RECOMBINANT VITAMIN K DEPENDENT PROTEINS WITH HIGH SIALIC ACID CONTENT AND METHODS OF PREPARING SAME
Methods of isolating highly sialylated recombinant vitamin K dependent proteins, particularly Factor IX, by chromatographic methods are described. The highly sialylated recombinant proteins are characterized. The improved Factor IX has at least 62% N-glycosylation with 3 or 4 sialic acid residues and improved bioavailability and pharmokinetic properties.
1 . A method of isolating recombinant Factor IX for treatment of hemophilia comprising:
providing a preparation of recombinant Factor IX; and
separating forms of recombinant Factor IX with 3 or more sialic acid residues per N-glycan;
collecting fractions enriched in recombinant Factor IX with 3 or more sialic acid residues per N-glycan; and
pooling the fractions having recombinant Factor IX, wherein the percentage of N-linked glycans with 3 or more sialic acid residues is at least 50%.
2 . The method of claim 1 , wherein the separation is carried out by chromatography.
3 . The method of claim 2 , wherein the chromatography is carried out in the presence of calcium.
4 . The method of claim 1 , wherein the Factor IX is fully gamma-carboxylated.
5 . The method of claim 1 , wherein the percentage of N-linked glycans with 3 or more sialic acid residues is at least 62%.
6 .- 19 . (canceled)
20 . The method of claim 1 , wherein the percentage of N-linked glycans with 3 or more sialic acid residues is at least 65%.
21 . The method of claim 1 , wherein the percentage of N-linked glycans with 3 or more sialic acid residues is at least 75%.
22 . The method of claim 1 , wherein the percentage of N-linked glycans with 3 or more sialic acid residues is at least 85%.
23 . The method of claim 1 , wherein the percentage of N-linked glycans with 3 or more sialic acid residues is at least 95%.
24 . The method of claim 1 , wherein the percentage of N-linked glycans with 3 or more sialic acid residues is 100%.