Erythropoietin composition

• US 7,169,754 B2
• Filed: 05/11/2001
• Issued: 01/30/2007
• Est. Priority Date: 05/15/2000
• Status: Expired due to Term

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Papadimitriou, Apollon
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US09/853,731
• Publication Number:
  US 20020037841A1
• Time in Patent Office: 2,090 Days
• Field of Search: 514/8, 514/12, 514/21, 514/970, 530/350, 530/397, 435/69.1, 435/320.1, 435/325
• US Class Current: 514/7.7
• CPC Class Codes: A61K 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/183 Amino acids, e.g. glycine, EDTA or aspartame
• CPC Class Codes: A61K 47/20 containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/08 Solutions composition of solutions A61K47/00
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics

Abstract

The present invention relates to a liquid pharmaceutical composition comprising an erythropoietin protein, a multiple charged inorganic anion in a pharmaceutically acceptable buffer suitable to keep the solution pH in the range from about 5.5 to about 7.0, and optionally one or more pharmaceutically acceptable excipients. This composition is especially useful for the prophylaxis and treatment of diseases related to erythropoiesis.

Claims

1. A liquid pharmaceutical composition in the form of an aqueous solution comprising:

(a) an erythropoietin glycoprotein conjugate comprising an erythropoietin protein having at least one free amino group and having the sequence SEQ ID NO;

1 or SEQ ID NO;

2, or having either of said sequences modified by the addition of from 1 to 6 glycosylation sites or by rearrangement of at least one glycosylation site;

said erythropoietin protein being covalently linked to from one to three lower-alkoxy poly(ethylene glycol) groups with each poly(ethylene glycol) group being covalently linked to the erythropoietin via a linker of the formula —

C(O)—

X—

S—

Y—

with the C(O) of the linker forming an amide bond with one of said amino groups;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

;

k is from 1 to 10;

Y is selected from

2. The composition of claim 1 wherein said conjugate has the formula:

3. The composition of claim 1 wherein the conjugate has the formula:

4. The composition of claim 1 wherein said solution is an isotonic solution.

5. The composition of claim 1 wherein the anion is an anion of a multiple charged strong inorganic acid.

6. The composition of claim 5 wherein the anion is selected from the group consisting of sulfate or phosphate.

7. The composition of claim 6 wherein the anion is a sulfate anion.

8. The composition of claim 7 wherein the pH is 5.8 to 6.7.

9. The composition of claim 8 wherein the pH is about 6.2.

10. The composition of claim 7 wherein the pH is 6.0 to 6.5.

11. The composition of claim 1 wherein the buffer is a phosphate buffer.

12. The composition of claim 1 wherein the erythropoietin has the amino acid sequence SEQ ID NO:

1 or SEQ ID NO;

2.

13. The composition of claim 1 wherein the erythropoietin glycoprotein has the sequence SEQ ID NO:

1 or SEQ ID NO;

2 that is modified by the addition of from 1 to 6 glycosylation sites.

14. The composition of claim 13 wherein the sequence of modification is

Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰.

15. The composition of claim 1 wherein said erythropoietin glycoprotein has the sequence SEQ ID NO:

1 or SEQ ID NO;

2 that is modified by a rearrangement of at least one glycosylation site.

16. The composition of claim 15, wherein the rearrangement comprises deletion of any of the N-linked glycosylation sites in human erythropoietin with the addition of an N-linked glycosylation site at position 88 of the sequence of human erythropoietin.

17. The composition of claim 1 wherein said solution contains 10 μ

g to 10,000 μ

g erythropoietin protein per ml of solution, from 10 to 200 mmol/liter of solution of a sulfate as the multiple charged inorganic anion, and 10 to 50 mmol/liter of solution of a phosphate as the pharmaceutically acceptable buffer, said solution having a pH of from about 6.0 to about 6.5.

18. The composition of claim 17 further comprising up to 20 mM of methionine, and 1–

5% of a polyol (w/v).

19. The composition of claim 18 comprising 10 μ

g to 10,000 μ

g erythropoietin protein per ml of solution, 40 mmol/liter of solution of the sulfate, 10 mmol/liter of said solution of the phosphate, 10 mM methionine, said composition having a pH of about 6.2, and wherein the polyol is mannitol which is present in the solution at 3% (w/v).

20. The composition of claim 1 wherein said solution contains 10 μ

g to 10,000 μ

g erythropoietin protein per ml of solution, the buffer is phosphate which is present at 10 to 50 mmol/liter of solution, said solution further comprising NaCl which is present at 10 to 100 mmol/liter of solution and having a pH of from about 6.0 to about 7.0.

21. The composition of claim 20 wherein the NaCl is present at 100 mmol/liter of solution, the phosphate is present at 10 mmol/l, said solution further comprising 10 mM methionine and having a pH of about 7.0.

22. The composition of claim 1 wherein the amount of erythropoietin protein is 50, 100, 400, 800 or 2,500 μ

g/ml of solution.

23. The composition of claim 22 comprising 10 mM sodium phosphate and 40 mM sodium sulfate and further comprising 3% mannitol, 10 mM methionine and 0.01% poloxamer 188, and having a pH of about 6.2.

24. The composition of claim 22 comprising 30 mM sodium sulfate and further comprising 3% mannitol, 40 mM arginine, 10 mM methionine, 0.01% poloxamer 188, and a pH of about 6.2.

25. A liquid pharmaceutical composition in the form of an aqueous solution comprising a pegylated erythropoietin glycoprotein conjugate of formula

P—

[NHCO—

(CH₂)_x—

(OCH₂CH₂)_m—

OR]_n 

 

(I)

wherein

P is an erythropoietin glycoprotein having the sequence SEQ ID NO;

1, SEQ ID NO;

2, or either of these sequences modified by the addition of from 1 to 6 glycosylation sites or by rearrangement of at least one glycosylation site, minus the n amino group of said glycoprotein,

R is lower alkyl,

x is 2 or 3,

m is from about 450 to about 900;

n is from 1 to 3; and

wherein the values of n and m are such that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons, and wherein the amount of the erythropoietin glycoprotein is from about 10 μ

g per ml to about 10,000 μ

g per ml of solution; and

from about 10 to about 200 mmol per liter of solution of a multiple charged inorganic anion and from about 10 to about 50 mmol per liter of solution of a pharmaceutically acceptable buffer, said anion and said buffer being present in said solution in an amount such that the pH of the solution is from about 5.5 to about 7.0.

26. The composition of claim 25 wherein x is 2, m is 650 to 750, n is 1, and R is methyl.

27. The liquid pharmaceutical composition of claim 26 wherein the pegylated erythropoietin conjugate is present in an amount such as to provide about 100.0 μ

g of erythropoietin protein per mL of solution, the multiple charged inorganic anion is sodium sulfate which is present in an amount of about 5.68 mg/mL, the pharmaceutically acceptable buffer is sodium phosphate which is present in an amount of about 1.38 mg/mL, and wherein the pH of the solution is 6.2±

0.2.

28. The liquid pharmaceutical composition of claim 27 further comprising methionine in an amount of about 1.49 mg/mL, mannitol in an amount of about 30.0 mg/mL and poloxamers type 188 in an amount of 0.1 mg/mL.

29. The liquid pharmaceutical composition of claim 26 wherein the pegylated erythropoietin conjugate is present in an amount such as to provide about 400 μ

g of erythropoietin protein per mL of solution, the multiple charged inorganic anion is sodium sulfate which is present in an amount of about 5.68 mg/mL, the pharmaceutically acceptable buffer is sodium phosphate which is present in an amount of about 1.38 mg/mL, and wherein the pH of the solution is 6.2±

0.2.

30. The liquid pharmaceutical composition of claim 29 further comprising methionine in an amount of about 1.49 mg/mL, mannitol in an amount of about 30.0 mg/mL and poloxamers type 188 in an amount of 0.1 mg/mL.

31. The liquid pharmaceutical composition of claim 26 wherein the pegylated erythropoietin conjugate is present in an amount such as to provide about 800.0 μ

g of erythropoietin protein per mL of solution, the multiple charged inorganic anion is sodium sulphate which is present in an amount of about 5.68 mg/mL, the pharmaceutically acceptable buffer is sodium phosphate which is present in an amount of about 1.38 mg/mL, and wherein the pH of the solution is 6.2±

0.2.

32. The liquid pharmaceutical composition of claim 31 further comprising methionine in an amount of about 1.49 mg/mL, mannitol in an amount of about 30.0 mg/mL and poloxamers type 188 in an amount of 0.1 mg/mL.

33. The composition of claim 25 wherein the erythropoietin protein is present at about 25 to about 2,500 μ

g/ml, the buffer is sodium or potassium phosphate which is present in an amount of about 10 mM, said composition further comprising NaCl which is present in an amount of about 100 mM and having a pH of about 7.0.

34. The composition of claim 25 wherein the solution is an isotonic solution.

35. The composition of claim 25 wherein the anion is an anion of a multiple charged strong inorganic acid.

36. The composition of claim 25 wherein the anion is selected from the group consisting of sulfate or phosphate.

37. The composition of claim 25 wherein the anion is a sulfate anion.

38. The composition of claim 25 wherein the pH is from 5.8 to 6.7.

39. The composition of claim 38 wherein the pH is from 6.0 to 6.5.

40. The composition of claim 39 wherein the pH is about 6.2.

41. The composition of claim 25 wherein the buffer is a phosphate buffer.

42. The composition of claim 25 wherein the erythropoietin glycoprotein has the amino acid sequence SEQ ID NO:

1 or SEQ ID NO;

2.

43. The composition of claim 42 wherein the erythropoietin glycoprotein has the amino acid sequence SEQ ID NO:

1.

44. The composition of claim 25 wherein said solution contains from 10 μ

g to 10,000 μ

g erythropoietin protein per ml of solution, from 10 to 200 mmol/liter of solution of a sulfate as the multiple charged inorganic anion, and 10 to 50 mmol/liter of solution of a phosphate as the pharmaceutically acceptable buffer, said solution having a pH of from about 6.0 to about 6.5.

45. The composition of claim 44 further comprising up to 20 mM of methionine, and 1–

5% of a polyol (w/v).

46. The composition of claim 45 comprising from 10 μ

g to 10,000 μ

g erythropoietin protein per ml of solution, 40 mmol/liter of solution of the sulfate, 10 mmol/liter of said solution of the phosphate, 10 mM methionine, said composition having a pH of about 6.2, and wherein the polyol is mannitol which is present in the solution at 3% (w/v).

47. The composition of claim 25 wherein the solution contains from 10 μ

g to 10,000 μ

g erythropoietin protein per ml of solution, the buffer is phosphate which is present at 10 to 50 mmol/liter of solution, said solution further comprising NaCl which is present at 10 to 100 mmol/liter of solution and having a pH of from about 6.0 to about 7.0.

48. The composition of claim 47 wherein the NaCl is present at 100 mmol/liter of solution, the phosphate is present at 10 mmol/l, said solution further comprising 10 mM methionine and having a pH of about 7.0.

49. The composition of claim 25 wherein the amount of erythropoietin protein is 50, 100, 400, 800 or 2,500 μ

g/ml of solution.

50. The composition of claim 49 comprising 10 mM sodium phosphate and 40 mM sodium sulfate and further comprising 3% mannitol, 10 mM methionine and 0.01% poloxamer 188, and having a pH of about 6.2.

51. The composition of claim 49 comprising 30 mM sodium sulfate and further comprising 3% mannitol, 40 mM arginine, 10 mM methionine, 0.01% poloxamer 188, and a pH of about 6.2.

52. A liquid pharmaceutical composition in the form of an aqueous solution comprising from 10 μ

g to 10,000 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, from 10 to 100 mmol/liter of solution of sodium sulfate, a buffer, and from 10 to 50 mmol/liter of said solution of arginine, said solution having a pH of from about 6 to about 6.5 and being stable at room temperature.

53. The composition of claim 52 wherein said solution comprises 40 mmol/liter of solution of arginine, 30 mmol/liter of solution of sodium sulfate, said solution further comprising 3% mannitol, 10 mM methionine, and 0.01% poloxamer 188, and having a pH of about 6.2.

54. A liquid pharmaceutical composition in the form of an aqueous solution comprising from about 25 μ

g to about 2,500 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, about 120 mM of sodium sulfate, and about 10 mM of sodium phosphate, said solution having a pH of about 6.2 and being stable at room temperature.

55. A liquid pharmaceutical composition in the form of an aqueous solution comprising from about 25 μ

g to about 2,500 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, about 40 mM of sodium sulfate, and about 10 mM sodium phosphate, said solution having a pH of about 6.2 and being stable at room temperature.

56. A liquid pharmaceutical composition in the form of an aqueous solution comprising from about 25 μ

g to about 2,500 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, about 40 mM of sodium sulfate, about 10 mM sodium phosphate, 3% mannitol and 10 mM methionine, and having a pH of about 6.2 and being stable at room temperature.

57. A liquid pharmaceutical composition in the form of an aqueous solution comprising from about 25 μ

g to about 2,500 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, about 30 mM of sodium sulfate, about 40 mM arginine, a buffer, and 3% mannitol, said solution having a pH of about 6.2 and being stable at room temperature.

58. A liquid pharmaceutical composition in the form of an aqueous solution comprising from about 25 μ

g to about 2,500 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, about 30 mM of sodium sulfate, about 40 mM arginine, and about 3% mannitol, said solution having a pH of about 6.2 and being stable at room temperature.