title: Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.
abstract: 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 96 h, 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 240 h with a 4-12-fold increase in production levels, depending on the product type considered.
authors.affiliation: Grup d'Enginyeria Cellular i Tissular, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193, Barcelona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Cervera
authors.forename: Sonia
authors.initials: S
authors.lastname: Gutiérrez-Granados
authors.forename: Nicholas Simon
authors.initials: NS
authors.lastname: Berrow
authors.forename: Maria Mercedes
authors.initials: MM
authors.lastname: Segura
authors.forename: Francesc
authors.initials: F
authors.lastname: Gòdia
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D011094:Polyethyleneimine / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics*; D014162:Transfection; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA
keywords: HEK 293; VLPs; production optimization; recombinant proteins; transient transfection