Mono- and di-PEG IL-10 production; and uses
Provided are methods of producing a mixture of mono- and di-pegylated IL-10.
1. A method of producing a mixture of mono- and di-pegylated interleukin-10 (IL-10), wherein at least one PEG molecule is covalently attached to at least one amino acid residue of at least one subunit of IL-10, comprising:
a) reacting 1 mg/ml to 12 mg/ml of IL-10 with an activated PEG-linker such that the IL-10 to PEG-linker ratio is 1:3.5, in the presence of 25 mM to 35 mM of a reducing agent; and
b) purifying the mixture of mono- and di-pegylated IL-10.
2. The method of claim 1 , wherein the PEG-linker is selected from the group consisting of succinimidylcarbonate-PEG, PEG-butyraldehyde, PEG-pentaldehyde, PEG-amido-propionaldehyde, PEG-urethano-propioaldehyde, and PEG-propylaldehyde.
3. The method of claim 2 , wherein the PEG-linker is PEG-propylaldehyde.
4. The method of claim 1 , wherein the molecular mass of PEG comprising the PEG-linker is from 5,000 daltons to 20,000 daltons.
5. The method of claim 4 , wherein the molecular mass of PEG comprising the PEG linker is 5,000 daltons.
6. The method of claim 1 , wherein the reducing agent is selected from the group consisting of borohydride, sodium cyanoborohydride, amine borane, and picoline borane.
7. The method of claim 6 , wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride and picoline borane.
8. The method of claim 1 , wherein the mixture of mono- and di-PEG is purified by chromatography selected from the group consisting of cation exchange, anion exchange, size exclusion, and hydrophobic interaction.
9. The method of claim 8 , wherein the mixture of mono- and di-PEG is purified by size exclusion chromatography.
10. The method of claim 1 , wherein the reacting is at a pH of about 5.0-7.4.
11. The method of claim 10 , wherein the pH is 6.3.
12. The method of claim 1 , wherein the reacting is at a temperature of 5° C. to 30° C.
13. The method of claim 12 , wherein the temperature is 15° C.
14. The method of claim 1 , wherein the reacting is for 3-24 hours.
15. The method of claim 14 , wherein the reacting is for 12-19 hours.
16. The method of claim 14 , wherein the reacting is for 12-18 hours.
17. The method of claim 14 , wherein the reacting is for 15-19 hours.
18. The method of claim 14 , wherein the reacting is for 15-18 hours.
19. The method of claim 1 , wherein the reacting is in the presence of 25 mM of a reducing agent.
20. A method of producing a mixture of mono- and di-pegylated interleukin-10 (IL-10), wherein at least one PEG molecule is covalently attached to at least one amino acid residue of at least one subunit of IL-10, comprising:
a) reacting 1 mg/ml to 12 mg/ml of IL-10 with an activated PEG-linker in the presence of 25 mM to 35 mM of a reducing agent; and
b) purifying the mixture of mono- and di-pegylated IL-10.
21. The method of claim 20 , wherein the ratio of IL-10 to PEG linker is 1:1-1:7.7.
22. The method of claim 21 , wherein the ratio is 1:3.5.
23. The method of claim 20 , wherein the reacting is at a pH of about 5.0-7.4.
24. The method of claim 23 , wherein the pH is 6.3.
25. The method of claim 20 , wherein the reacting is at a temperature of 5° C. to 30° C.
26. The method of claim 25 , wherein the temperature is 15° C.
27. The method of claim 20 , wherein the reacting is for 3-24 hours.
28. The method of claim 27 , wherein the reacting is for 12-19 hours.
29. The method of claim 27 , wherein the reacting is for 12-18 hours.
30. The method of claim 27 , wherein the reacting is for 15-19 hours.
31. The method of claim 27 , wherein the reacting is for 15-18 hours.
32. The method of claim 20 , wherein the PEG-linker is PEG-propylaldehyde.
33. The method of claim 20 , wherein the molecular mass of PEG comprising the PEG-linker is from 5,000 daltons to 20,000 daltons.
34. The method of claim 33 , wherein the molecular mass of PEG comprising the PEG linker is 5,000 daltons.
35. The method of claim 20 , wherein the reducing agent is selected from the group consisting of borohydride, sodium cyanoborohydride, amine borane, and picoline borane.
36. The method of claim 35 , wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride and picoline borane.
37. The method of claim 20 , wherein the mixture of mono- and di-PEG is purified by chromatography selected from the group consisting of cation exchange, anion exchange, size exclusion, and hydrophobic interaction.
38. The method of claim 37 , wherein the mixture of mono- and di-PEG is purified by size exclusion chromatography.
39. The method of claim 20 , wherein the method produces a 1:1 ratio of mono-pegylated IL-10 to di-pegylated IL-10.
40. The method of claim 20 , wherein the reacting is in the presence of 25 mM of a reducing agent.
41. A method of producing a mixture of mono- and di-pegylated interleukin-10 (IL-10), comprising:
a) 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; and
b) purifying the mixture of mono- and di-pegylated IL-10.
42. The method of claim 41 , wherein the ratio of IL-10 to PEG linker is 1:1-1:7.7.
43. The method of claim 42 , wherein the ratio of IL-10 to PEG linker is 1:3.5.
44. The method of claim 41 , wherein the reacting is at a pH of about 5.0-7.4.
45. The method of claim 44 , wherein the pH is 6.3.
46. The method of claim 41 , wherein the reacting is at a temperature of 5° C. to 30° C.
47. The method of claim 46 , wherein the temperature is 15° C.
48. The method of claim 41 , wherein the reacting is for 3-24 hours.
49. The method of claim 48 , wherein the reacting is for 12-19 hours.
50. The method of claim 48 , wherein the reacting is for 12-18 hours.
51. The method of claim 48 , wherein the reacting is for 15-19 hours.
52. The method of claim 48 , wherein the reacting is for 15-18 hours.
53. The method of claim 41 , wherein the PEG-linker is PEG-propylaldehyde.
54. The method of claim 41 , wherein the molecular mass of PEG comprising the PEG-linker is from 5,000 daltons to 20,000 daltons.
55. The method of claim 54 , wherein the molecular mass of PEG comprising the PEG linker is 5,000 daltons.
56. The method of claim 41 , wherein the reducing agent is selected from the group consisting of borohydride, sodium cyanoborohydride, amine borane, and picoline borane.
57. The method of claim 56 , wherein reducing agent is selected from the group consisting of sodium cyanoborohydride and picoline borane.
58. The method of claim 41 , 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.
59. The method of claim 58 , wherein the mixture of mono- and di-pegylated IL-10 is purified by size exclusion chromatography.
60. The method of claim 41 , wherein the method produces a 1:1 ratio of mono-pegylated IL-10 to di-pegylated IL-10.
61. The method of claim 41 , wherein the molar ratio of IL-10 to reducing agent is 0.0009-0.01.
62. The method of claim 61 , wherein the reacting is at a pH of about 5.0-7.4.
63. The method of claim 62 , wherein the pH is 6.3.
64. The method of claim 61 , wherein the reacting is at a temperature of 5° C. to 30° C.
65. The method of claim 64 , wherein the temperature is 15° C.
66. The method of claim 61 , wherein the reacting is for 3-24 hours.
67. The method of claim 66 , wherein the reacting is for 12-19 hours.
68. The method of claim 66 , wherein the reacting is for 12-18 hours.
69. The method of claim 66 , wherein the reacting is for 15-19 hours.
70. The method of claim 66 , wherein the reacting is for 15-18 hours.
71. The method of claim 41 , wherein the molar ratio of IL-10 to reducing agent is 0.006-0.04.
72. The method of claim 41 , wherein the molar ratio of IL-10 to reducing agent is 0.006-0.01.
73. The method of claim 41 , wherein the molar ratio of IL-10 to reducing agent is 0.008.