LARGE-SCALE PREPARATION OF HIGHLY PURIFIED HUMAN C1-INHIBITOR FOR THERAPEUTIC USE

A two-step chromatographic procedure has been developed to purify huma n C1- inhibitor from cryoprecipitate-poor plasma after removal of vita min K-dependent proteins and antithrombin III. The procedure, which is fully compatible with modern plasma fractionation schemes, includes a nion-exchange chromatography on DMAE-Fractogel EMD, viral inactivation by solvent-detergent treatment, adsorption on SO3-Fractogel EMD and v iral removal by nanofiltration on 35- and 15-nm pore size membranes. O verall yields were about 45% and 58% for antigen and activity, respect ively, providing 60-70 mg of highly purified inhibitor per litre of pl asma. The purified inhibitor had a specific activity of 6.5 +/- 0.5 un its/mg protein, representing a more than 400-fold increase in purity c ompared with plasma. C1-inhibitor purity with respect to total protein was greater than 80%. The main contaminant was complement component C 3 which accounted for 4-10% of the total protein. Minor contaminants i ncluded low amounts of IgM,IgG, IgA, fibrinogen and albumin. Complemen t component C4 was undetectable. The purified inhibitor was stable thr oughout the purification process and for more than 24 h at room temper ature after reconstitution of the freeze-dried material. Animal tests in rats and mice demonstrated that the C1-inhibitor concentrate was we ll tolerated at relatively high doses.