A technique for specific removal of factor IX alloantibodies from human plasma: partial characterization of the alloantibodies

A method for specific removal of large amounts of factor IX:C alloantibodies by a resin to which highly purified factor IX was linked (factor IX CH-Sepharose) is described. Factor IX was isolated from human plasma by a three-step procedure, including barium citrate adsorption and elution, DEAE-Sepharose CL-6B chromatography, and dextran sulfate agarose chromatography. Approximately 100 mg factor IX was obtained from 60 liters of plasma. The preparation was about 95% pure as judged by SDS-PAA gel electrophoresis. Its specific coagulant activity was 160 U/mg (IX) and its factor IX clotting antigen (IX:Ag) 500–600 U/mg. Essentially quantitative coupling of the factor IX preparation to activated CH-Sepharose 4B was obtained (4 mg factor IX/ml gel; 2300–3000 U/IX:Ag/ml). This resin bound 1500–2000 U factor IX inhibitor/ml gel and could be re-used at least 5 times without any loss in binding capacity. The binding capacity was dependent on the flow rate. No signs of activation of the coagulation, fibrinolytic, or complement system were observed in vitro. Using this factor IX resin, factor IX alloantibodies were isolated and found to consist of two portions, one minor bound to the resin only in the presence of Ca2+ and another major portion Ca2+ independent. The specific inhibitory activity/milligram IgG of the Ca2+-dependent alloantibodies was about 5 times higher in the presence of Ca2+. It is concluded that 25 ml of the factor IX resin described can remove about 40,000 factor IX inhibitor units (comparable to 120,000 Bethesda U) in one run, provided the flow rate does not exceed 20 ml/hr. By using such a technique for removal of antibodies it seems feasible to convert hemophilia-B patients complicated with inhibitors against factor IX into ordinary hemophilia- B patients for treatment at an emergency or in association with major surgery.

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