O-Linked Glycoforms Of Polypeptides And Method To Manufacture Them

• US 20080305518A1
• Filed: 05/03/2005
• Published: 12/11/2008
• Est. Priority Date: 05/04/2004
• Status: Abandoned Application

• Current Assignee: Novo Nordisk Health Care AG (Novo Nordisk A/S)
• Original Assignee: NOVO Nordisk Biotech Incorporated (Novo Nordisk A/S)
• Inventors: Klausen, Niels Kristian, Rasmussen, Daniel
• Application Number:
  US11/579,401
• Publication Number:
  US 20080305518A1
• Time in Patent Office: Days
• US Class Current: 435/68.1
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/745 Blood coagulation or fibrinolysis factors

Abstract

The present invention relates to compositions comprising glycoproteins having altered patterns of O-linked glycosylation, in particular Factor VII, Factor IX, and methods for making these.

Claims

1. A preparation of a glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr covalent bond, X1 and X2 each represent any amino acid residue and the preparation has a substantially uniform serine/threonine-linked glycosylation pattern.

2. The preparation according to claim 1, wherein the glycosylation pattern is at least 80% uniform.

3. The preparation according to claim 1, wherein the serine/threonine-linked sugar chain is Xyl-Xyl-Glc-.

4. The preparation according to claim 1, wherein the serine/threonine-linked sugar chain is Xyl-Glc-.

5. The preparation according to claim 1, wherein the serine/threonine-linked sugar chain is Glc-.

6. The preparation according to claim 1, wherein the glycoprotein is selected from the group consisting of Factor VII polypeptides, Factor VII-related polypeptides, Factor IX polypeptides, Factor IX-related polypeptides, Factor X polypeptides, Factor X-related polypeptides, Factor XII polypeptides, and protein Z polypeptides.

7. The preparation according to claim 6, wherein the glycoprotein is human Factor VII.

8. A preparation according to claim 6, wherein the glycoprotein is a variant of Factor VII and wherein the ratio between the activity of the Factor VII-variant and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the “

In Vitro Hydrolysis Assay”

or in the “

In vitro Proteolysis Assay”

.

9. The preparation according to claim 6, wherein the glycoprotein is human Factor IX or a human Factor IX sequence variant.

10. A method for making a preparation according to claim 5 comprising:

(a) obtaining a preparation of a precursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by Glc-O-Ser/Thr covalent bond and X1 and X2 each represent any amino acid residue; and

(b) contacting the preparation obtained in step (a) with an α

-xylosidase under conditions appropriate for removing xylose residues from the precursor glycoprotein, thereby producing the glycoprotein.

11. The method according to claim 10, further including the step of isolating the glycoprotein prepared in step (b).

12. The method according to claim 10, wherein the glycosylation is a serine glycosylation.

13. The method according to claim 1010, further including the step of analysing the structure of the sugar chain linked to the glycoprotein to determine a glycoform pattern, and, optionally, repeating step (b) until the desired glycoform pattern is achieved.

14. A method for making a preparation according to in claim 6 comprising:

(a) obtaining a preparation of a polypeptide containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein X1 and X2 each represent any amino acid residue; and

(b) contacting the preparation obtained in step (a) with a O-glucosyltransferase and an activated glucose donor under conditions appropriate for transferring a glucose residue from the glucose donor moiety to the serine/threonine thereby producing the glycoprotein.

15. The method according to claim 14, further including the step of isolating the glycoprotein prepared in step (b).

16. The method according to claim 14, wherein the glycosylation is a serine glycosylation.

17. The method according to claim 14, further including the step of analyzing the structure of the sugar chain linked to glycoproteins in the preparation to determine if the glycoproteins have a desired glycoform pattern, and, optionally, repeating step (b) until the desired glycoform pattern is achieved.

18. A method for making a preparation according to claim 4 comprising:

(a) obtaining a preparation of a precursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr covalent bond and X1 and X2 each represent any amino acid residue; and

(b) contacting the preparation obtained in step (a) with (i) UDP-D-xylose;

β

-D-glucoside α

-1,3-D-xylosyltransferase and (ii) an activated xylosyl donor under conditions appropriate for transferring a xylose residue from an xylose donor moiety to an acceptor moiety, thereby producing the glycoprotein.

19. The method according to claim 18, further including the step of isolating the glycoprotein prepared in step (b).

20. The method according to claim 18, wherein the glycosylation is a serine glycosylation.

21. The method according to claim 18, further including the step of analyzing the structure of the sugar chain linked to glycoproteins in the preparation to determine if the glycoproteins have a desired glycoform pattern, and, optionally, repeating step (b) until the desired glycoform pattern is achieved.

22. The method according to claim 18, further including the step of removing terminal xylose-residues by subjecting the preparation obtained in step (a) to the method described in claim 10 prior to step (b).

23. A method for making a preparation according to claim 3 comprising:

(a) obtaining a preparation of a precursor glycoprotein containing a Cys-X1-Ser/Thr-X2-Pro-Cys motif, wherein the serine/threonine is linked to a sugar chain by a Glc-O-Ser/Thr covalent bond and X1 and X2 each represent any amino acid residue;

(b) contacting the preparation obtained in step (a) with UDP-D-xylose;

β

-D-glucoside α

-1,3-D-xylosyltransferase and an activated xylosyl donor under conditions appropriate for transferring a xylose residue from a xylose donor moiety to an acceptor moiety; and

(c) contacting the preparation obtained in step (b) with UDP-D-xylose;

α

-D-xyloside α

-1,3-xylosyltransferase and an activated xylosyl donor under conditions appropriate for transferring a xylose residue from a xylose donor moiety to an acceptor moiety, thereby producing the glycoprotein.

24. The method according to claim 23, further including the step of isolating the preparation obtained in step (b) prior to subjecting the preparation to step (c).

25. The method according to claim 23, further including the step of isolating the glycoprotein prepared in step (c).

26. The method according to claim 23, wherein the glycosylation is a serine glycosylation.

27. The method according to claim 23, further including the step of analyzing the structure of the sugar chain linked to the glycoproteins in the preparation to determine if the glycoproteins have a desired glycoform pattern, and, optionally, repeating step (b) and/or step (c) until the desired glycoform pattern is achieved.

28. The method according to claim 23, further including the step of removing terminal xylose-residues by subjecting the preparation obtained in step (a) to the method described in claim 10 prior to step (b).

29. The preparation according to claim 2, wherein the glycosylation pattern is at least 90% uniform.

30. The preparation according to claim 29, wherein the glycosylation pattern is at least 98% uniform.