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High-level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene-switch

 
 
Affiliation:
National Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Language:
English
Type:
Article
Published in:
Biotechnology and Bioengineering
Date:
2010
Pages :
203-215
NRCC #:
52758
NPArC #:
16225332
Keywords:
Acids; Adenovirus; Alkaline Phosphatase; analysis; Animal; bio; Biotechnology; blood; Cell Culture; Cell Line; Cells; chemistry; CHO cells; Chromatography; Cumate gene-switch; Dna; Gels; Gene Expression; Gene Therapy; Genomics; Glycosylation; HEK-293; Hek-293 cells; Hepatocytes; Human; I; Igg; immunology; insect cell; Kinetics; Large-scale production; Lentiviral vectors; Lentivirus; Macrophages; Mass Spectrometry; methods; Molecular Weight; Nucleic Acids; Oxygen; phosphatase; Pichia; Protein; protein production; Proteins; Purification; Recombinant protein; Recombinant protein production; Recombinant Proteins; Reduced temperature; serum-free medium; Solutions; Stable expression; Suspension culture; synthesis; T-Lymphocytes; Temperature; therapy; Transfection; virology; Yeast
Program(s):
Bioprocesses Development Program; Programme de développement de bioprocédés
Group(s):
Bioprocess Center; Centre Bioprocédés
Abstract:
Fast and efficient production of recombinant proteins for structural and functional studies is a crucial issue for research and for industry. To this end, we have developed an efficient system to generate in less than 2 months, starting from the cDNA, pools of CHO cells stably expressing high-level of recombinant proteins. It is based on lentiviral vectors (LVs) for stable transduction coupled with the cumate gene-switch for inducible and efficient gene expression. Transcription is initiated upon binding of the cumate transactivator (cTA) or the reverse cTA (rcTA) to the CR5 promoter. Binding of cTA or rcTA is prevented or induced by addition of cumate respectively. We first validated the CHO/LV production system with an LV carrying the secreted alkaline phosphatase (SEAP), whose expression was linked to the green fluorescent protein (GFP) through an internal ribosome entry site (IRES). CHO cells stably expressing the cTA (CHO-cTA) were transduced at various multiplicity of infection (MOI). Pools of cells were incubated at 37 and 30-¦C during 10 days. Optimal SEAP production (65++g/mL) was achieved at 30-¦C with a MOI of 200. The pool stability was demonstrated for 48 days of culture by GFP expression analysis. The system was also evaluated using LV expressing three typical therapeutic proteins (a protein made up of the extracellular domain of CD200 fused to IgG Fc region [CD200Fc], a chimeric antibody [chB43], and erythropoietin [EPO]). CHO cells expressing rcTA (CHO-Cum2) were transduced with these LVs at a MOI of 200 and production was tested at 30-¦C. After 13 days of culture, 235, 160, and 206mg/mL of CD200Fc, chB43, and EPO were produced, respectively. The ON/OFF ratio of these pools was equal to 6 for CD200Fc, 16 for chB43, and 74 for EPO. In conclusion, this system should be very useful to produce mg quantities of recombinant proteins in a timely manner in serum free suspension culture of CHO cells for preclinical studies.
 
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