2. Academic Validation
  3. Optimized production and coagulation activity of rhFVII in HEK293 cells for bleeding disorders

Optimized production and coagulation activity of rhFVII in HEK293 cells for bleeding disorders

  • Protein Expr Purif. 2025 Dec 3:239:106867. doi: 10.1016/j.pep.2025.106867.
Sen Zou 1,  Xiaoxiao Li 2,  Jiajun Liu 2,  Shuai Fan 3,  Zhaoyong Yang 4
Affiliations

Affiliations

  • 1 NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
  • 2 North China University of Science and Technology, Tangshan, 063210, Hebei, China.
  • 3 NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China. Electronic address: [email protected].
  • 4 NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China. Electronic address: [email protected].
PMID: 41349861 DOI: 10.1016/j.pep.2025.106867
Abstract

Hemophilia, a genetic disorder characterized by impaired blood clotting, necessitates innovative treatments. This study optimized the expression of recombinant human coagulation factor VII (rhFVII) in HEK293 cells to enhance its therapeutic efficacy. The human FVII gene was cloned into the pcDNA3.1 vector, and transient transfection was performed with an optimized DNA-to-PEI ratio (1:2) at 70 % cell density. Stable FVII-expressing cell lines were selected with G418, confirmed via RT-PCR, and adapted to suspension culture. rhFVII expression was analyzed using SDS-PAGE, Western blot, and ELISA, while its coagulation activity was evaluated by prothrombin time tests. Transient transfection yielded rhFVII concentrations of 10.22 μg/L and clotting activity of 232.46 %. Stable monoclonal HEK293 cells in suspension produced rhFVII at 11.22 mg/L with maximum coagulation activity of 563.17 % and high stability and safety. The findings underscore the successful optimization of rhFVII expression in HEK293 cells, paving the way for advancements in biopharmaceutical production and improved treatment options for bleeding disorders. Future research should focus on in vivo validation and further refinement of culture conditions to maximize protein yield and stability.

Keywords

Bleeding disorders; Coagulation activity; Coagulation factor VII; HEK293; Hemophilia.

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