Aedes densonucleosis virus analysis and examinations in bioprocessing

Aedes densonucleosis virus (AeDNV) is a mosquito specific parvovirus that infects the Aedes aegypti mosquito. There is potential to develop AeDNV as a vector control agent. This work presents several different topics centered on AeDNV. In particular, the ultrafiltration of AeDNV, expression of AeDNV structural protein in Pichia pastoris and identification of virus binding proteins were explored. The final portion of this work presents the investigation of performance differences between an anion exchange resin and membrane adsorbers.; Purification of AeDNV using tangential flow ultrafiltration was examined. The performance of ultrafiltration membranes, with molecular weight cut-offs (MWCO) of 300, 100, 50, and 30 kDa, was investigated. The virus was grown using two cell culture medias, with and without serum. The ideal ultrafiltration membrane will reject the AeDNV while allowing the host cell proteins (HCP) from the mosquito tissue culture to pass through its pores. The 300 kDa membrane does not reject all AeDNV, leading to a decrease in membrane flux and loss of virus particles. The 50 and 30 kDa MWCO membranes reject virus but also reject HCP. The 100 kDa MWCO membrane rejects virus and allows the HCP from the serum free cell culture to pass through the pores. The ultrafiltration concentrates the virus 10-fold.; To examine the role of structural proteins in virus assembly, the AeDNV major capsid protein was expressed in the methylotrophic yeast Pichia pastoris. High cell density fermentation was carried out in a three-liter bioreactor. The protein spontaneously assembles into virus-like particles (VLPs) as determined by electron microscopy. The VLPs are antigenically similar to AeDNV native capsids as determined by enzyme-linked immunosorbant assay. AeDNV establishes a persistent infection in C6/36 Aedes albopictus mosquito cells, with only a small percentage (1-2%) of cells producing viral antigen. When VLPs are incubated with C6/36 cells, only a similar small percentage of cells bind antigen. Thus, it seems that only a small percentage of C6/36 cells have exposed cellular receptors located on their surface. To identify the molecules responsible for binding AeDNV, a virus overlay protein blot assay and a pull-down assay were performed with biotinylated densovirus-like particles. Three cellular proteins, with approximate molecular weights of 220, 110 and 67 kDa, that specifically bind AeDNV were identified in both assays as putative virus receptors. Binding of the AeDNVLPs was blocked when blots were first incubated with AeDNV with a virus to cell ratio of 10,000.; The performance differences between anion exchange resin and membrane adsorbers for trace impurity removal in biomanufacturing processes were investigated. The ability of anion exchange membranes and resins to remove HCP from monoclonal antibody feedstocks was evaluated and compared based on several factors: load density, HCP level in the feedstock, and residence time. Strength of binding was determined to be an indicator of HCP removal performance.