Abstract
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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Acknowledgments
The authors wish to thank Dr. Amine Kamen (McGill University, Montreal, Canada) for the valuable discussions and for providing the HEK 293 cell line used in this study. They would also like to thank Dr. Julià Blanco at IRSI Caixa (Badalona, Spain) for providing the plasmid construct for Gag-GFP and for the helpful comments. The contribution of Manuela Costa (Institut de Biotecnología i Biomedicina UAB) with FACS analyses is deeply appreciated. The support of Dr. Salvador Bartolomé (Departament de Bioquímica i de Biología Molecular UAB) in fluorometry analysis is recognized. Gavin Whissell provided generous help in the revision of the manuscript. This work is supported by a grant of SEIDI - Ministerio de Economía y Competitividad of Spain (BIO2012-31251) and Generalitat de Catalunya (2009 SGR 1038). Laura Cervera and Javier Fuenmayor are recipients of PIF scholarships from UAB. Sònia Gutiérrez-Granados is a recipient of a FPU grant from the Ministerio de Educación y Deportes of Spain.
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Cervera, L., Fuenmayor, J., González-Domínguez, I. et al. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures. Appl Microbiol Biotechnol 99, 9935–9949 (2015). https://doi.org/10.1007/s00253-015-6842-4
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DOI: https://doi.org/10.1007/s00253-015-6842-4