Double-stranded polyethylene glycol modified growth hormone, preparation method and application thereof

Abstract

The growth hormone with high biological activity modified by the double-stranded polyethylene glycol at a single site and the preparation method thereof are provided. The PEGylated growth hormone has a higher biological activity and a longer half-life than the unmodified growth hormone. The composition comprising the PEGylated growth hormone is useful in the treatment of the growth or development disorder such as growth hormone deficiency, Turner syndrome etc.

Claims

1. A method for preparing a PEGylated growth hormone preparation, the method comprising: a) bringing U-shaped or Y-shaped branched double-stranded PEG into contact with a growth hormone in a solution with a pH not lower than 8.0 to produce a product modified by the double-stranded PEG at a single site; b) assaying the product modified by the double-stranded PEG at a single site obtained in step a) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of an appropriate concentration, wherein the product shows two bands; and c) separating and recovering the product modified by the double-stranded PEG at a single site; wherein the recovered product is a mixture predominantly containing the product of lower apparent molecular weight modified by the double-stranded PEG at a single site, wherein the SDS-PAGE content of the product of lower apparent molecular weight modified by the double-stranded PEG at a single site is not lower than 70%.

2. The method of claim 1, wherein the double-stranded PEG is Y-shaped branched PEG of the following structural formula (I), ##STR00021## wherein, P.sub.a and P.sub.b are same or different PEG; j is an integer from 1 to 12; R.sub.i is H, substituted or unsubstituted C.sub.1-12 alkyl, substituted aryl, aralkyl, or heteroalkyl; X.sub.1 and X.sub.2 are independently linking group, wherein X.sub.1 is (CH.sub.2).sub.n, X.sub.2 is selected from the group consisting of: (CH.sub.2)n, (CH.sub.2).sub.nOCO, (CH.sub.2).sub.nNHCO, (CH.sub.2).sub.nCO, wherein n is an integer from 1 to 10; F is a terminal group selected from the group consisting of: hydroxyl, carboxyl, ester group, acyl chloride, hydrazide, maleimide, pyridine disulfide, capable of reacting with an amino, hydroxyl or hydrosulfide group of a therapeutic agent or substrate to form a covalent bond.

3. The method of claim 2, wherein the Y-shaped branched PEG is of the following structural formula (II): ##STR00022## wherein R and R′ are independently low molecular weight alkyl and m and m′ denote the degree of polymerization and can be any integer.

4. The method of claim 3, wherein the Y-shaped branched PEG is of the following structural formula (III): ##STR00023##

5. The method of claim 1, wherein the double stranded PEG is U-shaped branched PEG of the following structural formula (IV), ##STR00024## wherein, R and R′ are independently low molecular weight alkyl; n and n′ denote the degree of polymerization and can be any integer; n+n′ is from 600 to 1500; the average molecular weight of the U-shaped branched PEG is about from 26 kiloDaltons (kDa) to 66 kDa.

6. A method for preparing a PEGylated growth hormone preparation, comprising the following steps: a) in a solution having a pH of 9.0 or 10.5, bringing a double-stranded PEG of the following structural formula (III) into contact with human growth hormone, wherein the molar ratio of the growth hormone to the double stranded PEG is about 1:2; ##STR00025##  wherein m+m′ is 910, the average total molecular weight of the PEG is about 40 kDa; b) assaying the product modified by the double-stranded PEG at a single site obtained in step a) in 12% SDS-PAGE; c) separating and recovering the product modified by the double-stranded PEG at a single site using gel chromatography selected from Q Sepharose FF chromatography, DEAE Sepharose FF chromatography or MacroCap SP chromatography, wherein the SDS-PAGE content of the product of lower apparent molecular weight in the recovered product modified by the double-stranded PEG at a single site is not lower than 70%.

7. A PEGylated growth hormone preparation prepared according to the method of claim 1, wherein the growth hormone is extracted from a natural source or obtained by recombinant biotechnology, and further wherein at least 70% of the PEGylated growth hormone preparation comprises a growth hormone polypeptide modified with a double-stranded PEG moiety linked to an ε-NH.sub.2 group of a single Lys residue present within the growth hormone polypeptide.

8. The PEGylated growth hormone preparation of claim 7, wherein the product modified by the PEG at a single site is shown as the following formula (VII): ##STR00026## wherein, R and R′ are independently low molecular weight alkyl, m+m′ is 910; and j is an integer from 1 to 12.

9. A method for treating a patient with a disease in need of growth hormone treatment comprising administering a therapeutically effective amount of the PEGylated growth hormone preparation of claim 7 to the patient.

10. The PEGylated growth hormone preparation of claim 7, wherein the recombinant growth hormone is artificially synthesized or expressed by an expression system selected from the group consisting of a prokaryotic system; a eukaryotic system; an insect cell system; and a mammalian cell system.

11. The PEGylated growth hormone preparation of claim 8, wherein the recombinant growth hormone is artificially synthesized or expressed by an expression system selected from the group consisting of a prokaryotic system; a eukaryotic system; an insect cell system; and a mammalian cell system.

12. A composition comprising a pharmaceutically effective amount of the PEGylated growth hormone preparation of claim 7 and a pharmaceutically acceptable carrier or excipient.

13. A composition comprising a pharmaceutically effective amount of the PEGylated growth hormone preparation of claim 8 and a pharmaceutically acceptable carrier or excipient.

14. A method for treating a patient with a disease in need of growth hormone treatment comprising administering a therapeutically effective amount of the PEGylated growth hormone preparation of claim 8 to the patient.

15. A composition comprising a pharmaceutically effective amount of the PEGylated growth hormone preparation of claim 10 and a pharmaceutically acceptable carrier or excipient.

16. A composition comprising a pharmaceutically effective amount of the PEGylated growth hormone preparation of claim 11 and a pharmaceutically acceptable carrier or excipient.

17. A method for treating a patient with a disease in need of growth hormone treatment comprising administering a therapeutically effective amount of the composition of claim 12 to the patient.

18. A method for treating a patient with a disease in need of growth hormone treatment comprising administering a therapeutically effective amount of the composition of claim 13 to the patient.

19. A method for treating a patient with a disease in need of growth hormone treatment comprising administering a therapeutically effective amount of the composition of claim 15 to the patient.

20. A method for treating a patient with a disease in need of growth hormone treatment comprising administering a therapeutically effective amount of the composition of claim 16 to the patient.

21. The PEGylated growth hormone preparation of claim 7, wherein the growth hormone comprises an amino acid sequence comprising SEQ ID NO: 1.

22. The PEGylated growth hormone preparation of claim 8, wherein R and R′ are independently C.sub.1-C.sub.4 alkyl.

23. The PEGylated growth hormone preparation of claim 8, wherein R or R′ is methyl.

24. The PEGylated growth hormone preparation of claim 7, wherein at least 80% of the PEGylated growth hormone preparation comprises a growth hormone polypeptide modified with a double-stranded PEG moiety linked to an ε-NH.sub.2 group of a single Lys residue present within the growth hormone polypeptide.

25. The PEGylated growth hormone preparation of claim 7, wherein at least 90% of the PEGylated growth hormone preparation comprises a growth hormone polypeptide modified with a double-stranded PEG moiety linked to an ε-NH.sub.2 group of a single Lys residue present within the growth hormone polypeptide.

26. The PEGylated growth hormone preparation of claim 10, wherein the prokaryotic system is E. coli.

27. The PEGylated growth hormone preparation of claim 10, wherein the eukaryotic system is Pichia.

28. The PEGylated growth hormone preparation of claim 10, wherein the mammalian cell system is CHO cell.

29. The PEGylated growth hormone preparation of claim 11, wherein the prokaryotic system is E. coli.

30. The PEGylated growth hormone preparation of claim 11, wherein the eukaryotic system is Pichia.

31. The PEGylated growth hormone preparation of claim 11, wherein the mammalian cell system is CHO cell.

32. The composition of claim 12, wherein the pharmaceutically acceptable carrier or excipient comprises one or more of mannitol, an amino acid, sodium chloride, acetic acid, and sodium acetate.

33. The composition of claim 13, wherein the pharmaceutically acceptable carrier or excipient comprises one or more of mannitol, an amino acid, sodium chloride, acetic acid, and sodium acetate.

34. The composition of claim 15, wherein the pharmaceutically acceptable carrier or excipient comprises one or more of mannitol, an amino acid, sodium chloride, acetic acid, and sodium acetate.

35. The composition of claim 16, wherein the pharmaceutically acceptable carrier or excipient comprises one or more of mannitol, an amino acid, sodium chloride, acetic acid and sodium acetate.

36. The method of claim 1, wherein the solution has a pH of not lower than 9.0.

37. The method of claim 36, wherein the solution has a pH of not lower than 9.5.

38. The method of claim 37, wherein the solution has a pH of not lower than 10.0.

39. The method of claim 38, wherein the solution has a pH of not lower than 10.5.

40. The method of claim 1, wherein the growth hormone is human growth hormone.

41. The method of claim 1, wherein the growth hormone is present in a ratio of 1:2 with respect to the double stranded PEG.

42. The method of claim 1, wherein the SDS-PAGE content of the product of lower apparent molecular weight modified by the double-stranded PEG at a single site is not lower than 80%.

43. The method of claim 42, wherein the SDS-PAGE content of the product of lower apparent molecular weight modified by the double-stranded PEG at a single site is not lower than 90%.

44. The method of claim 1, further comprising a purification step subsequent to the separating and recovering step.

45. The method of claim 44, wherein the purification step employs gel chromatography.

46. The method of claim 3, wherein R and R′ are independently C.sub.1-C.sub.4 alkyl.

47. The method of claim 46, wherein R and R′ are independently methyl.

48. The method of claim 3, wherein m+m′ is from 600 to 1500.

49. The method of claim 48, wherein m+m′ is 910.

50. The method of claim 4, wherein the Y-shaped branched PEG has an average molecule weight of about 26 kilodaltons (kDa) to 60 kDa.

51. The method of claim 50, wherein the Y-shaped branched PEG has an average molecule weight of about 40 kDa.

52. The method of claim 4, wherein the Y-shaped branched PEG is of equal-arm.

53. The method of claim 5, wherein R and R′ are independently C.sub.1-C.sub.4 alkyl.

54. The method of claim 5, wherein n+n′ is 910.

55. The method of claim 5, wherein the U-shaped branched PEG has an average molecule weight of about 40 kDa.

56. The method of claim 5, wherein the U-shaped branched PEG is of equal-arm.

57. The method of claim 6, wherein the SDS-PAGE content of the product of lower apparent molecular weight in the recovered product modified by the double-stranded PEG at a single site is not lower than 80%.

58. The method of claim 57, wherein the SDS-PAGE content of the product of lower apparent molecular weight in the recovered product modified by the double-stranded PEG at a single site is not lower than 90%.

59. The method of claim 9, wherein the disease in need of growth hormone treatment is selected from the group consisting of dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, acquired immunodeficiency syndrome (AIDS), endogenous growth hormone deficiency dwarfism, Turner syndrome, anabolic disorder, and adult growth hormone deficiency.

60. The method of claim 14, wherein the disease in need of growth hormone treatment is selected from the group consisting of dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, acquired immunodeficiency syndrome (AIDS), endogenous growth hormone deficiency dwarfism, Turner syndrome, anabolic disorder, and adult growth hormone deficiency.

61. The method of claim 17, wherein the disease in need of growth hormone treatment is selected from the group consisting of dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, acquired immunodeficiency syndrome (AIDS), endogenous growth hormone deficiency dwarfism, Turner syndrome, anabolic disorder, and adult growth hormone deficiency.

62. The method of claim 37, wherein the disease in need of growth hormone treatment is selected from the group consisting of dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, acquired immunodeficiency syndrome (AIDS), endogenous growth hormone deficiency dwarfism, Turner syndrome, anabolic disorder, and adult growth hormone deficiency.

63. The method of claim 19, wherein the disease in need of growth hormone treatment is selected from the group consisting of dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, acquired immunodeficiency syndrome (AIDS), endogenous growth hormone deficiency dwarfism, Turner syndrome, anabolic disorder, and adult growth hormone deficiency.

64. The method of claim 20, wherein the disease in need of growth hormone treatment is selected from the group consisting of dwarfism, burn, wound, bone fracture, bleeding ulcer, renal failure, acquired immunodeficiency syndrome (AIDS), endogenous growth hormone deficiency dwarfism, Turner syndrome, anabolic disorder, and adult growth hormone deficiency.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: The results of non-reductive SDS-PAGE of the rHuGH samples modified by YPEG-NHS 40 kDa or UPEG-NHS 40 kDa at pH 8.0. The concentration of the separation gel is 12%, and silver staining is used for visualization. Lane 1: marker, LMW, GE Healthcare; Lane 2: rHuGH sample modified by YPEG-NHS at pH 8.0, loading amount 2 μg; Lane 3: rHuGH sample modified by YPEG-NHS at pH 8.0, loading amount 5 μg; Lane 4: rHuGH sample modified by UPEG-NHS at pH 8.0, loading amount 5 μg.

(2) FIG. 2: The results of non-reductive SDS-PAGE of the rHuGH samples modified by YPEG-NHS 40 kDa at different pHs. The concentration of the separation gel is 12%, and silver staining is used for visualization. Lane 1: rHuGH sample modified by YPEG-NHS at pH 6.0; Lane 2: rHuGH sample modified by YPEG-NHS at pH 7.0; Lane 3: rHuGH sample modified by YPEG-NHS at pH 8.0; Lane 4: rHuGH sample modified by YPEG-NHS at pH 9.0; Lane 5: rHuGH sample modified by YPEG-NHS at pH 9.5; Lane 6: rHuGH sample modified by YPEG-NHS at pH 10.0; Lane 7: rHuGH sample modified by YPEG-NHS at pH 10.5; Lane 8: marker, LMW, GE Healthcare. The loading amount of all samples is 5 μg.

(3) FIG. 3: The results of non-reductive SDS-PAGE of the rHuGH samples modified by UPEG-NHS at different pHs. The concentration of the separation gel is 12%, and silver staining is used for visualization. Lane 1: rHuGH sample modified by UPEG-NHS at pH 6.0; Lane 2: rHuGH sample modified by UPEG-NHS at pH 7.0; Lane 3: rHuGH sample modified by UPEG-NHS at pH 8.0; Lane 4: rHuGH sample modified by UPEG-NHS at pH 9.0; Lane 5: rHuGH sample modified by UPEG-NHS at pH 9.5; Lane 6: rHuGH sample modified by UPEG-NHS at pH 10.0; Lane 7: rHuGH sample modified by UPEG-NHS at pH 10.5; Lane 8: marker, LMW, GE Healthcare. The loading amount of all samples is 5 μg.

(4) FIG. 4: The results of non-reductive SDS-PAGE of the purified rHuGH modification products modified by YPEG-NHS 40 kDa or UPEG-NHS 40 kDa at a single site at pH 6.0 or pH 10.5. The concentration of the separation gel is 12%, and silver staining is used for visualization. Lane 1: UPEG-rHuGH U10.5, loading amount 10 μg; Lane 2: UPEG-rHuGH U10.5, loading amount 2 μg; Lane 3: UPEG-rHuGH U6.0, loading amount 10 μg; Lane 4: UPEG-rHuGH U6.0, loading amount 2 μg; Lane 5: marker, LMW, GE Healthcare; Lane 6: YPEG-rHuGH Y10.5, loading amount 10 μg; Lane 7: YPEG-rHuGH Y10.5, loading amount 2 μg; Lane 8: YPEG-rHuGH Y6.0, loading amount 10 μg; Lane 9: YPEG-rHuGH Y6.0, loading amount 2 μg; Lane 10: rHuGH, loading amount 100 ng; Lane 11: rHuGH, loading amount 50 ng.

(5) FIG. 5: The assay results of the cellular activity of the purified rHuGH modification products modified by UPEG-NHS 40 kDa at a single site at pH 6.0 or pH 10.5, duplicate plates.

(6) FIG. 6: The assay results of the cellular activity of the purified rHuGH modification products modified by YPEG-NHS 40 kDa at a single site at pH 6.0 or pH 10.5, duplicate plates.

(7) FIG. 7: The molecular weights of the purified rHuGH modification products modified by YPEG-NHS 40 kDa or UPEG-NHS 40 kDa at a single site at pH 6.0, pH 9.0 or pH 10.5, detected by MALDI-TOF MS. a: YPEG-rHuGH, Y6; b: YPEG-rHuGH, Y9; c: YPEG-rHuGH, Y10.5; d: YPEG-rHuGH, Y6-1; e: UPEG-rHuGH, U6; f: UPEG-rHuGH, U9; g: UPEG-rHuGH, U10.5; h: UPEG-rHuGH, U6-1; i: YPEG-NHS, 40 kDa; j: UPEG-NHS, 40 kDa; k: Protein Calibrate Standard II, BRUKER; I: rHuGH; m: Protein Calibrate Standard I, BRUKER.

(8) FIG. 8: The results of non-reductive SDS-PAGE of the purified rHuGH modification product of higher apparent molecular weight modified by PEG-NHS 40 kDa at a single site at pH 6.0, and of the purified rHuGH modification product modified by PEG-NHS 40 kDa at a single site at pH 10.5. The concentration of the separation gel is 12%, and silver staining is used for visualization. Lane 1: YPEG-rHuGH Y10.5, loading amount 2 μg; Lane 2: YPEG-rHuGH Y6.0-1, loading amount 2 μg; Lane 3: UPEG-rHuGH U10.5, loading amount 2 μg; Lane 4: UPEG-rHuGH U6.0-1, loading amount 2 μg; Lane 5: rHuGH, loading amount 50 ng; Lane 6: rHuGH, loading amount 100 ng; Lane 7: marker, LMW, GE Healthcare; Lane 8: YPEG-rHuGH Y10.5, loading amount 10 μg; Lane 9: YPEG-rHuGH Y6.0-1, loading amount 10 μg; Lane 10: UPEG-rHuGH U10.5, loading amount 10 μg; Lane 11: UPEG-rHuGH U6.0-1, loading amount 10 μg.

(9) FIG. 9: The apparent molecular weights detected by non-reductive SDS-PAGE of the purified rHuGH modification products (Y6-1, U6-1) of higher apparent molecular weight modified by PEG-NHS 40 kDa at a single site at pH 6.0, and of the purified rHuGH modification products (Y10.5, U10.5) modified by PEG-NHS 40 kDa at a single site at pH 10.5. Lane 1: YPEG-rHuGH Y6-1+YPEG-rHuGH Y10.5, each 25 ng; Lane 2: YPEG-rHuGH Y6-1, 50 ng; Lane 3: YPEG-rHuGH Y10.5, 50 ng; Lane 4, 6: blank; Lane 5: marker, HMW, GE Healthcare; Lane 7: UPEG-rHuGH U6-1+UPEG-rHuGH U10.5, each 25 ng; Lane 8: UPEG-rHuGH U6-1, 50 ng; Lane 9: UPEG-rHuGH U10.5, 50 ng.

(10) FIG. 10: The curve of average serum drug concentration vs. time of single subcutaneous injection in crab-eating macaque of 300 μg.Math.kg.sup.−1 of rHuGH and YPEG-rHuGH (Y10.5) respectively.

CONCRETE EMBODIMENTS TO CARRY OUT THE INVENTION

(11) The present invention will be further described through the following examples, but any examples or combinations thereof should not be considered as limiting the scope and embodiments of this invention. The scope of this invention is only limited by the appended claims. Combining this description and prior art in the art, a person skilled in the art can clearly understand the scope limited by the claims.

Example 1

The Modification of the Recombinant Human GH by U-Shaped or Y-Shaped Branched PEG

(12) 200 mg of each of UPEG-NHS and YPEG-NHS (average M.W. 40 kDa, equal-arm; lot. Nos. ZZ004P182 and ZZ004P167, respectively) (Beijing JenKem Technology Co., Ltd.) were weighted and dissolved in 2 ml of 2 mM HCl (Guangdong Guanghua Chemical Factory Co., Ltd.) respectively. 50 mg rHuGH (Xiamen Amoytop Biotech Co., Ltd.) and 50 mM boric acid-borax buffer, pH 8.0 (Sinopharm Shanghai Chemical Reagent Co., Ltd.) were added respectively to a final total reaction volume of 10 ml. In the reaction system, the final reaction concentration of rHuGH was 5 mg/ml, and the reaction molar ratio of rHuGH to PEG-NHS was about 1:2. The incubation was done at <10° C. for 2h with shaking, and glacial acetic acid (Shantou Xilong Chemical Co., Ltd.) was added to make pH <4.0 to stop the reaction. A sample was taken for SDS-PAGE, and silver staining was used for visualization. The SDS-PAGE results are shown in FIG. 1. From the SDS-PAGE results in FIG. 1, the modification products at pH 8.0 show two main bands, and the samples modified by UPEG-NHS and YPEG-NHS show the same SDS-PAGE electrophoresis characteristics.

Example 2

The Modifications of the Recombinant Human GH by U-Shaped and Y-Shaped Branched PEG at Different pHs

(13) 200 mg of each of UPEG-NHS and YPEG-NHS (average M.W. 40 kDa, equal-arm; lot. Nos. ZZ004P182 and ZZ004P167 respectively) (Beijing JenKem Technology Co., Ltd.) were weighted and dissolved in 2 ml of 2 mM HCl (Guangdong Guanghua Chemical Factory Co., Ltd.) respectively. 50 mg rHuGH (Xiamen Amoytop Biotech Co., Ltd.) and the corresponding buffer were added respectively to a final total reaction volume of 10 ml. 10 mM PBNa buffer (Sinopharm Shanghai Chemical Reagent Co., Ltd.) of the corresponding pH for the reaction at pH 6.0, 7.0 or 8.0 was used, and 50 mM borax buffer (Sinopharm Shanghai Chemical Reagent Co., Ltd.) of the corresponding pH for the reaction at pH 9.0, 9.5, 10.0 or 10.5 was used. In the reaction system, the final reaction concentration of rHuGH was 5 mg/ml, and the reaction molar ratio of rHuGH to PEG-NHS was about 1:2. Incubation was done at <10° C. for 2h with shaking, and glacial acetic acid (Shantou Xilong Chemical Co., Ltd.) was added to make pH<4.0 to stop the reaction. A sample was taken for SDS-PAGE, and silver staining was used for visualization. The gel visualization system (Model No.: FR-200, Shanghai FURI Science & Technology Co., Ltd.) was used to analyze the electrophoresis results. SDS-PAGE electrophoresis results are shown in FIG. 2 and FIG. 3, and the analysis results by the gel visualization system are shown in table 1. From the electrophoresis results, the modified products at pH 6.0-9.5 show clearly two main bands, and as the reaction pH increases, the content of the band of lower apparent molecular weight also increases correspondingly. The modified products at pH 10.0 and 10.5 are substantially the band of lower apparent molecular weight. The samples modified UPEG-NHS and YPEG-NHS show the same SDS-PAGE electrophoresis characteristics.

(14) TABLE-US-00001 TABLE 1 The analysis of the gel visualization system on SDS- PAGE results of the recombinant human GHs modified by U-shaped and Y-shaped branched PEG at different pHs Modification reaction pH 6.0 7.0 8.0 9.0 9.5 10.0 10.5 YPEG Band 1 content (%) 53.9 48.6 27.7 25.7 6.9 5.6 7.9 Band 2 content (%) 46.1 51.4 72.3 74.3 93.1 94.4 92.1 UPEG Band 1 content (%) 47.5 41.8 30.1 27.4 24.4 18.9 7.5 Band 2 content (%) 52.5 58.2 69.9 72.6 75.6 81.1 92.5 Note: “content” refers to the relative percentage content of band 1 (higher apparent M.W.) to band 2 (lower apparent M.W.) of the rHuGH products modified by PEG at a single site.

Example 3

The Preparation, Cellular Activity and M.W. of the Recombinant Human GH Modified by U-Shaped or Y-Shaped Branched PEG at a Single Site at pH6.0, pH9.0 or 10.5

(15) 1 Modification

(16) Three samples of 1200 mg of each of UPEG-NHS and YPEG-NHS (average M.W. 40 kDa, equal-arm; lot. Nos. ZZ004P182 and ZZ004P167 respectively) (Beijing JenKem Technology Co., Ltd.) were weighted and dissolved in 12 ml of 2 mM HCl (Guangdong Guanghua Chemical Factory Co., Ltd.) respectively. 300 mg of rHuGH (Xiamen Amoytop Biotech Co., Ltd.) and 50 mM borax buffer (pH10.5) or 50 mM boric acid/borax buffer (pH9.0) or 10 mM PBNa (pH6.0) (Sinopharm Shanghai Chemical Reagent Co., Ltd.) were added respectively to a final total reaction volume of 60 ml. In the reaction system, the final reaction concentration of rHuGH was 5 mg/ml, the reaction molar ratio between the rHuGH and the PEG-NHS was about 1:2, and the reaction pHs were 10.5, 9.0 and 6.0 respectively. Incubation was done at <10° C. for 2h with shaking, and glacial acetic acid (Shantou Xilong Chemical Co., Ltd.) was added to make pH<4.0 to stop the reaction. A sample was taken for SDS-PAGE, and silver staining was used for visualization.

(17) 2 Purification

(18) 2.1 Q Sepharose FF Chromatography Purification

(19) The PEG modification sample of rHuGH was diluted 3 times using ultrapure water, and the pH of the diluted sample was adjusted to 9.0 with NaOH or HCl.

(20) 2.1.1 The Q Sepharose FF Chromatography Purification of the PEG Modification Samples (pH6.0) of rHuGH

(21) The chromatography column (Shanhai Jinhua Chromatography Equipment Factory) was Φ18 mm×400 mm, the packing specification of the Q Sepharose FF packing material (GE Healthcare) was Φ18 mm×240 mm, and the volume of the column bed (CV) was 61 ml. The Q Sepharose FF chromatography column was cleaned-in-place using 0.5M NaOH at 5 ml/min for 30 min, eluted with 3 CV of ddH.sub.2O at 5 ml/min, regenerated with 3 CV of 1M NaCl at 5 ml/min, and eluted with κCV of 20 mM boric acid/borax-17 mM NaCl (pH 9.0, solution A) at 5 ml/min. The ultrapure water diluted samples of the PEG modified rHuGH was loaded at a flow rate of 3 ml/min, and the eluent was detected at 280 nm (AKTA Basic100, GE Healthcare). Elution was done using solution A at 5 ml/min until the first peak was completely detected; and 20 mM boric acid/borax-100 mM NaCl (pH 9.0, solution B) was then used to elute at 5 ml/min until the second peak was completely detected. 20 mM boric acid/borax-200 mM NaCl (pH 9.0, solution C) was then used to elute at 5 ml/min until the third peak was completely detected. The sample from the second peak was collected as the target sample. The buffer system of the target sample was changed to 20 mM boric acid/borax (pH9.0) through ultrafiltration with 5K ultrafilter (Millipore, polyethersulfone material).

(22) 2.1.2 The Q Sepharose FF Chromatography Purification of PEG Modification Samples (pH9.0 or 10.5) of rHuGH

(23) The chromatography column (Shanhai Jinhua Chromatography Equipment Factory) was Φ18 mm×400 mm, the packing specification of the Q Sepharose FF packing material (GE Healthcare) was φ18 mm×240 mm, and the volume of the column bed (CV) was 61 ml. The Q Sepharose FF chromatography column was cleaned-in-place using 0.5M NaOH at 5 ml/min for 30 min, eluted with 3 CV of ddH.sub.2O at 5 ml/min, regenerated with 3 CV of 1M NaCl at 5 ml/min, and eluted with 5 CV of 20 mM boric acid/borax-17 mM NaCl (pH 9.0, solution A) at 5 ml/min. The ultrapure water diluted sample of PEG modified rHuGH was loaded at a flow rate of 3 ml/min, and the eluent was detected at 280 nm (AKTA Basic100, GE Healthcare). Elution was done using solution A at 5 ml/min until the first peak was completely detected, and 20 mM boric acid/borax-40 mM NaCl (pH 9.0, solution B) was used to elute at 5 ml/min until the second peak was completely detected. 20 mM boric acid/borax-100 mM NaCl (pH 9.0, solution C) was then used to elute at 5 ml/min until the third peak was completely detected, and 20 mM boric acid/borax-200 mM NaCl (pH 9.0, solution D) was used to elute at 5 ml/min until the fourth peak was completely detected. The sample from the third peak was collected as the target sample. The buffer system of the target sample was changed to 20 mM boric acid/borax (pH9.0) through ultrafiltration with 5K ultrafilter (Millipore, polyethersulfone material).

(24) 2.2 DEAE Sepharose FF Chromatography Purification

(25) The chromatography column (Shanhai Jinhua Chromatography Equipment Factory) was Φ18 mm×400 mm, the packing specification of the DEAE Sepharose FF packing material (GE Healthcare) was Φ18 mm×235 mm, 1 CV=60 ml.

(26) The DEAE Sepharose FF chromatography column was cleaned-in-place using 0.5M NaOH at 5 ml/min for 30 min, eluted with 3 CV of ddH.sub.2O at 5 ml/min, regenerated with 3 CV of 1M NaCl at 5 ml/min, and eluted with 3 CV of 20 mM boric acid/borax (pH 9.0, solution A) at 5 ml/min. The Q Sepharose FF purified PEG-rHuGH sample was loaded at a flow rate of 3 ml/min, eluted with 3 CV of solution A at 5 ml/min, and eluted with 6 CV of 20 mM boric acid/borax-30 mM NaCl (pH 9.0, solution B) at 5 ml/min. 20 mM boric acid/borax-100 mM NaCl (pH 9.0, solution C) was used to elute at 5 ml/min until the first and second peaks were completely detected. The eluent was detected at 280 nm (AKTA Basic100, GE Healthcare). The sample from the second peak was collected as the target sample. The buffer system of the target sample was changed to 5 mM PBNa (pH 8.5) and appropriately concentrated through ultrafiltration with 5K ultrafilter (Millipore, polyethersulfone material).

(27) 2.3 Refined Purification using Q Sepharose FF Chromatography

(28) The chromatography column (Shanhai Jinhua Chromatography Equipment Factory) was Φ25 mm×400 mm, the packing specification of the Q Sepharose FF packing material (GE Healthcare) was Φ25 mm×200 mm, 1 CV=98 ml.

(29) The Q Sepharose FF chromatography column was cleaned-in-place using 0.5M NaOH at 10 ml/min for 30 min, eluted with 3 CV of ddH.sub.2O at 10 ml/min, regenerated with 3 CV of 1M NaCl at 10 ml/min, and eluted with 3 CV of 5 mM PBNa (pH 8.5, solution A) at 10 ml/min. The DEAE Sepharose FF purified PEG-rHuGH sample was loaded at a flow rate of 6 ml/min, and eluted with 3 CV of solution A at 10 ml/min. 5 mM PBNa-90 mM NaCl (pH 8.5, solution B) was used to elute at 10 ml/min until the first peak was completely detected, and 5 mM PBNa-300 mM NaCl (pH 8.5, solution C) was used to elute at 10 ml/min until the second peak was completely detected. The eluent was detected at 280 nm (AKTA Basic100, GE Healthcare). The sample from the first peak was collected as the target sample. The buffer system of the target sample was changed to 3 mM NaAc/HAc-7 mM NaCl-5 mM Lys (pH 5.0) through ultrafiltration using 5K ultrafilter (Millipore, polyethersulfone material), and mannitol was supplemented to the final concentration of 45 mg/ml. The sample was sterilized through 0.2 μm filtration. A sample was taken for SDS-PAGE electrophoresis, and silver staining was used for visualization. The remaining sample was stored at −70° C. The modification product modified at pH6.0 was designated as Y6 or U6, wherein the band of higher apparent M.W. was designated as Y6-1 or U6-1, whereas the band of lower apparent M.W. was designated as Y6-2 or U6-2. The modification product at pH9.0 was designated as Y9 or U9, and the modification product at pH10.5 was designated as Y10.5 or U10.5.

(30) The SDS-PAGE results are shown in FIG. 4. From the electrophoresis results, the modification products at pH6.0 show clearly two bands, but the modification products at pH10.5 were mainly the band of lower apparent M.W. with a SDS-PAGE content of not lower than 80%.

(31) 3 Cellular Activity

(32) Using GH national standard as the control, HuGH dependent rat lymphoma cell line Nb2-11 was employed to assay the cellular activity of each PEG-rHuGH sample.

(33) Nb2-11 cells were diluted to the final concentration of 5×10.sup.4 cells/ml. GH national standard (lot. No.: 35-20002, 1 mg/ml/tube, 3 IU/tube; purchased from National Institute for the Control of Pharmaceutical and Biological Products) were pre-diluted to 100 ng/ml (0.0003 IU/ml), and each PEG-rHuGH sample to be assayed was pre-diluted to 0.0003 IU/ml according to the results from pre-experiments. Based on the pre-dilution, each sample was assayed after one and half times' gradient dilution. The activity of the sample was calculated according to the following equation:
Activities of samples for examination=Activities of Standards×C.sub.1/C.sub.2×D.sub.1/D.sub.2
wherein: C.sub.1 is the dilution folds of the sample to be assayed equivalent to half-effect amount of the standard C.sub.2 is the dilution folds of the half-effect standard D.sub.1 is the pre-dilution folds of the sample to be assayed D.sub.2 is the pre-dilution folds of the standard

(34) Assay Method:

(35) (1) The cells in logarithmic growth phase were taken, repeatedly pipetted, centrifuged and washed. The cells were resuspended in the diluent, and were adjusted to a concentration of 5×10.sup.4 cells/ml.

(36) (2) Each pre-diluted sample to be assayed was double gradient diluted respectively on cell plate (96-well plate, Corning), 10 gradients in total, and duplicate wells were made for each gradient, 50 μl/well. The positive control was made in 8 gradients in the same manner. The diluent was used as the negative control.

(37) (3) Cells were added in a density of 100 μl/well, placed in CO.sub.2 incubator and incubated at 37° C. for about 70 hours. ALAMARBLUE® solution (BioSource) was added at 30 μl/well, blended with shaking for 1 min. The incubation was done in CO.sub.2 incubator at 37° C. for 5 hours. After shaking at room temperature for 5 min, the plate was read (wavelength of excited light 530 nm; wavelength of emission light 590 nm).

(38) (4) Four-parameter regression method was used to plot the standard and the sample to be assayed. The titres of each sample to be assayed were calculated according to the equation of the plots of the standard and the samples to be assayed.

(39) The results of the cellular activity are shown in table 2 and FIG. 5 as well FIG. 6. Duplicate samples were assayed for each sample. The cellular specific activity of YPEG-NHS modified rHuGH at pH6.0 (Y6) is 1.08×10.sup.−1 IU/mg, the cellular specific activity of the modified product at pH9.0 (Y9) is 1.66×10.sup.−1 IU/mg, and the cellular specific activity of the modified product at pH10.5 (Y10.5) is 2.09×10.sup.−1 IU/mg, wherein the cellular specific activity of Y10.5 is about 2 times of that of Y6. The cellular specific activity of UPEG-NHS modified rHuGH at pH6.0 (U6) is 8.85×10.sup.−2 IU/mg, the cellular specific activity of the modified product at pH9.0 (U9) is 1.42×10.sup.−1 IU/mg; and the cellular specific activity of the modified product at pH10.5 (U10.5) is 1.82×10.sup.−1 IU/mg, wherein the cellular specific activity of U10.5 is about two times of that of U6. As the pH of modification reaction increases, the cellular activity of the product modified by PEG at a single site (2 major bands) also increases correspondingly.

(40) TABLE-US-00002 TABLE 2 The cellular activity of each sample of YPEG-rHuGH and UPEG-rHuGH * PEG M.W. Cellular specific activity (×10.sup.−1 IU/mg) Sample PEG type (kDa) Plate 1 Plate2 average YPEG-rHuGH, Y6 Y branched 40 1.04 1.12 1.08 YPEG-rHuGH, Y9 Y branched 40 1.58 1.74 1.66 YPEG-rHuGH, Y10.5 Y branched 40 2.06 2.12 2.09 UPEG-rHuGH, U6 U branched 40 0.88 0.89 0.88 UPEG-rHuGH, U9 U branched 40 1.38 1.46 1.42 UPEG-rHuGH, U10.5 U branched 40 1.84 1.80 1.82 Note: * GH national standard was used as the standard. Lot. No. of the standard: 35-20002, 1 mg/ml/tube, 3 IU/tube, purchased from National Institute for the Control of Pharmaceutical and Biological Products.
4 The Molecular Weight Determined by MALDI-TOF MS

(41) Using AUTOFLEX™ III TOF/TOF mass spectroscope (BRUKER, Germany), MALDI-TOF MS method was employed to determine the molecular weight of each sample of PEG-rHuGH. Sinapinic acid (SA, C.sub.11H.sub.12O.sub.5, M.W. 224.22, lot number: 2006 236870 002, BRUKER) was used as the matrix, Protein Calibration Standard I (Part No. 206355) and Protein Calibration Standard II (Part No. 207234) from BRUKER were used as protein molecular weight standard, and the analysis software was FLEXANALYSIS™ Ver.3.0.54.0. Results are shown in FIG. 7.

(42) The YPEG-NHS modified rHuGHs at pH 6.0 (Y6), pH 9.0 (Y9) and pH 10.5 (Y10.5) all have a MS molecular weight in a range of 62012 Dalton±10%, which is consistent with the theoretical molecular weight of the rHuGH modified by YPEG at a single site (the molecular weight of YPEG-NHS is 40 kDa±10%), indicating that Y6, Y9 and Y10.5 are the rHuGH modification product modified by YPEG at a single site. The UPEG-NHS modified rHuGHs at pH 6.0 (U6), pH 9.0 (U9) and pH 10.5 (U10.5) all have a MS molecular weight in a range of 62012 Dalton±10%, which is consistent with the theoretical molecular weight of the rHuGH modification products modified by UPEG at a single site (the molecular weight of UPEG-NHS is 40 kDa±10%), indicating that U6, U9 and U10.5 are the rHuGH modification product modified by UPEG at a single site.

Example 4

The Preparation as Well the Cellular Activity and M.W. Assay of the Recombinant Human GH Modification Product of Higher Apparent Molecular Weight Modified by U-Shaped or Y-Shaped Branched PEG at a Single Site at pH6.0 (Y6-1, U6-1)

(43) 1 Modification

(44) Two samples of 1200 mg of each UPEG-NHS and YPEG-NHS (average M.W. 40 kDa, equal-arm; lot. Nos. are ZZ004P182, ZZ004P167 respectively) (Beijing JenKem Technology Co., Ltd.) were weighted and dissolved in 12 ml of 2 mM HCl (Guangdong Guanghua Chemical Factory Co., Ltd.) respectively. 300 mg rHuGH (Xiamen Amoytop Biotech Co., Ltd.) and 10 mM PBNa (pH6.0) (Sinopharm Shanghai Chemical Reagent Co., Ltd.) were added respectively to a final total reaction volume of 60 ml. In the reaction system, the final reaction concentration of rHuGH was 5 mg/ml, the reaction molar ratio of the rHuGH to PEG-NHS was about 1:2, and the reaction pH was 6.0. Incubation was done at <10° C. for 2h with shaking, and glacial acetic acid (Shantou Xilong Chemical Co., Ltd.) was added to make pH<4.0 to stop the reaction.

(45) 2 The Purification of the Product of Higher Apparent Molecular Weight Modified by PEG at a Single Site (Y6-1, U6-1)

(46) 2.1 Q Sepharose FF Chromatography Purification

(47) The rHuGH sample modified by PEG (pH 6.0) was diluted 3 times using ultrapure water, and the pH was adjusted to 9.0 using NaOH.

(48) The chromatography column (Shanhai Jinhua Chromatography Equipment Factory) was Φ25 mm×500 mm, and the packing specification of the Q Sepharose FF packing material (GE Healthcare) was φ25 mm×310 mm, 1 CV=152 ml. The Q Sepharose FF chromatography column was cleaned-in-place using 0.5M NaOH at 10 ml/min for 30 min, eluted with 3 CV of ddH.sub.2O at 10 ml/min, regenerated with 3 CV of 1M NaCl at 10 ml/min, and eluted with 5 CV of 20 mM boric acid/borax-17 mM NaCl (pH 9.0, solution A) at 10 ml/min. The ultrapure water diluted sample of the PEG modified rHuGH was loaded at a flow rate of 6 ml/min, and eluted using solution A at 10 ml/min until the first peak was completely detected. 20 mM boric acid/borax-100 mM NaCl (pH 9.0, solution B) was then used to elute at 10 ml/min until the second peak was completely detected, and 20 mM boric acid/borax-200 mM NaCl (pH 9.0, solution C) was used to elute at 10 ml/min until the third peak was completely detected. The eluent was detected at 280 nm (AKTA Basic100, GE Healthcare). The sample from the second peak was collected as the target sample. The buffer system of the target sample was changed to 5 mM NaAc/HAc (pH 4.5) through ultrafiltration with 5K ultrafilter (Millipore, polyethersulfone material).

(49) 2.2 MacroCap SP Chromatography Purification

(50) The chromatography column (Shanhai Jinhua Chromatography Equipment Factory) was Φ12 mm×300 mm, and the packing specification of MacroCap SP packing material (GE Healthcare) was Φ12 mm×180 mm, 1 CV=20 ml. The MacroCap SP chromatography column was cleaned-in-place using 0.5M NaOH at 1 ml/min for 30 min, eluted with 3 CV of ddH.sub.2O at 1 ml/min, regenerated with 3 CV of 1M NaCl at 1 ml/min, and eluted with 5 CV of 5 mM NaAc/HAc (pH 4.5, solution A) at 1 ml/min. The Q Sepharose FF purified PEG-rHuGH sample was loaded at a flow rate of 1 ml/min, and eluted with 3 CV of solution A at 1 ml/min. 5 mM NaAc/HAc-100 mM NaCl (pH 4.5, solution B) was used to elute with 5 CV in a gradient of 0%-30% solution B at 1 ml/min, eluted with 10 CV in a gradient of 30%-45% B, and then eluted with 5 mM NaAc/HAc-1M NaCl (pH 4.5, solution C) at 1 ml/min until the first and the second peaks were completely detected. The eluent was detected at 280 nm (AKTA Basic100, GE Healthcare). The eluent between the fifth and the eighth CV during the elution with a gradient of 30%-45% solution B was collected as the target sample. The buffer system of the target sample was changed to 3 mM NaAc/HAc-7 mM NaCl-5 mM Lys (pH 5.0) through ultrafiltration with 5K ultrafilter (Millipore, polyethersulfone material), and mannitol was supplemented to a final concentration of 45 mg/ml. The sample was sterilized through 0.2 μm filtration. A sample was taken for SDS-PAGE electrophoresis, and silver staining was used for visualization. The remaining sample was stored at −70° C. The sample numbers were: U6-1, Y6-1. The results of the SDS-PAGE electrophoresis are shown in FIG. 8, and the apparent molecular weight results of the SDS-PAGE electrophoresis are shown in FIG. 9.

(51) In the case of loading 10 μg PEG-rHuGH sample, a small amount of the product modified at more than one site was detected in each sample of PEG-rHuGH, and the content of the substrate protein (rHuGH) in each sample was not more than 0.5% (FIG. 8), wherein the content of the major band is not lower than 80%. The apparent molecular weight of each PEG-rHuGH sample determined by the SDS-PAGE electrophoresis shows one major band, wherein the apparent molecular weight of Y6-1 is clearly higher than that of Y10.5, and the apparent molecular weight of U6-1 is clearly higher than that of U10.5 (FIG. 9).

(52) 3 The Molecular Weight Detected by MALDI-TOF MS

(53) Using AUTOFLEX™ TOF/TOF mass spectroscope (BRUKER, Germany), MALDI-TOF MS method was used to assay the molecular weight of each PEG-rHuGH sample. The detection method was the same as that in Example 3. The results are shown in FIG. 7.

(54) The MS molecular weights of Y6-1 and U6-1 are both in a range of 62012 Dalton±10%, which is consistent with the theoretical molecular weight of the rHuGH modified by PEG at a single site (the molecular weights of YPEG-NHS and UPEG-NHS are 40 kDa±10%), indicating that both are the products modified by PEG at a single site.

(55) Using GH national standard as the control, HuGH dependent rat lymphoma cell line Nb2-11 was used to assay the cellular activity of each PEG-rHuGH sample, comparing the cellular activity difference between Y6-1 and Y10.5, U6-1 and U10.5. The assay method was the same as that in Example 3. The results are shown in table 3, triplicate for each sample.

(56) The average cellular specific activity of Y10.5 is 2.08×10.sup.−1 IU/mg, the average cellular specific activity of Y6-1 is 5.50×10.sup.−2 IU/mg; the average cellular specific activity of U10.5 is 2.28×10.sup.−1 IU/mg, and the average cellular specific activity of U6-1 is 5.00×10.sup.−2 IU/mg. The average cellular specific activity of Y10.5/U10.5 is clearly higher than that of Y6-1/U6-1, and can reach up to 3 times of the latter.

(57) TABLE-US-00003 TABLE 3 The cellular activity of each YPEG-rHuGH or UPEG-rHuGH sample.sup.1 Cellular Number of specific PEG PEG activity M.W. modification (×10.sup.−1 IU/ Sample PEG type (kDa) sites mg).sup.2 YPEG-rHuGH, Y10.5 Y branched 40 single 2.08 ± 0.10 YPEG-rHuGH, Y6-1 Y branched 40 single 0.55 ± 0.06 UPEG-rHuGH, U10.5 U branched 40 single 2.28 ± 0.14 UPEG-rHuGH, U6-1 U branched 40 single 0.50 ± 0.06 Note: .sup.1using GH national standard as the standard. Lot. No. of the standard: 35-20002, 1 mg/ml/tube, 3 IU/tube, purchased from National Institute for the Control of Pharmaceutical and Biological Products. .sup.2average of triplicate samples.

Example 5

The In Vivo Biological Activity Assay of YPEG-rHuGH (Y10.5) and UPEG-rHuGH (U10.5)

(58) Using rats with the pituitary glands removed as animal models, the in vivo animal growth promoting biological activity of YPEG-rHuGH (Y10.5) and UPEG-rHuGH (U10.5) were assayed according to the growth hormone bioassay as described in Pharmacopoeia of the People's Republic of China, version 2005, Volume 3, Appendix XII P, i.e. observing the effect on the growth and development of rats with the pituitary glands removed (no endogenous GH) one week after a single administration.

(59) Wistar rats, SPF level, male, born 26-28d, body weight of 60-80 g, provided by the experiment animal center of National Institute for the Control of Pharmaceutical and Biological Products (animal certification No.: SCXK (Jing)2005-0004), were used. 2-3 weeks before the experiment, the pituitary glands of rats were removed aseptically by surgery, and the rats were then normally raised in a S-2 laboratory to recover for further experiment. The qualified rats with pituitary glands removed were selected, and divided evenly into 10 groups of 10 rats according to body weight, specifically: negative control (blank solvent) group; positive control rHuGH (GH national standard, prepared by National Institute for the Control of Pharmaceutical and Biological Product, 3 IU.Math.mg.sup.−1.Math.tube.sup.−1), low dose (2.7 IU.Math.kg.sup.−1), medium dose (5.3 IU.Math.kg.sup.−1) and high dose (10.7 IU.Math.kg.sup.−1) groups, administered in 6 times, once per day, 6 consecutive administrations; low dose (2.7 IU.Math.kg.sup.−1), medium dose (5.3 IU.Math.kg.sup.−1) and high dose (10.7 IU.Math.kg.sup.−1) groups of the testing sample Y10.5, low dose (2.7 IU.Math.kg.sup.−1), medium dose (5.3 IU.Math.kg.sup.−1) and high dose (10.7 IU.Math.kg.sup.−1) groups of the testing sample U10.5, single administration once in the first day when the standard was administered. Y10.5 and U10.5 were formulated according to the estimated titre of 3 IU/mg. Administration was performed by subcutaneous injection of 0.5 ml to the neck of the animal. The negative control group was only administered the solvent, once per day, 6 times in total. The rats were sacrificed 24 h after the last administration in the positive control group, and the body weights and the width of tibial growth plates were measured. The data were processed according to the growth hormone assay in Appendix XII P and the statistic method for biological assay in Appendix XIV of the Pharmacopoeia of the People's Republic of China, version 2005.

(60) The biological titre of YPEG-rHuGH (Y10.5) is 5.0 IU.Math.mg.sup.−1, the biological titre of UPEG-rHuGH (U10.5) is 5.2 IU.Math.mg.sup.−1, both more than 1.5 times of the normal rHuGH. Single administration of YPEG-rHuGH (Y10.5) or UPEG-rHuGH (U10.5) has a higher biological activity for promoting the body growth in animal and a longer pharmaceutical effect than the sum of daily injected rHuGH.

Example 6

The Serum Drug Metabolic Half-Life of YPEG-rHuGH (Y10.5) in Crab-Eating Macaque

(61) 6 crab-eating macaques were selected, 3 female and 3 male, body weight of 3.24-5.48 kg (Guangxi Beihai Yu Qi Experiment Animal technology co. Ltd., certification No.: SCXK (Gui)2005-0005). The experiment included two groups of 3 crab-eating macaques: one group with subcutaneous injection of YPEG-rHuGH (Y10.5) at 300 μg.Math.kg.sup.−1 (2♂, 1♀) and the other group with subcutaneous injection of rHuGH (Saizen, Laboratoires Serono S.A. Switzerland) at 300 μg.Math.kg.sup.−1 (1♂, 2♀), single administration. After administration, the venous blood was taken regularly from the hind leg opposite to the injected side, and the serum was separated. Human Growth Hormone ELISA kit from R&D was used to assay the blood drug concentration through ELISA, and the curve of blood drug concentration was plotted to calculate drug metabolic half-life. The results are shown in FIG. 10.

(62) After subcutaneous injection of YPEG-rHuGH (Y10.5) at 300 μg.Math.kg.sup.−1 in crab-eating macaques, the time-to-peak of drug concentration in serum is 8-24 h. The drug was eliminated slowly. The average drug metabolic half-life in serum is 41.33 h. After subcutaneous injection of rHuGH (Saizen) at 300 μg.Math.kg.sup.−1 in crab-eating macaques, the time-to-peak of drug concentration in serum is 1-2 h, and by 24 h the concentration decreases to the level before administration. The elimination is clearly faster than YPEG-rHuGH (Y10.5). The average drug metabolic half-life in serum is 1.80 h. The average drug metabolic half-life in serum of YPEG-rHuGH (Y10.5) is more than 20 times of rHuGH.