Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement

Laura Cervera, Sonia Gutiérrez-Granados, Nicholas Simon Berrow, Maria Mercedes Segura, Francesc Gòdia

Research output: Contribution to journalArticleResearchpeer-review

16 Citations (Scopus)


© 2014 Wiley Periodicals, Inc. Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240h with a 4-12-fold increase in production levels, depending on the product type considered.
Original languageEnglish
Pages (from-to)934-946
JournalBiotechnology and Bioengineering
Issue number5
Publication statusPublished - 1 May 2015


  • HEK 293
  • Production optimization
  • Recombinant proteins
  • Transient transfection
  • VLPs


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