Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 99, Issue 23, Pages 9935-9949Publisher
SPRINGER
DOI: 10.1007/s00253-015-6842-4
Keywords
VLPs; Production additives; HEK 293; Transient transfection; Design of experiments
Categories
Funding
- SEIDI - Ministerio de Economia y Competitividad of Spain [BIO2012-31251]
- Generalitat de Catalunya [2009 SGR 1038]
- PIF scholarships from UAB
- FPU grant from Ministerio de Educacion y Deportes of Spain
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The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94 %.
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