Development and optimization of an in vitro process for the production of Oryctes nudivirus in insect cell cultures
Thesis DisciplineChemical Engineering
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The coconut rhinoceros beetle, an economically important pest of coconut and oil palms, is effectively managed by application of its natural pathogen, the Oryctes nudivirus (OrNV), which act as a bioinsecticide. While this approach offers an environment-friendly alternative to chemical pesticides, the current method of production in infected larvae suffers from inconsistencies in virus productivity and purity. While the anchorage-dependent DSIR-HA-1179 insect cell line has been identified as a susceptible and permissive host for OrNV and therefore would be suitable for the in vitro mass production of the virus, no attempts have been made toward the mass production of the virus, because of the technological challenges that working with DSIR-HA-1179 cells represent. Thus, the main objective of this research was to develop processes for the in vitro production of OrNV in the DSIR-HA-1179 cell line.
Knowledge of the growth kinetics and metabolic properties of the host cell line in a chosen culture medium, as well as the selection of an appropriate infection strategy, form the basis for the rational development of bioreactor-based virus production processes. However, characterization of these properties in the DSIR-HA-1179 cell line has been virtually precluded, due to its strongly adherent growth characteristics and the lack of a reliable method to accurately dissociate and count cells grown in monolayers. Using TrypLE™ Express enzyme, a technique allowing the precise counting of cells was developed.
The cell line was adapted to grow in four serum-supplemented culture media: TC-100, IPL-41, Sf-900 II and Sf-900 III, which were then individually screened for cell growth and virus production in 25 cm2 attached T-flask cultures. TC-100 supplemented with 10% fetal bovine serum was chosen as a suitable culture medium, based on its capacity for achieving a high cell yield and OrNV production. The cell line metabolism was characterized with respect to nutrient consumption and metabolites production in this culture medium. Glucose, along with glutamine were found to be the nutrients that were consumed faster and to a greater extent, while other amino acids were not consumed to a significant degree. The production of metabolites was characterized by non-production of lactate and ammonia, and production of alanine, as a non-toxic alternative to ammonia.
The influence of cell density (CD) at time of infection (TOI) and multiplicity of infection (MOI) on OrNV production was evaluated in T-flask cultures that were infected at different CDs at the TOI and a range of MOIs. The CD at TOI was found to significantly influence OrNV yields, while MOI influenced the dynamics of infection. The cell density effect was found to exist for the DSIR-HA-1179/OrNV system with the progressive decline in cell-specific yield beginning at low cell densities. It was found that in order to maximize OrNV volumetric yield, a combination of MOI and CD at TOI should be selected that allows to keep the maximum cell density reached by the infected culture within a range between 5.0 and 7.0 x 105 viable cells/ml.
The roller bottle system was evaluated for its potential to scale-up DSIR-HA-1179 cell growth and OrNV production, and culture parameters were optimized for the improvement of cell and virus yields. An inoculum density of 3.3 x 105 cells/ml and culture volume of 60 ml resulted in the highest cell yield of 1.5 x 106 cells/ml, in 490 cm2 roller bottles. It was found that an optimal infection strategy for roller bottle cultures, which represented the most efficient use of viral inoculum, involved infecting cells at a density of 5.0 x 105 cells/ml and at a MOI of 1. The resulting OrNV volumetric yield of 2.5 x108 TCID50/ml, improved significantly the viral yields obtained in attached T-flask cultures infected under similar conditions (6.8 x 107 TCID50/ml).
The microcarrier system was also evaluated for culturing DSIR-HA-1179 cells and producing OrNV in spinner flask bioreactors. Three types of microcarriers (Cytodex-1, Cytodex-3 and Cultispher-G microcarriers) were screened for their ability to support DSIR-HA-1179 growth. Cells attached to Cytodex-1 and 3, but failed to attach to Cultispher-G microcarriers. The final cell density reached in microcarrier culture was dependent on bead type and concentration, and the cell to bead ratio. At an optimal bead concentration of 1 mg/ml and cell to bead ratio of 30, cells grew to a maximum density of 1.7 x 106 cells/ml on Cytodex-1, but only to 1.3 x 106 cells/ml on Cytodex-3 microcarriers. Since it supported higher cell yields, Cytodex-1 was chosen to study the kinetics of OrNV production in this system. Microcarrier cultures infected at a cell density of 5.0 x 105 cells/ml and a MOI of 1, produced OrNV at 1.4 x 108 TCID50/ml, which was higher than the yield obtained in T-flask cultures infected under similar conditions.
A framework of knowledge on the physiology, metabolism and growth kinetics of the DSIR-HA-1179 insect cell line has been developed in this thesis. In addition, the feasibility of using roller bottles and microcarrier systems for the in vitro production of the virus has been ascertained. It is envisaged that these findings will contribute to the future development of a large-scale industrial process for the production of the OrNV biopesticide.