Pegylated interleukin-10

1. A mono-pegylated interleukin-10 (mono-PEG-IL-10) comprising one or more polyethylene glycol (PEG) molecules covalently attached via a linker to a single amino acid residue of a single subunit of IL-10, wherein said amino acid residue is the alpha amino group of the N-terminal amino acid residue or the epsilon amino group of a lysine residue.

2. The mono-PEG-IL-10 of claim 1 , wherein one or two PEG molecules are attached via said linker to said single amino acid residue.

3. The mono-PEG-IL-10 of claim 1 , wherein said subunit of said IL-10 has the formula:

(PEG) b -L-NH-IL-10

wherein b is 1–9 and L is a C 2-12 alkyl linker moiety covalently attached to a nitrogen (N) of said single amino acid residue.

4. The mono-PEG-IL-10 of claim 3 , wherein b is 1 and L is —CH 2 CH 2 CH 2 —.

5. The mono-PEG-IL-10 of claim 1 , wherein said PEG molecule is covalently attached via said linker to the nitrogen of the alpha amino group of the N-terminal amino acid residue.

6. The mono-PEG-IL-10 of claim 1 , wherein said subunit of said IL-10 has the formula:

[X—O(CH 2 CH 2 O) n ] b -L-NH-IL-10,

wherein X is H or C 1-4 alkyl, n is 20 to 2300, b is 1 to 9 and L is a C 1-11 alkyl linker moiety which is covalently attached to the nitrogen (N) of the alpha amino group at the amino terminus of one IL-10 subunit; provided that when b is greater than 1, the total of n does not exceed 2300.

7. The mono-PEG-IL-10 of claim 6 , wherein L is —CH 2 CH 2 CH 2 —.

8. The mono-PEG-IL-10 according to claim 1 , wherein said mono-PEG-IL-10 has greater than 30% of the activity of unconjugated IL-10.

9. A composition of pegylated IL-10 comprising the mono-PEG-IL-10 according to claim 1 , wherein the population of mono-PEG-IL-10 is at least 80% of a positional isomer in which the PEG is conjugated to the N-terminal amino acid of one subunit of IL-10.

10. A process for preparing a pharmaceutical composition comprising the no-PEG-IL-10 according to claim 1 , comprising mixing the mono-PEG-IL-10 with a pharmaceutically acceptable carrier.

11. The mono-PEG-IL-10 according to claim 1 , wherein the IL-10 is human IL-10.

12. A pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 11 and a pharmaceutical carrier.

13. The mono-PEG-IL-10 according to claim 11 , wherein the PEG molecule has a molecular weight of 12,000 or 20,000 daltons.

14. A pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 13 and a pharmaceutical carrier.

15. A pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 1 in combination with a pharmaceutically acceptable carrier.

16. A method of treating inflammation in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 15 .

17. A process, for preparing the mono-PEG-IL-10 according to claim 1 , comprising the step of:

reacting IL-10 with an activated PEG-aldehyde linker in the presence of a reducing agent to form the mono-PEG-IL-10 under conditions in which the linker is covalently attached to one amino acid residue of the IL-10.

18. The process according to claim 17 wherein:

(a) the reducing agent is sodium cyanoborohydride;

(b) the activated PEG-aldehyde linker is PEG-propionaidehyde;

(c) the PEG is a methoxy-PEG;

(d) the linker is multi-armed;

(e) the ratio of IL-10 to the sodium cyanoborohydride is from about 1:0.5 to 1:50;

(f) the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 3,000 daltons to 60,000 daltons; or

(g) the reacting step is performed at a pH of 5.6 to 7.8.

19. The process according to claim 17 , wherein the ratio of IL-10 to the sodium cyanoborohydride is 1:5 to 1:15.

20. The process according to claim 17 , wherein the total molecular mess of all PEG comprising the PEG-aldehyde linker is from 10,000 daltons to 36,000 daltons.

21. The process according to claim 17 , wherein the reacting step is performed at a pH of 6.3 to 7.5.

22. The process according to claim 17 , further comprising a step selected from:

incubating the mono-PEG-IL-10 product in a buffer at pH 5.0 to 9.0; or

treating the mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.