IN-SOLUTION ACTIVATION OF FACTOR VII
The present invention is directed to a method for activation of Factor VII to FVIIa in solution.
1 . A method for activating FVII to FVIIa in solution, comprising
(a) obtaining a solution comprising a substantially purified preparation of scFVII;
(b) adding to the solution an amine compound, Ca 2+ to a final concentration of about 2 mM to about 50 mM, and adjusting the final pH of the solution to about pH 7.2 to 8.6;
(c) incubating the resulting activation mixture at between about 2° C. and about 25° C. for an amount of time sufficient to convert at least 90% of the scFVII to FVIIa; and
(d) optionally, isolating the FVIIa from the activation mixture.
2 . The method of claim 1 wherein the solution comprising the substantially purified preparation of scFVII contains at least 85% scFVII relative to FVIIa or other FVII-derived fragments.
3 . The method of claim 1 wherein the amine compound is Tris, lysine, arginine, phosphorylcholine, or betaine.
4 . The method of claim 1 wherein the amine compound is added to a final concentration of about 50 mM to about 500 mM.
5 . The method of claim 1 wherein the final pH of the solution is pH 7.6 to 8.2
6 . The method of claim 5 wherein the final pH of the solution is about pH 8.
7 . The method of claim 1 wherein the activation mixture has an initial concentration of scFVII of at least 4 mg/ml.
8 . The method of claim 1 wherein the activation mixture has an initial concentration of scFVII of about 1 mg/ml to 4 mg/ml, and the activation mixture further comprises an activation enhancer.
9 . The method of claim 8 wherein the activation enhancer is polyethylene glycol, glycerol, or ethylene glycol.
10 . The method of claim 9 wherein the activation enhancer is PEG4000, PEG8000, or glycerol.
11 . The method of claim 1 wherein the amount of time sufficient to convert at least 90% of the scFVII to FVIIa is between about 4 hours and 24 hours.
12 . The method of claim 1 wherein the activation mixture has an initial concentration of scFVII of about 4 mg/ml to about 10 mg/ml, the amine compound is Tris or lysine at a final concentration of about 0.1 M, the Ca 2+ concentration is 10 mM to 30 mM, the pH of the activation mixture is about pH 8, and the solution is incubated at about 4° C. for 8-24 hrs.
13 . The method of claim 1 wherein the FVII is a FVII variant which differs in 1-15 amino acid residues from the amino acid sequence of human FVII (SEQ ID NO:1).
14 . The method of claim 13 wherein the FVII variant comprises the substitutions P10Q and K32E.