Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification

Area of Science:

• Biotechnology
• Biopharmaceutical Manufacturing
• Hematology

Background:

• Hemophilia A treatment necessitates regular Factor VIII (FVIII) administration.
• Recombinant FVIII is widely used but biosimilar development requires optimized production.
• Key challenges include creating high-yield cell lines and efficient purification strategies.

Purpose of the Study:

• To develop a highly productive cell line for truncated FVIII variants.
• To generate monoclonal antibodies for efficient FVIII purification.
• To establish upstream and downstream processes for biosimilar FVIII production.

Main Methods:

• Methotrexate-driven gene amplification to enhance FVIII expression.
• Selection of stable clonal cell lines producing truncated FVIII.
• Generation and screening of monoclonal antibodies against FVIII heavy chain.

Main Results:

• Truncated FVIII variant showed 6.5x higher expression than full-length.
• Transgene amplification increased expression twofold; clonal line produced 0.52 IU/ml.
• Four monoclonal antibodies effectively purified truncated FVIII with >85% yield and preserved activity.

Conclusions:

• Optimized cell line and monoclonal antibodies are suitable for biosimilar FVIII production.
• This work supports the development of upstream and downstream processes.
• Future work will focus on stronger promoters and shorter FVIII cDNA for enhanced productivity.