Monopegylated growth hormone proteins
The present invention relates to novel methods of making soluble proteins having free cysteines in which a host cell is exposed to a cysteine blocking agent. The soluble proteins produced by the methods can then be modified to increase their effectiveness. Such modifications include attaching a PEG moiety to form pegylated proteins.
1. An isolated, monoPEGylated growth hormone protein, wherein said monoPEGylated growth hormone protein comprises a polyethylene glycol attached to an amino acid selected from the group consisting of amino acid 2, 3, 5, 38, 40, 55, 57, 99, 101, 102, 103, 132, 133, 134, 137, 140, 141, 143, 144, 147, 148, 149, 186, and 187 of growth hormone, wherein said monoPEGylated growth hormone protein stimulates proliferation in vitro of a cell line that proliferates in response to growth hormone.
2. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said monoPEGylated growth hormone protein has an EC 50 of less than about 400 ng/ml.
3. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said monoPEGylated growth hormone protein has an EC 50 of less than about 110 ng/ml.
4. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said monoPEGylated growth hormone protein has an EC 50 of less than about 50 ng/ml.
5. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said monoPEGylated growth hormone protein has an EC 50 of less than about 11 ng/ml.
6. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said monoPEGylated growth hormone protein contains less than 5% unPEGylated growth hormone protein.
7. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said polyethylene glycol has a molecular mass of between 20 kDA and 40 kDA.
8. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said polyethylene glycol is selected from the group consisting of a linear polyethylene glycol and a branched polyethylene glycol.
9. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said polyethylene glycol is a cysteine-reactive polyethylene glycol.
10. The isolated, monoPEGylated growth hormone protein of claim 9 , wherein said polyethylene glycol is selected from the group consisting of a maleimide-polyethylene glycol, a vinylsulfone-polyethylene glycol, and an iodoacetyl-polyethylene glycol.
11. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein said monoPEGylated growth hormone protein comprises at least one added sulfur group attached to at least one amino acid in said growth hormone protein.
12. The isolated, monoPEGylated growth hormone protein of claim 11 , wherein said added sulfur group is attached to at least one lysine residue in said growth hormone protein.
13. The monoPEGylated growth hormone protein of claim 11 , wherein said monoPEGylated growth hormone protein comprises a polyethylene glycol attached to at least one added sulfur group in said growth hormone protein.
14. A mixture comprising two or more of the monoPEGylated growth hormone proteins of claim 1 .
15. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 133 of growth hormone.
16. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 2 of growth hormone.
17. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 3 of growth hormone.
18. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 5 of growth hormone.
19. A method for treating a condition treatable with growth hormone, comprising administering to a patient a composition comprising the monoPEGylated growth hormone protein of claim 1 .
20. The isolated, monoPEGylated growth hormone protein of claim 1 , obtained by:
a) exposing the growth hormone protein to a polyethylene glycol under conditions such that a polyethylene glycol attaches to at least one amino acid in said growth hormone protein; and
b) isolating monoPEGylated growth hormone protein from unPEGylated growth hormone protein by column chromatography.
21. The method of claim 20 , wherein said column chromatography in step (b) is selected from the group consisting of ion-exchange column chromatography and size-exclusion column chromatography.
22. The isolated, monoPEGylated growth hormone protein of claim 1 , wherein the monoPEGylated growth hormone protein has an apparent molecular weight of greater than about 70 kDA, as measured by size exclusion column chromatography.
23. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 38 of growth hormone.
24. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 40 of growth hormone.
25. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 55 of growth hormone.
26. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 57 of growth hormone.
27. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 99 of growth hormone.
28. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 101 of growth hormone.
29. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 102 of growth hormone.
30. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 103 of growth hormone.
31. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 132 of growth hormone.
32. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 134 of growth hormone.
33. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 137 of growth hormone.
34. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 140 of growth hormone.
35. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 141 of growth hormone.
36. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 143 of growth hormone.
37. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 144 of growth hormone.
38. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 147 of growth hormone.
39. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 148 of growth hormone.
40. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 149 of growth hormone.
41. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 186 of growth hormone.
42. The isolated monoPEGylated growth hormone protein of claim 1 , wherein a polyethylene glycol is attached to amino acid 187 of growth hormone.