Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

• Published in: Biotechnology letters 2006-06, Vol.28 (11), p.843-848
• Main Authors: Sun, Xiangming, Goh, Peen Ern, Wong, Kathy T. K, Mori, Tetsushi, Yap, Miranda G. S
• Format: Article
• Language: English
• Subjects: Amino acids; Batch culture; Biological and medical sciences; Bioreactors; Biotechnology; Cell culture; Cell Culture Techniques - methods; Cell Line; Culture Media, Serum-Free - metabolism; Erythropoietin; Erythropoietin - biosynthesis; Erythropoietin - genetics; Fed batch; Fed-batch culture; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression - physiology; Gene Expression Regulation; Genes, Reporter - physiology; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; HEK 293 EBNA1 cells; Humans; Kidney - cytology; Kidney - embryology; Polyethylenimine; Proteins; Recombinant Proteins - biosynthesis; Serum-free medium; Transfection; Transfection - methods; Transient gene expression
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-006-9010-1

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.