Large-scale preparation of thrombin from human plasma

Area of Science:

• Biochemistry
• Biotechnology
• Plasma Protein Purification

Background:

• Thrombin is a critical enzyme in hemostasis, essential for fibrin clot formation.
• Efficient and safe large-scale production of thrombin is vital for clinical applications like fibrin sealants.

Purpose of the Study:

• To develop and characterize a cost-effective, scalable manufacturing process for human thrombin from plasma.
• To ensure high purity and viral safety of the final thrombin product.

Main Methods:

• Prothrombin isolation using cryoprecipitation, ion-exchange chromatography (DEAE-IEX), heparin affinity chromatography, and immobilized metal-affinity chromatography (IMAC).
• Prothrombin activation to thrombin, followed by purification via hydrophobic interaction chromatography (HIC) and ultrafiltration.
• Viral reduction achieved through solvent/detergent (S/D) treatment, pasteurization, and nanofiltration.

Main Results:

• A scalable process yielding thrombin with approximately 75% purity and specific activity of ~2400 IU/mg protein.
• Demonstrated cost-effectiveness by utilizing a waste fraction from heparin affinity chromatography for Factor IX production.
• Validated viral safety through multiple independent reduction techniques.

Conclusions:

• The described manufacturing process is suitable for large-scale production of high-purity, virus-safe human thrombin.
• This method offers a cost-effective approach by integrating with existing plasma-derived Factor IX production.
• The final thrombin product meets specifications for use in fibrin glue formulations.