Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters

Area of Science:

• Biochemistry
• Protein Purification
• Affinity Chromatography

Background:

• Human coagulation factor VIII (FVIII) is crucial for hemostasis.
• Current FVIII extraction methods have limitations in recovery and purity.
• Development of efficient FVIII purification processes is essential for therapeutic applications.

Purpose of the Study:

• To optimize a novel process for human coagulation factor VIII (FVIII) extraction from plasma.
• To evaluate the efficacy of a tailor-made affinity matrix, dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix), for FVIII purification.
• To compare the performance of the new process with established methods like cryoprecipitation.

Main Methods:

• Plasma pre-treatment using DEAE-Sephadex A-50 anion exchanger to remove prothrombin complex proteins.
• Contacting the FVIII-containing unbound fraction with the C3-C5 affinity matrix.
• Optimization of affinity chromatography parameters: ligand density, adsorption mode (batch vs. column), matrix-to-plasma ratio, pH, and conductivity for washing and desorption.

Main Results:

• Scale-up experiments processed 20 L of plasma.
• The new process achieved higher recovery (340 U VIII:C/kg plasma) compared to cryoprecipitation (300 U VIII:C/kg plasma).
• The specific activity (s.a.) of purified FVIII was significantly improved (1.2 U VIII:C/mg protein) versus cryoprecipitation (0.3 U VIII:C/mg protein).

Conclusions:

• The developed process using the C3-C5 affinity matrix is effective for FVIII purification.
• This novel method offers superior recovery and specific activity over cryoprecipitation.
• The process shows significant potential for industrial-scale purification of FVIII.