Mono- and di-PEG IL-10 production; and uses

• US 9,259,478 B2
• Filed: 02/18/2014
• Issued: 02/16/2016
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Inventors: Blaisdell, Steven J., Cutler, Collette M., Paporello, Brittany C., Ambrogelly, Alexandre
• Primary Examiner(s): Saoud, Christine J
• Assistant Examiner(s): Seharaseyon, Jegatheesan
• Application Number:
  US14/183,302
• Publication Number:
  US 20150086505A1
• Time in Patent Office: 728 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 31/506 not condensed and containing further heterocyclic rings
• CPC Class Codes: A61K 31/513 having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• CPC Class Codes: A61K 33/243 Platinum; Compounds thereof
• CPC Class Codes: A61K 38/2066 IL-10
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 35/00 Antineoplastic agents

Abstract

Provided are methods of producing mono- and di-pegylated IL-10.

Claims

1. A pharmaceutical composition comprising:

a mixture of mono-pegylated interleukin-10 (IL-10) and di-pegylated IL-10, wherein at least one polyethylene glycol (PEG) molecule is covalently attached via a linker to a single amino acid residue of one subunit of the mono-pegylated IL-10 and wherein at least one PEG molecule is covalently attached via a linker to a single amino acid residue of each subunit of the di-pegylated IL-10; and

a pharmaceutically acceptable carrier;

wherein the mixture is produced by a method comprising;

reacting IL-10 with an activated PEG-linker in the presence of a reducing agent, wherein the IL-10 and the reducing agent are present in a molar ratio of 0.0009 to 0.04 to produce the mixture of mono-pegylated IL-10 and di-pegylated IL-10.

2. The pharmaceutical composition of claim 1, wherein the molar ratio of IL-10 to reducing agent is 0.0009-0.01.

3. The pharmaceutical composition of claim 1, wherein the molar ratio of IL-10 to reducing agent is 0.006-0.04.

4. The pharmaceutical composition of claim 1, wherein the molar ratio of IL-10 to reducing agent is 0.006-0.01.

5. The pharmaceutical composition of claim 1, wherein the molar ratio of IL-10 to reducing agent is 0.008.

6. The pharmaceutical composition of claim 1, wherein the di-pegylated IL-10 is 50% of pegylated IL-10 in the mixture.

7. The pharmaceutical composition of claim 1, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

8. The pharmaceutical composition of claim 1, wherein the at least one PEG molecule of the mono-pegylated IL-10 is attached via a linker to the alpha amino group of the N-terminal amino acid of the IL-10 subunit.

9. The pharmaceutical composition of claim 8, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

10. The pharmaceutical composition of claim 1, wherein the at least one PEG molecules of the di-pegylated IL-10 are each attached via a linker to the alpha amino groups of the N-terminal amino acid of each of the IL-10 subunits.

11. The pharmaceutical composition of claim 10, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

12. The pharmaceutical composition of claim 1, wherein the PEG is succinimidylcarbonate-PEG, PEG-butyraldehyde, PEG-pentaldehyde, PEG-amido-propionaldehyde, PEG-urethano-propioaldehyde, or PEG-propylaldehyde.

13. The pharmaceutical composition of claim 12, wherein the PEG-linker is PEG-propylaldehyde.

14. The pharmaceutical composition of claim 12, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

15. The pharmaceutical composition of claim 13, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

16. The pharmaceutical composition of claim 1, wherein the average molecular weight of the PEG is from 5,000 daltons to 50,000 daltons.

17. The pharmaceutical composition of claim 16, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

18. The pharmaceutical composition of claim 16, wherein the average molecular weight of the PEG is from 5,000 daltons to 20,000 daltons.

19. The pharmaceutical composition of claim 18, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

20. The pharmaceutical composition of claim 1, wherein the IL-10 is human IL-10.

21. The pharmaceutical composition of claim 20, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

22. The pharmaceutical composition of claim 1, wherein the ratio of IL-10 to PEG linker is 1:

1-1;

7.7.

23. The pharmaceutical composition of claim 22, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

24. The pharmaceutical composition of claim 1, wherein the ratio of IL-10 to PEG linker is 1:

3.5.

25. The pharmaceutical composition of claim 24, wherein the ratio of mono-pegylated IL-10 to di-pegylated IL-10 in the mixture is 1:

1.

26. The pharmaceutical composition of claim 1, wherein the reacting is at a pH of 5.0-7.4.

27. The pharmaceutical composition of claim 26, wherein the pH is 6.3.

28. The pharmaceutical composition of claim 1, wherein the reacting is at a temperature of 5°

C. to 30°

C.

29. The pharmaceutical composition of claim 28, wherein the temperature is 15°

C.

30. The pharmaceutical composition of claim 1, wherein the reacting is for 3-24 hours.

31. The pharmaceutical composition of claim 30, wherein the reacting is for 12-19 hours.

32. The pharmaceutical composition of claim 30, wherein the reacting is for 12-18 hours.

33. The pharmaceutical composition of claim 30, wherein the reacting is for 15-19 hours.

34. The pharmaceutical composition of claim 33, wherein the reacting is for 15-18 hours.

35. The pharmaceutical composition of claim 1, wherein the reducing agent is selected from the group consisting of borohydride, sodium cyanoborohydride, amine borane, and picoline borane.

36. The pharmaceutical composition of claim 35, wherein reducing agent is selected from the group consisting of sodium cyanoborohydride and picoline borane.

37. The pharmaceutical composition of claim 1, wherein the mixture is produced by a method further comprising purifying the mixture of mono- and di-pegylated IL-10.

38. The pharmaceutical composition of claim 37, wherein the mixture of mono- and di-pegylated IL-10 is purified by chromatography selected from the group consisting of cation exchange, anion exchange, size exclusion, and hydrophobic interaction.

39. The pharmaceutical composition of claim 38, wherein the mixture of mono- and di-pegylated IL-10 is purified by size exclusion chromatography.