title: Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium.
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 5.4×10(6)cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×10(6)cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×10(6)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 2.8μg of Gag-GFP/mL or 2.7×10(9)VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively.
authors.affiliation: Departament d'Enginyeria Química, Universitat Autònoma de Barcelona, Campus Bellaterra, Cerdanyola del Vallès 08193, Barcelona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Cervera
authors.forename: Sonia
authors.initials: S
authors.lastname: Gutiérrez-Granados
authors.forename: Marta
authors.initials: M
authors.lastname: Martínez
authors.forename: Julià
authors.initials: J
authors.lastname: Blanco
authors.forename: Francesc
authors.initials: F
authors.lastname: Gòdia
authors.forename: María Mercedes
authors.initials: MM
authors.lastname: Segura
mesh_terms: D000818:Animals; D002138:Calibration; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards; D016895:Culture Media, Serum-Free / Q000737:chemistry / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D006801:Humans; D013535:Suspensions; D014162:Transfection / Q000379:methods*; D054928:Validation Studies as Topic; D014771:Virion / Q000235:genetics*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; D013535:Suspensions; C485184:enhanced green fluorescent protein; D054301:gag Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins
keywords: Animal cell culture; Design of experiments (DoE); HIV-1 vaccine; Media supplementation; Transient gene expression (TGE); Virus-like particles (VLP)