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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23489 43443 2013 14 PDF Available
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

•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.

Keywords
Animal cell culture; Media supplementation; Design of experiments (DoE); Virus-like particles (VLP); HIV-1 vaccine; Transient gene expression (TGE)
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Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 166, Issue 4, 20 July 2013, Pages 152–165
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us