title: Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension.
abstract: Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 x amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.
authors.affiliation: Bioprocessing Technology Institute, Centros, 138668 Singapore. sun_xiang_ming@bti.a-star.edu.sg
authors.forename: Xiangming
authors.initials: X
authors.lastname: Sun
authors.forename: Peen Ern
authors.initials: PE
authors.lastname: Goh
authors.forename: Kathy T K
authors.initials: KT
authors.lastname: Wong
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Mori
authors.forename: Miranda G S
authors.initials: MG
authors.lastname: Yap
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000502:physiology; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods
chemical_list: D016895:Culture Media, Serum-Free; C485191:GFP10 Protein; D011994:Recombinant Proteins; D004921:Erythropoietin; D049452:Green Fluorescent Proteins