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Immunoaffinity purification of factor VIII complex.

V S Hornsey, B D Griffin, D S Pepper

    Thrombosis and Haemostasis
    |February 3, 1987
    PubMed
    Summary
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    Researchers developed a novel immunoaffinity chromatography method to purify the Factor VIII complex. This method effectively isolates both Factor VIII procoagulant activity and von Willebrand factor ristocetin cofactor activity in a single product.

    Area of Science:

    • Biochemistry
    • Immunology
    • Hematology

    Background:

    • Von Willebrand factor (vWf) and Factor VIII (F VIII) are crucial proteins in hemostasis.
    • Purification of the F VIII complex, containing both F VIII procoagulant activity (VIII:C) and vWf ristocetin cofactor activity (R. cof.), is essential for therapeutic applications.

    Purpose of the Study:

    • To develop a novel immunoaffinity chromatography method for purifying the F VIII complex.
    • To achieve high-purity F VIII complex with retained biological activities from plasma and cryoprecipitate.

    Main Methods:

    • Immobilization of murine monoclonal antibodies to human vWf on Sephacryl S-1000.
    • Screening of solutes for elution of 125I-vWf and assessment of retention of VIII:C and R. cof.
    • Purification of F VIII complex using immunoaffinity chromatography under selected conditions.

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    Main Results:

    • A 4000-fold purification of the F VIII complex from plasma was achieved.
    • The purified product exhibited specific activities of 45 units VIII:C/mg protein and 60 units R. cof./mg.
    • The final product comprised 60% vWf, with <4% fibrinogen and fibronectin, and minimal murine antibody contamination (<300 ng/mg).

    Conclusions:

    • A novel and efficient method for purifying the F VIII complex using immunoaffinity chromatography was established.
    • The method yields a high-purity product containing both VIII:C and R. cof. activities, suitable for clinical use.
    • This single-step purification process utilizes a specific monoclonal antibody to isolate the complete F VIII complex.