Direct capture of factor IX from unclarified human plasma by IMEBAC

The method development for direct capture of clotting factor IX (FIX) from unclarified human plasma using immobilized metal expanded bed affinity chromatography (IMEBAC) has been described. Cu(II), Ni(II), Zn(II) and Co(II) metal ions were evaluated in immobilized metal affinity chromatography for the recovery of FIX. Immobilized Cu(II) chelating adsorbent was found to be the most effective for capture of general protein and FIX. The adsorption characteristic of Cu(II)-STREAMLINE chelating adsorbent used in this work for general protein and FIX in unclarified plasma has been assessed by the measurement of equilibrium isotherm. The influence of liquid velocities between 100 and 300 cm/h on the selective adsorption of FIX from clarified plasma was investigated in small packed bed experiments. Optimal conditions for the elution of FIX were also investigated in packed bed experiments conducted with clarified plasma. On the basis of the results, IMEBAC for direct capture of FIX from unclarified plasma was established. The purification results showed that FIX was directly recovered using 20 mM glycine buffer (pH 7.0) from unclarified plasma with a purification factor of 83 and yield of 16% in a single step. Other contaminants and FIX were completely eluted by using 25 mM EDTA (pH 7.0).