| Author(s): |
Karina Klafke
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Escola Politécnica (EP/BC) |
| Defense date: | 2020-06-09 |
| Examining board members: |
Aldo Tonso;
Soraia Attie Calil Jorge;
Ângela Maria Moraes
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| Advisor: | Aldo Tonso; Marlinda Lobo de Souza |
| Abstract | |
Brazil is one of the world biggest producers and exporters of corn crop, hence cultivation damage can bring a substantial prejudice. Spodoptera frugiperda is America\'s most common corn pest and baculoviruses are natural pathogens of this pest and can act as a biopesticide. Currently, in vivo strategies for commercial scale are employed by multiplying the virus in the host insect in biofactory facilities, however, in vitro large-scale production alternative can bring economic advantages. This study pursued to develop the process of in vivo production of SfMNPV through the study of different multiplicities of infection (MOI) and nutritional supplements, morphological and molecular analysis of the infection and tests of the effect of decreasing shear stress on the growth and production of virus. This project was developed through a partnership between the Polytechnic School of University of São Paulo (GenBio-USP) with EMBRAPA Genetic Resources and Biotechnology (Cenargen) and the United States Department of Agriculture (NRS-FS/USDA). The determinations were accomplished in Sf-9 insect cells in Sf-900 III and ExCell 420 culture media and performed in flasks and STR with aspersion, STR bubble free and Wave bioreactor models. At first, a series of supplementations were tested in the ExCell 420 culture medium and it was defined that the best condition among those evaluated was the combination of the addition of 2 g/L of glucose and 200 mg/L of cysteic acid, which increased cell density by 37.5%, resulting in a maximum cell concentration of 8.8x106 cell/mL in shaker flasks. As for the Sf-900 III culture medium, 1 g/L of glutamine was added. Batches in sparged STR bioreactor had an average maximum specific growth rate (µmax) of 0.60 day-1. Tests in STR bubble free showed a significant impact on the maximum cell concentration (Xvmax) value, reaching values of 10x106 cell/mL, representing a 42.8% increase compared to the sparged STR approach, which was accompanied by a drop in µmax to 0.30 day-1. Likewise, cultures in the Wave reactor with ExCell 420 medium showed, on average, a 42.5% increase in Xvmax compared to the equivalent in STR and µmax around 0.45 day-1. These data corroborate the effect of shear as an important source of cellular stress. The metabolic analysis of the batches with Sf-900 III culture medium met with data from the literature, confirming the metabolic efficiency via TCA cycle in Sf-9 cells, however, due to slightly increased values of ammonium concentration, it is assumed that a phenomenon reported as density effect, in which there is loss of metabolic efficiency due to inhibition by high cell concentration, may be occurring. In addition, an increase in glucose consumption was also observed in a post-infection manner. Finally, the optimization of the cell growth stage led to a significant improvement in the viral production stage. In bioreactor tests, a MOI of 1.0 was confirmed as an optimized parameter without viral genetic alterations. Batches in sparged STR bioreactor with ExCell 420 medium yielded 6x1010 pol/L, compared to its Wave equivalent of 43x1010 pol/L, which had the best reproducibility in the study. However, the sparged STR approach with Sf-900 III culture medium combined with the MOI of 1.0 and glutamine supplementation showed the best viral production performance with specific productivity above 300 pol/cell and volumetric productivity of 9x1011 pol/L. (AU) | |
| FAPESP's process: | 17/06371-1 - Development of production process of baculovirus biopesticide by Sf9 insect cells in bioreactor |
| Grantee: | Karina Klafke |
| Support Opportunities: | Scholarships in Brazil - Master |