title: Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.
abstract: HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 10(10) VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.
authors.affiliation: Departament d'Enginyeria Química, Biològica i Ambiental. Universitat Autònoma de Barcelona. Edifici Q, Carrer de les Sitges, Bellaterra, 08193, Cerdanyola del Vallès, Barcelona, Spain. sonia.gutierrez@uab.cat.
authors.forename: Sonia
authors.initials: S
authors.lastname: Gutiérrez-Granados
authors.affiliation: Departament d'Enginyeria Química, Biològica i Ambiental. Universitat Autònoma de Barcelona. Edifici Q, Carrer de les Sitges, Bellaterra, 08193, Cerdanyola del Vallès, Barcelona, Spain.|Department of Bioengineering, McGill University, 817 Sherbrooke Street West, Room 270. Macdonald Engineering Building, Montreal, QC, H3A 0C3, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: Cervera
authors.affiliation: Bluebird Bio Pharmaceutical Sciences, Process Development group, 150 Second St., Cambridge, MA, 02141, USA.
authors.forename: María de Las Mercedes
authors.initials: Mde L
authors.lastname: Segura
authors.affiliation: CEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Straße 62, 51105, Cologne, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Wölfel
authors.affiliation: Departament d'Enginyeria Química, Biològica i Ambiental. Universitat Autònoma de Barcelona. Edifici Q, Carrer de les Sitges, Bellaterra, 08193, Cerdanyola del Vallès, Barcelona, Spain.
authors.forename: Francesc
authors.initials: F
authors.lastname: Gòdia
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000821:virology*; D014162:Transfection; D014773:Virology / Q000379:methods; D022701:Virosomes / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D022701:Virosomes
keywords: CAP-T; Design of experiments; HIV-1 virus-like particles; Transient gene expression