One pot desialylation and glycopegylation of therapeutic peptides

• US 8,911,967 B2
• Filed: 05/20/2010
• Issued: 12/16/2014
• Est. Priority Date: 08/19/2005
• Status: Active Grant

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: DeFrees, Shawn, Zopf, David A., Taudte, Susann, Willett, Walter Scott, Bayer, Robert J., Kalo, Matthew
• Primary Examiner(s): Lukton, David
• Application Number:
  US12/784,323
• Publication Number:
  US 20100330645A1
• Time in Patent Office: 1,671 Days
• Field of Search: 435/68.1, 530/322, 530/345
• US Class Current: 435/68.1
• CPC Class Codes: A61K 38/4846 Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

The present invention provides conjugates between peptides and PEG moieties. The conjugates are linked via an intact glycosyl linking group that is interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from both glycosylated and unglycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto either an amino acid or glycosyl residue on the peptide. Also provided are pharmaceutical formulations including the conjugates. Methods for preparing the conjugates are also within the scope of the invention.

Claims

1. A method of synthesizing a peptide conjugate comprising combining:

(a) sialidase;

(b) an enzyme capable of catalyzing the transfer of a glycosyl linking group;

(c) a modified sugar; and

(d) a peptide,

wherein the method comprises

(i) a desialylation step in which the sialidase removes a sialic acid residue from the peptide, thereby forming a desialyated peptide, and

(ii) a conjugation step in which the enzyme capable of catalyzing the transfer of a glycosyl linking group transfers a modified sugar moiety from the modified sugar to an amino acid or glycosyl residue of the peptide,

which desialylation and conjugation steps are performed in the same vessel, thereby synthesizing the peptide conjugate.

2. The method of claim 1, wherein a covalent bond is formed between the modified sugar and the peptide.

3. The method of claim 1, wherein the desialyated peptide is not purified prior to the conjugation step.

4. The method of claim 3, wherein the enzyme capable of catalyzing the transfer of a glycosyl linking group is a glycosyltransferase.

5. The method of claim 1, wherein the peptide is in contact with components (a)-(c) simultaneously.

6. The method of claim 2, wherein the peptide is in contact with components (a)-(c) simultaneously.

7. The method of claim 3, wherein the peptide is in contact with components (a)-(c) simultaneously.

8. The method of claim 4, wherein the peptide is in contact with components (a)-(c) simultaneously.

9. The method of claim 1, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.

10. The method of claim 2, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.

11. The method of claim 3, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.

12. The method of claim 4, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.

13. The method of claim 1, wherein the enzyme capable of catalyzing the transfer of a glycosyl linking group is selected from the group consisting of glycosyltransferase, exoglycosidase, and endogylcosidase.

14. The method of claim 13, wherein the combining of components (a)-(d) is for a time less than ten hours.

15. The method of claim 1, wherein the modified sugar comprises a sugar moiety, and the sugar moiety is a nucleotide sugar.

16. The method of claim 15, wherein the nucleotide sugar comprises poly(ethylene glycol).

17. The method of claim 16, wherein at least 50% of the peptide conjugate includes at least two poly(ethylene glycol) moieties.

18. The method of claim 16, wherein at least 50% of the peptide conjugate includes at most one poly(ethylene glycol) moiety.

19. The method of claim 15, wherein the nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, and CMP-NeuAc.

20. The method of claim 19, wherein the nucleotide sugar comprises poly(ethylene glycol).

21. The method of claim 20, wherein at least 50% of the peptide conjugate includes at least two poly(ethylene glycol) moieties.

22. The method of claim 20, wherein at least 50% of the peptide conjugate includes at most one poly(ethylene glycol) moiety.

23. The method of claim 1, wherein the modified sugar comprises a sugar moiety, and the sugar moiety comprises poly(ethylene glycol).

24. The method of claim 23, wherein at least 50% of the peptide conjugate includes at least two poly(ethylene glycol) moieties.

25. The method of claim 23, wherein at least 50% of the peptide conjugate includes at most one poly(ethylene glycol) moiety.

26. The method of claim 1, wherein the ratio of the sialidase to the peptide is 0.1 U/L:

2 mg/mL to 10 U/L;

1 mg/mL.

27. The method of claim 1, further comprising a capping step involving sialylation of the peptide conjugate.

28. The method of claim 27, wherein the capping step is performed in the same reaction vessel in which the desialylation and conjugation steps are performed.

29. The method of claim 28, wherein the enzyme capable of catalyzing the transfer of a glycosyl linking group is a sialyltransferase.