IP Library › Granted Patent US 7,897,734
Granted Patent B2
US 7,897,734 · App. 11/229,428 · Granted Mar 1, 2011

Method for the production of proteins

Assignee: Novo Nordisk Healthcare AG
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Quick Facts
Patent No.
US 7,897,734
App. No.
11/229,428
Granted
Mar 1, 2011
Kind
B2
Abstract

The present invention relates to a process for the purification of a protease.

Claims (12)

1. A method for the purification of a gamma-carboxyglutamic (GLA)-residue containing serine protease, said method comprising subjecting a solution comprising the protease to one or more purification steps, wherein (i) the GLA-residue containing serine protease is a human Factor VII polypeptide variant wherein the ratio between the activity of the Factor VII polypeptide variant and the activity of wild type human Factor VIIa (SEQ ID NO: 1) is at least about 1.25 when tested in an in vitro hydrolysis assay; and (ii) the pH to which the protease is subjected during at least one of the purification steps has a value between 4.5 and 6.9 or has a value between 8.8 and 9.8.

2. The method according to claim 1 , wherein at least one of the purification steps is performed using a free calcium ion concentration higher than 1.2 mM.

3. The method according to claim 2 , wherein the free calcium ion concentration in at least one of the purification steps is higher than 2.0 mM.

4. The method according to claim 1 , wherein the free calcium ion concentration in at least one of the purification steps is lower than 0.10 mM.

5. The method according claim 4 , wherein the free calcium ion concentration in at least one of the purification steps is lower than 0.05 mM.

6. The method according to claim 1 , wherein, at least one of the purification steps is performed using a free ion concentration of a divalent metal cation other than a zinc ion and a calcium ion higher than 0.025 mM.

7. The method according to claim 6 , wherein the divalent metal cation is selected from the list consisting of Mg 2+ , Cu 2+ , Mn 2+ , Co 2+ , Fe 2+ , Sm 2+ , Ni 2+ , Cd 2+ , Hg 2+ and Sm 2+ .

8. The method according to claim 6 , wherein the free ion concentration of a divalent metal cation other than a zinc ion and a calcium ion is higher than 0.1 mM.

9. The method according to claim 1 , wherein the GLA-residue containing serine protease is purified in at least one of the purification steps in the presence of a divalent metal ion chelator.

10. The method according to claim 1 , wherein the GLA-residue containing serine protease is purified at a pH between 4.5 and 6.9.

11. The method according to claim 1 , wherein the GLA-residue containing serine protease is purified at a pH between 8.8 and 9.8.

12. The method according to claim 1 , wherein said Factor VII polypeptide is a variant of wild-type human Factor VII (SEQ ID NO: 1) selected from the group consisting of: L305V-FVII, L305V/M306D/D309S-FVII, L3051-FVII, V158T/M298Q-FVII, V158D/E296 V/M298Q-F VII, K337A-FVII, M298Q-F VII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296 V/M298Q/K33 7A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, E296 V-F VII, E296 V/M298Q-F VII, L305V/K337A-FVII, L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V 1 58T-FVII, L305V/K337A/V158T-F VII, L305V/K337A/M298Q-F VII, L305 V/K337A/E296 V-FVII, L305V/K337A/V158D-F VII, L305V/V158D/M298Q-F VII, L305 V/V158D/E296 V-FVII, L305V/V158T/M298Q-FVII, L305 V/V158T/E296 V-FVII, L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-F VII,L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-F VII,L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII,L305V/V158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII, S314E/K316Q-FVII,S314E/L305V-FVII, S314E/K337A-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/L305V/K337A-FVII, S314E/L305V/M298Q-FVII, S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296 V-FVII, S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296 V/M298Q-FVII, S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII, S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII, S314E/L305V/V158D/E296V/K337A-FVII, S314E/L305V/V158D/E296V/M298Q/K337A-FVII, S314E/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/L305 V-FVII, F374Y/L305V/K337A-FVII, F374Y/L305V/314E-FVII, F374Y/K337A/314E-FVII, F374Y/L305V/K337A/S314E-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII, and F374Y/L305V/E296V/M298Q/K337A/V158T/S314E.

Assignments (3)
CHANGE OF ADDRESS OF ASSIGNEE Recorded Nov 15, 2021
From: NOVO NORDISK HEALTHCARE A/G
To: NOVO NORDISK HEALTHCARE AG
Reel/Frame 058122/0546 →
CHANGE OF ADDRESS Recorded Jun 19, 2013
From: NOVO NORDISK HEALTHCARE A/G
To: NOVO NORDISK HEALTHCARE AG
Reel/Frame 030653/0189 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 25, 2009
From: KRARUP, JANUS; PERSSON, EGON
To: NOVO NORDISK HEALTHCARE AG
Reel/Frame 022875/0480 →
Continuity (3)
Continuation PCTDK2004000183 · Mar 18, 2004
Provisional Application 60457809 · Mar 26, 2003
Related Publication 20060051854A1 · Mar 9, 2006