LARGE-SCALE PREPARATION AND BIOCHEMICAL-CHARACTERIZATION OF A NEW HIGH-PURITY FACTOR-IX CONCENTRATE PREPARED BY METAL CHELATE AFFINITY-CHROMATOGRAPHY

Metal chelate affinity chromatography on copper-charged Chelating Seph arose has been used to purify a factor IX concentrate from 4 000- to 5 000-kg pools of human plasma, with an overall yield of 194 IU/kg. Unw anted proteins and solvent-detergent reagents added to inactivate lipi d-enveloped viruses were removed during the chromatographic step. The freeze-dried product was > 80% pure factor IX with a mean specific act ivity of > 160 IU/mg protein. The concentrate showed no evidence of cl otting factor activation by in vitro tests for potential thrombogenici ty or by direct assay for activated factor IX. The concentrate did not exhibit proteolytic activity against a range of synthetic peptide chr omogenic substrates. Full functional factor IX activity was retained a nd there was no evidence of protein degradation. Metal chelate affinit y chromatography therefore appears to present less physicochemical cha llenge to the protein than other factor IX purification methods, while allowing the preparation of a clinical factor IX concentrate at a lar ge scale.