Resumen de Bioprocess engineering and characterization of hiv virus-like particle production in insect cells
Virus-like particles (VLPs) have emerged as an interesting alternative to conventional vaccines based on live-attenuated or inactivated viruses. Their capacity for self-assembling upon expression of the core protein and the lack of viral genomic material make them excellent candidates for a variety of purposes. Gag VLPs from the human immunodeficiency virus (HIV) are a type of enveloped VLPs that have drawn special attention due to their structural properties with applications in gene therapy, nanobiotechnology and multivalent vaccine development. Insect cell lines are a reference system to produce these types of nanoparticles since they provide the ideal conditions for their production and assembly. In this work, the production of HIV-1 GageGFP VLPs is assessed in Sf9 and High Five insect cells with the baculovirus expression vector system (BEVS) and transient gene expression (TGE). A rational approach based on the combination of Design of Experiments (DoE) and desirability functions is used to optimize the VLP production conditions. Advanced measurement techniques are implemented to monitor and quantify the production process and for final VLP characterization.
In the first chapter, the characteristics of both insect cell lines as platforms for GageGFP VLP production with the BEVS are analyzed. In both cases, similar VLP sizes for both cells are measured by cryogenic electron microscopy (cryo-EM) and other nanoparticle populations are identified. The analysis of baculovirus production levels results in a 23-fold increase of budded virus in Sf9 cells while a larger amount of occlusion-derived virus is detected in High Five cells. The presence of this baculovirus phenotype evidences a shift in the cellular complexity of High Five cells upon baculovirus infection. Finally, the combination of analytical ultracentrifugation with flow virometry reveals a higher sedimentation coefficient for High Five-derived VLPs, indicating their possible association with other cellular compounds.
In the second chapter, the optimal conditions for VLP production in Sf9 and High Five cells with the BEVS are determined by means of DoE and desirability functions. Different methodologies based on direct nanoparticle quantification are used to gain insight into these systems. Two objective situations are defined, one targeting the maximization of the VLP titer (Quantity) and the second one aiming to find a balance between production and assembled VLPs (Quality). Final VLP production levels in the quality condition are 4.5-fold higher for Sf9 cells while similar VLP concentrations are found for both insect cells in the quantity condition.
In the third chapter of this thesis, a baculovirus-free VLP production strategy is optimized for both insect cells based on plasmid-mediated TGE with polyethylenimine (PEI). As in chapter 2, a systematic approach combining DoE and desirability functions is implemented. In both cases, medium exchange before transfection proves to be beneficial to achieve the highest transgene expression yields. Then, the optimal conditions for viable cell concentration at transfection, DNA and PEI concentrations are determined and the correct formation of the VLPs produced is corroborated using cryo-EM. In this case, Sf9 cells achieve a 8.4-fold increase in VLP production compared to High Five cells. In the last chapter, stable Sf9 and High Five cell pools to produce VLPs are developed by random integration and selection of the high producer cells using fluorescence-activated cell sorting. In terms of VLP production, a 3.7-fold increase in VLP titer is achieved in High Five over Sf9 stable pools. Finally, cell pool stability is successfully corroborated during the course of a month.
