Mono- and di-peg IL-10 production; and uses
Provided are methods of producing 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 in the presence of 25 mM to 35 mM of a reducing agent, 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; and
b) purifying the mixture of mono- and di-pegylated IL-10.
2. The method of claim 1 , wherein the PEG-linker is PEG-propylaldehyde.
3. The method of claim 1 , wherein the molecular mass of PEG comprising the PEG-linker is from 5,000 daltons to 20,000 daltons.
4. The method of claim 1 , wherein the molecular mass of PEG comprising the PEG linker is 5,000 daltons.
5. The method of claim 1 , wherein the reducing agent is selected from the group consisting of borohydride, sodium cyanoborohydride, amine borane, and picoline borane.
6. The method of claim 5 , wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride and picoline borane.
7. 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.
8. The method of claim 7 , wherein the mixture of mono- and di-PEG is purified by size exclusion chromatography.
9. The method of claim 1 , wherein the ratio of IL-10 to PEG linker is 1:1-1:7.7.
10. The method of claim 9 , wherein the ratio is 1:3.5.
11. The method of claim 1 , wherein the reacting is at a pH of about 5.0-7.4.
12. The method of claim 1 , wherein the reacting is at a temperature of 5° C. to 30° C.
13. The method of claim 1 , wherein the reacting is for 3-24 hours.
14. The method of claim 13 , wherein the reacting is for 15-18 hours.
15. The method of claim 1 , wherein the method produces a 1:1 ratio of mono-pegylated IL-10 to di-pegylated IL-10.
16. The method of claim 1 , wherein the reacting is in the presence of 25 mM of a reducing agent.