Alpha Therapeutic Corporation

Steven W Herring

Yahiro Uemura

Munehiro Noda

Kenneth T Shitanishi

The present invention relates to a process for purifying Factor IX from an impure protein fraction containing Factor IX. The purification process comprises the steps of adding a solvent and a detergent to an impure protein fraction and incubating the solvent/detergent protein solution to inactivate any viral contaminants. Factor IX is purified from the solvent/detergent protein solution by chromatography on a sulfated polysaccharide resin without first removing the solvent and detergent prior to the purification on the sulfated polysaccharide resin. The Factor IX, purified by the process has a specific activity of at least 85 units/mg.

Purification of factor IX

The present invention describes a process for activating Factor II to Factor II.sub.a by incubating Factor II in the presence of Factor V, Factor X.sub.a, phospholipids, and calcium ions. Each of the factors is prepared from a single impure protein fraction which includes Factors II, V and X. The Factor II, V and X purification procedure comprises the steps of DEAE ligand chromatography and precipitation by the addition of barium chloride. Factor V is recovered from the barium chloride supernatant, and Factors II and X are contained in the barium chloride precipitate. The barium chloride precipitate is dissolved in an aqueous solution and is applied to a chromatographic resin coupled with a ligand which binds Factor X and Factor II weakly or not at all. Factor II is recovered from the fraction, which remains unbound or weakly bound to the Factor X binding ligand. Factor X.sub.a is prepared by recovering Factor X from the ligand during the Factor II preparation procedure and activating the Factor X to Factor X.sub.a by specific proteolytic cleavage.

Plasma fraction purification

There is provided, in accordance with practice of this invention, a process for separating Factor IX and/or Factor X from an impure protein fraction containing protein in addition to Factors IX and X. A silica resin coupled with a ligand capable of binding Factor IX and/or Factor X is provided. An aqueous solution of the impure protein fraction is applied to the ligand-coupled silica resin to thereby bind the Factor IX and/or Factor X to the resin. The Factor IX and/or Factor X is then recovered from the resin by elution.

Plasma fraction purification using silica resin bound to a ligand

A rapid and simple process for purifying human, bovine and porcine procoagulant protein Factor VIII on a large scale using sequential high performance size exclusion chromatography under, first, low salt concentration conditions and, second, under high salt concentration conditions from reconstituted commercial Factor VIII:C (complexed Factor VIII) concentrate. The chromatographic separation is carried out on a high performance size exclusion chromatographic column packed with porous beads having a particle size of from about 13 to about 35 microns, pore diameters of from about 500 to about 2000 Angstroms and a pore volume of from about 1.0 to about 1.8 ml per gram. The first chromatographic separation is carried in a buffered aqueous solution using the buffered aqueous solution as an eluant. The low molecular weight constituents (impurities) are separated from Factor VIII and the high molecular weight constituents (impurities). A second chromatographic separation may be carried out after Factor VIII has been dissociated in a buffered solution having a concentration of from about 0.25 to about 0.45M calcium ion. The second chromatographic column is packed with some packing as the first column and is eluted with a buffered aqueous solution containing 0.25 to 0.45M calcium ion. In a column of 2.5.times.60 cm, 4 gms of commercial Factor VIII concentrate can be purified in less than two hours. The process is amenable to scale up.

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San Dimas, CA, United States of America

Steven W Herring