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Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification.
MedLine Citation:
PMID:  22708069     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma-derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methotrexate-driven transgene amplification of genetic cassettes that code full-length and truncated variants of FVIII under the control of the CMV promoter was studied. It was shown that the expression level of the truncated variant of FVIII is 6.5 times higher than that of the full-length molecule. The transgene amplification procedure was sufficient for a twofold increase of the expression level in the transfected cells pool and subsequent selection of the clonal line, stably producing truncated FVIII at the level of 0.52 IU/ml during cultivation in a chemically defined protein-free culture medium. Four generated mouse monoclonal antibodies toward the heavy chain of FVIII were found suitable for binding the truncated variant of FVIII directly from the conditioned medium and elution of the FVIII with a more than 85% yield and normal pro-coagulant activity. The producer cell line and monoclonal antibodies obtained are sufficient for the development of upstream and downstream processes of biosimilar FVIII production. Generation of more productive cell lines by the use of stronger, nonviral promoters and shorter cDNA of FVIII will be the subject of further studies.
Authors:
N A Orlova; S V Kovnir; I I Vorobiev; A S Yuriev; A G Gabibov; A I Vorobiev
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Acta naturae     Volume:  4     ISSN:  2075-8251     ISO Abbreviation:  Acta Naturae     Publication Date:  2012 Jan 
Date Detail:
Created Date:  2012-06-18     Completed Date:  2012-08-23     Revised Date:  2013-05-30    
Medline Journal Info:
Nlm Unique ID:  101525823     Medline TA:  Acta Naturae     Country:  Russia (Federation)    
Other Details:
Languages:  eng     Pagination:  93-100     Citation Subset:  -    
Affiliation:
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences.
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