Mono- and di-PEG IL-10 production; and uses
Provided are methods of producing mono- and di-pegylated IL-10.
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.