Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium

• The production of HIV-1 VLPs in mammalian suspension cultures is reported.
• HEK 293 cell growth is optimized by addition of non-animal derived supplements using DoE.
• VLP titers were increased 2.4-fold obtaining 2.7 × 109 VLPs/mL.

Virus-like particles (VLPs) offer great promise as candidates for new vaccine strategies. Large-scale approaches for the manufacturing of HIV-1 Gag VLPs have mainly focused on the use of the baculovirus expression system. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in mammalian cell suspension cultures is reported. To facilitate process optimization, a Gag-GFP fusion construct enabling the generation of fluorescent VLPs was used. The great majority of Gag-GFP present in cell culture supernatants was shown to be correctly assembled into virus-like particles of the expected size and morphology consistent with immature HIV-1 particles. Medium optimization was performed using design of experiments (DoE). Culture medium supplementation with non-animal derived components including recombinant proteins and lipids of synthetic or non-animal-derived origin resulted in improved HEK 293 cell growth and VLP production. The maximum cell density attained using the optimized Freestyle culture medium was 5.4 × 106 cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9 × 106 cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2–3 × 106 cells/mL) with medium exchange at the time of transfection using standard amounts of plasmid DNA and polyethylenimine. By using an optimized production protocol, VLP titers were increased 2.4-fold obtaining 2.8 μg of Gag-GFP/mL or 2.7 × 109 VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively.