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

Laura Cervera, Sonia Gutiérrez-Granados, Marta Martínez, Julià Blanco, Francesc Gòdia, María Mercedes Segura

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    64 Citations (Scopus)

    Abstract

    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 5.4×106cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×106cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×106cells/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 2.8μg of Gag-GFP/mL or 2.7×109VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively. © 2013 Elsevier B.V.
    Original languageEnglish
    Pages (from-to)152-165
    JournalJournal of Biotechnology
    Volume166
    Issue number4
    DOIs
    Publication statusPublished - 1 Jul 2013

    Keywords

    • Animal cell culture
    • Design of experiments (DoE)
    • HIV-1 vaccine
    • Media supplementation
    • Transient gene expression (TGE)
    • Virus-like particles (VLP)

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