Polypeptide Purification

1 . A method for purifying a polypeptide having a desired content of gamma-carboxyglutamic acid from a sample comprising mixture of species of said polypeptide having different contents of gamma-carboxyglutamic acid, said method comprising the steps of:

(a) loading said sample onto an anion exchange chromatography material;

(b) eluting said polypeptide using a solution at a pH of less than pH 9.0 comprising at least one salt selected from ammonium acetate, ammonium chloride and sodium acetate; and

(c) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids.

2 . A method according to claim 1 comprising the following steps:

(a) equilibrating an anion exchange material with a buffer at a pH of less than 9.0;

(b) loading said sample onto the anion exchange material;

(c) optionally washing the anion exchange material with a buffer at a pH of less than 9.0;

(d) eluting said polypeptide from the ion exchange material using a solution at a pH of less than 9.0 comprising at least one salt selected from ammonium acetate, ammonium chloride and sodium acetate; and

(e) selecting a fraction obtained from said elution wherein the polypeptides in the fraction have the desired content of gamma-carboxyglutamic acids.

3 . A method according to claim 1 wherein the polypeptide to be purified is selected from Factor IX, Factor VII, Factor VIIa, Factor X, Prothrombin, Protein-S, Protein-C, Protein Z, Osteocalcin, Matrix-gla-protein, Growth arrest-specific-6, Proline-rich-Gla-1, Proline-rich-Gla-2, Proline-rich-Gla-3 and Proline-rich-Gla-4.

4 . A method according to claim 3 wherein the polypeptide is Factor IX.

5 . A method according to claim 4 wherein said method comprises selecting a fraction obtained from said elution which has an increase in the proportion of #1-11-Gla and/or #1-12-Gla forms of Factor IX compared with the proportion of #1-11-Gla and/or #1-12-Gla forms of Factor IX in the sample being purified.

6 . A method according to claim 4 wherein said method comprises selecting a fraction obtained from said elution which has an decrease in the proportion of #1-10-Gla form of Factor IX compared with the proportion of #1-10-Gla form of Factor IX in the sample being purified

7 . A method according to claim 3 wherein the polypeptide is Factor VII or Factor VIIa.

8 . A method according to claim 7 wherein said method comprises selecting a fraction obtained from said elution which has an increase in the proportion of #1-10-Gla and/or #1-11-Gla forms of Factor VII or Factor VIIa compared with the proportion of #1-10-Gla and/or #1-11-Gla forms of Factor VII or Factor VIIa in the sample being purified.

9 . A method according to claim 7 wherein said method comprises selecting a fraction obtained from said elution which has an decrease in the proportion of #1-9-Gla form of Factor VII or Factor VIIa compared with the proportion of #1-9-Gla form of Factor VII or Factor VIIa in the sample being purified.

10 . A method according to claim 1 wherein said elution buffer has a pH of between 5.0 and 8.5.

11 . A method according to claim 1 wherein ammonium acetate, ammonium chloride or sodium acetate is present in the elution buffer at between 0.1M and 2.0M

12 . A method according to claim 11 wherein ammonium acetate, ammonium chloride or sodium acetate is present in the elution buffer at about 0.6 M.

13 . A method according to claim 1 wherein said ion exchange chromatography utilises an equilibration buffer at a pH between 5.0 and 8.5.

14 . A polypeptide formulation obtained by a method according to claim 1 .