DEVELOPMENT OF AN IMMUNOAFFINITY PROCESS FOR FACTOR-IX PURIFICATION

Abstract. An immunoaffinity process based on monoclonal antibody (MAb) to factor IX (FIX) has been developed. Initially, vitamin‐K‐dependent proteins from cryoprecipitate‐poor plasma are isolated on DEAE‐Sephadex. The eluate is applied to an immunoaffinity column that utilizes a divalent metal‐ion‐dependent MAb directed against FIX. After washing the column with high salt in the presence of magnesium ion, the FIX is eluted using a citrate‐ or EDTA‐containing buffer. Coagulation assays and Western blots show no detectable amounts of any contaminating proteins. Purity of the FIX product is established using reduced and nonreduced Coomassie‐stanined SDS‐PAGE and HPLC. The N‐terminal 20 amino acids of the single peak of the HPLC were shown to be identical to those reported for FIX. The process shows no detectable leakage of monoclonal antibodies (MAb), efficient utilization of MAb, and provides yields greater than 95%. The use of solvent/detergent treatment as a potential viral inactivation method is incorporated in the process. Studies with tritiated Triton X‐100 indicate that the detergent can be washed out of the MAb column so that less than 1 ppm (total) Triton X‐100 coelutes with the FIX. © 1990 S. Karger AG, Basel