A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX

Area of Science:

• Biotechnology
• Molecular Biology
• Genetic Engineering

Background:

• Hemophilia B patients require factor IX replacement therapy due to a genetic blood-clotting defect.
• Recombinant human factor IX (rFIX) produced in Chinese hamster ovary (CHO) cells is a key therapeutic agent.
• Developing biosimilar rFIX necessitates high-yield, high-activity producer cell lines.

Purpose of the Study:

• To generate a clonal CHO cell line with enhanced specific productivity and procoagulant activity for biosimilar factor IX production.
• To optimize plasmid vectors and gene amplification strategies for efficient rFIX expression.
• To establish a robust purification process for industrial-scale manufacturing.

Main Methods:

• Cloning of the factor IX gene into a p1.1 vector for transfection into CHO DG44 cells.
• Methotrexate-driven gene amplification and co-transfection with p1.2 plasmids encoding PACE/furin and vitamin K oxidoreductase.
• Cell cloning, batch culturing, protein purification via chromatography, and activity assays.

Main Results:

• A producer cell line (3B12-86) achieved a specific productivity of 10.7 ± 0.4 pg/cell/day.
• Secreted factor IX exhibited restored procoagulant activity, reaching titers of 6 IU/ml in protein-free medium.
• Purified factor IX showed a specific activity of up to 230 IU/mg with >30% overall yield.

Conclusions:

• The developed clonal producer cell line and purification process are suitable for economically viable industrial-scale production of biosimilar factor IX.
• This advancement offers a promising therapeutic option for hemophilia B patients.
• The study demonstrates effective strategies for enhancing recombinant protein expression in mammalian cells.