Application of advanced quantification techniques in nanoparticle-based vaccine development with the Sf9 cell...

Nanoparticles generated by recombinant technologies are receiving increased interest in several applications, particularly the use of virus like particles (VLPs) for the generation of safer vaccines. The characterization and quantification of these nanoparticles with complex structures is very relevant for a better comprehension of the production systems and should circumvent the limitations of the most conventional quantification techniques often used. Here, we applied confocal microscopy, flow virometry and nanoparticle tracking analysis (NTA) to assess the production process of Gag virus-like particles (VLPs) in the Sf9 cell/baculovirus expression vector system (BEVS). These novel techniques were implemented in an optimization workflow based on Design of Experiments (DoE) and desirability functions to determine the best production conditions. A higher level of sensitivity was observed for NTA and confocal microscopy but flow virometry proved to be more accurate. Interestingly, extracellular vesicles were detected as an important source of contamination of this system. The synergistic interplay of viable cell concentration at infection (CCI), multiplicity of infection (MOI) and time of harvest (TOH) was assessed on five objective responses: VLP assembly, baculovirus infection, VLP production, cell viability and VLP productivity. Two global optimal conditions were defined, one targeting the maximal yield of VLPs and the other providing a balance between production and assembled VLPs. In both cases, a low MOI proved to be the best condition to achieve the highest VLP production and productivity yields. Cryo-EM analysis of nanoparticles produced in these conditions showed the typical size and morphology of HIV-1 VLPs. This study presents an integrative approach based on the combination of DoE and direct nanoparticle quantification techniques to comprehensively optimize the production of VLPs and other viral-based biotherapeutics.