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Appl Microbiol Biotechnol. 2016 May;100(9):3935-47. doi: 10.1007/s00253-015-7213-x. Epub 2015 Dec 19.

Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.

Author information

1
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.
2
Departament d'Enginyeria Química, Biològica i Ambiental. Universitat Autònoma de Barcelona. Edifici Q, Carrer de les Sitges, Bellaterra, 08193, Cerdanyola del Vallès, Barcelona, Spain.
3
Department of Bioengineering, McGill University, 817 Sherbrooke Street West, Room 270. Macdonald Engineering Building, Montreal, QC, H3A 0C3, Canada.
4
Bluebird Bio Pharmaceutical Sciences, Process Development group, 150 Second St., Cambridge, MA, 02141, USA.
5
CEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Straße 62, 51105, Cologne, Germany.

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.

KEYWORDS:

CAP-T; Design of experiments; HIV-1 virus-like particles; Transient gene expression

PMID:
26685677
DOI:
10.1007/s00253-015-7213-x
[Indexed for MEDLINE]

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