Formulations of VEG antagonist fusion proteins and method of manufacturing them
Formulations of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist are provided including a pre-lyophilized formulation, a reconstituted lyophilized formulation, and a stable liquid formulation. Preferably, the fusion protein has the sequence of SEQ ID NO:4.
1. A formulation comprising:
10-50 mg/ml of a vascular endothelial growth factor (VEGF) antagonist fusion protein comprising amino acids 27-457 of SEQ ID NO: 4,
a buffer comprising histidine,
an organic co-solvent comprising polysorbate, and
a stabilizing agent comprising a sugar, an amino acid or both,
wherein said VEGF antagonist fusion protein exhibits less than about 3% degradation after 15 months of storage at 5° C.
2. The formulation of claim 1 , wherein said buffer comprises 5-50 mM histidine.
3. The formulation of claim 1 , wherein said buffer comprises about 10 mM histidine.
4. The formulation of claim 1 , wherein said buffer is at a pH of about 6.0-6.5.
5. The formulation of claim 2 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
6. The formulation of claim 2 , wherein said formulation further comprises a tonicity agent.
7. The formulation of claim 6 , wherein said tonicity agent is sodium chloride.
8. The formulation of claim 2 , wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
9. The formulation of claim 2 , wherein said organic co-solvent comprises polysorbate 20.
10. The formulation of claim 2 , wherein said stabilizing agent comprises a sugar.
11. The formulation of claim 10 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
12. The formulation of claim 10 , wherein said sugar is 5-20% sucrose.
13. The formulation of claim 10 , wherein said sugar is 0.5-6.0% sucrose.
14. The formulation of claim 2 , wherein said stabilizing agent comprises an amino acid.
15. The formulation of claim 14 , wherein said amino acid is glycine.
16. The formulation of claim 14 , wherein said amino acid is 0.25-3.0% glycine.
17. The formulation of claim 9 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
18. The formulation of claim 9 , wherein said formulation further comprises a tonicity agent.
19. The formulation of claim 18 , wherein said tonicity agent is sodium chloride.
20. The formulation of claim 9 , wherein said stabilizing agent comprises a sugar.
21. The formulation of claim 20 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
22. The formulation of claim 20 , wherein said sugar is 5-20% sucrose.
23. The formulation of claim 20 , wherein said sugar is 0.5-6.0% sucrose.
24. The formulation of claim 9 , wherein said stabilizing agent comprises an amino acid.
25. The formulation of claim 24 , wherein said amino acid is glycine.
26. The formulation of claim 24 , wherein said amino acid is 0.25-3.0% glycine.
27. A formulation comprising:
10-50 mg/ml of a vascular endothelial growth factor (VEGF) antagonist fusion protein comprising amino acids 27-457 of SEQ ID NO: 4,
a buffer comprising histidine,
an organic co-solvent comprising polysorbate, and
a stabilizing agent comprising a sugar, an amino acid or both,
wherein said VEGF antagonist fusion protein is capable of inhibiting biological activity of human VEGF as measured by a mouse Baf/3 VEGFR1/EpoR cell line and achieving a percent relative potency of at least 75 relative to a reference VEGF IC 50 standard.
28. The formulation of claim 27 , wherein said buffer comprises 5-50 mM histidine.
29. The formulation of claim 27 , wherein said percent relative potency of at least 75 is achieved after 1 month of storage at 5° C.
30. The formulation of claim 27 , wherein said percent relative potency of at least 75 is achieved after 24 months of storage at 5° C.
31. A formulation comprising:
10-50 mg/ml of a vascular endothelial growth factor (VEGF) antagonist fusion protein comprising amino acids 27-457 of SEQ ID NO: 4,
a buffer comprising histidine,
an organic co-solvent comprising polysorbate, and
a stabilizing agent comprising a sugar, an amino acid or both,
wherein said VEGF antagonist fusion protein is capable of binding VEGF at a percent relative binding of at least 88 relative to a reference VEGF IC 50 standard, after storage at 5° C. for 3 months.
32. The formulation of claim 31 , wherein said buffer comprises 5-50 mM histidine.
33. A method of manufacturing a liquid formulation, said method comprising combining a vascular endothelial growth factor (VEGF) antagonist fusion protein that comprises amino acids 27-457 of SEQ ID NO: 4 with a buffer comprising histidine, an organic co-solvent comprising polysorbate, and a stabilizing agent comprising a sugar, an amino acid or both;
wherein the concentration of said VEGF antagonist fusion protein is 10-50 mg/ml in said liquid formulation; and
wherein said VEGF antagonist fusion protein exhibits less than about 3% degradation after 15 months of storage at 5° C.
34. The method of claim 33 , wherein said buffer comprises 5-50 mM histidine.
35. A method of manufacturing a liquid formulation, said method comprising combining a vascular endothelial growth factor (VEGF) antagonist fusion protein that comprises amino acids 27-457 of SEQ ID NO: 4 with a buffer comprising histidine, an organic co-solvent comprising polysorbate, and a stabilizing agent comprising a sugar, an amino acid or both;
wherein the concentration of said VEGF antagonist fusion protein is 10-50 mg/ml in said liquid formulation; and
wherein said VEGF antagonist fusion protein is capable of inhibiting biological activity of human VEGF as measured by a mouse Baf/3 VEGFR1/EpoR cell line and achieving a percent relative potency of at least 75 relative to a reference VEGF IC 50 standard.
36. The method of claim 35 , wherein said buffer comprises 5-50 mM histidine.
37. The method of claim 35 , wherein said percent relative potency of at least 75 is achieved after 1 month of storage at 5° C.
38. The formulation of claim 35 , wherein said percent relative potency of at least 75 is achieved after 24 months of storage at 5° C.
39. A method of manufacturing a liquid formulation, said method comprising combining a vascular endothelial growth factor (VEGF) antagonist fusion protein that comprises amino acids 27-457 of SEQ ID NO: 4 with a buffer comprising histidine, an organic co-solvent comprising polysorbate, and a stabilizing agent comprising a sugar, an amino acid or both;
wherein the concentration of said VEGF antagonist fusion protein is 10-50 mg/ml in said liquid formulation; and
wherein said VEGF antagonist fusion protein is capable of binding VEGF at a percent relative binding of at least 88 relative to a reference VEGF IC 50 standard, after storage at 5° C. for 3 months.
40. The method of claim 39 , wherein said buffer comprises 5-50 mM histidine.
41. The formulation of claim 2 , wherein said organic co-solvent comprises 0.05-0.15% polysorbate 20.
42. The formulation of claim 2 , wherein said organic co-solvent comprises 0.003-0.15% polysorbate.
43. The formulation of claim 29 , wherein said buffer comprises 5-50 mM histidine.
44. The formulation of claim 29 , wherein said buffer comprises about 10 mM histidine.
45. The formulation of claim 43 , wherein said buffer is at a pH of about 6.0-6.5.
46. The formulation of claim 43 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
47. The formulation of claim 46 , wherein said formulation further comprises a tonicity agent.
48. The formulation of claim 47 , wherein said tonicity agent is sodium chloride.
49. The formulation of claim 46 , wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
50. The formulation of claim 46 , wherein said organic co-solvent comprises 0.05-0.15% polysorbate 20.
51. The formulation of claim 46 , wherein said organic co-solvent comprises 0.003-0.15% polysorbate.
52. The formulation of claim 46 , wherein said stabilizing agent comprises a sugar.
53. The formulation of claim 52 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
54. The formulation of claim 52 , wherein said sugar is 5-20% sucrose.
55. The formulation of claim 52 , wherein said sugar is 0.5-6.0% sucrose.
56. The formulation of claim 46 , wherein said stabilizing agent comprises an amino acid.
57. The formulation of claim 56 , wherein said amino acid is glycine.
58. The formulation of claim 31 , wherein said buffer comprises about 10 mM histidine.
59. The formulation of claim 32 , wherein said buffer is at a pH of about 6.0-6.5.
60. The formulation of claim 32 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
61. The formulation of claim 60 , wherein said formulation further comprises a tonicity agent.
62. The formulation of claim 61 , wherein said tonicity agent is sodium chloride.
63. The formulation of claim 60 , wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
64. The formulation of claim 60 , wherein said organic co-solvent comprises 0.05-0.15% polysorbate 20.
65. The formulation of claim 60 , wherein said organic co-solvent comprises 0.003-0.15% polysorbate.
66. The formulation of claim 60 , wherein said stabilizing agent comprises a sugar.
67. The formulation of claim 66 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
68. The formulation of claim 66 , wherein said sugar is 5-20% sucrose.
69. The formulation of claim 66 , wherein said sugar is 0.5-6.0% sucrose.
70. The formulation of claim 60 , wherein said stabilizing agent comprises an amino acid.
71. The formulation of claim 70 , wherein said amino acid is glycine.
72. The method of claim 33 , wherein said buffer comprises about 10 mM histidine.
73. The method of claim 34 , wherein said buffer is at a pH of about 6.0-6.5.
74. The method of claim 34 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
75. The method of claim 74 , wherein said formulation further comprises a tonicity agent.
76. The method of claim 75 , wherein said tonicity agent is sodium chloride.
77. The method of claim 74 , wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
78. The method of claim 74 , wherein said organic co-solvent comprises 0.05-0.15% polysorbate 20.
79. The method of claim 74 , wherein said organic co-solvent comprises 0.003-0.15% polysorbate.
80. The method of claim 74 , wherein said stabilizing agent comprises a sugar.
81. The method of claim 80 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
82. The method of claim 80 , wherein said sugar is 5-20% sucrose.
83. The method of claim 80 , wherein said sugar is 0.5-6.0% sucrose.
84. The method of claim 74 , wherein said stabilizing agent comprises an amino acid.
85. The method of claim 84 , wherein said amino acid is glycine.
86. The method of claim 37 , wherein said buffer comprises 5-50 mM histidine.
87. The method of claim 37 , wherein said buffer comprises about 10 mM histidine.
88. The method of claim 86 , wherein said buffer is at a pH of about 6.0-6.5.
89. The method of claim 86 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
90. The method of claim 89 , wherein said formulation further comprises a tonicity agent.
91. The method of claim 90 , wherein said tonicity agent is sodium chloride.
92. The method of claim 89 , wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
93. The method of claim 89 , wherein said organic co-solvent comprises 0.05-0.15% polysorbate 20.
94. The method of claim 89 , wherein said organic co-solvent comprises 0.003-0.15% polysorbate.
95. The method of claim 89 , wherein said stabilizing agent comprises a sugar.
96. The method of claim 95 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
97. The method of claim 95 , wherein said sugar is 5-20% sucrose.
98. The method of claim 95 , wherein said sugar is 0.5-6.0% sucrose.
99. The method of claim 89 , wherein said stabilizing agent comprises an amino acid.
100. The method of claim 99 , wherein said amino acid is glycine.
101. The method of claim 39 , wherein said buffer comprises about 10 mM histidine.
102. The method of claim 40 , wherein said buffer is at a pH of about 6.0-6.5.
103. The method of claim 40 , wherein said formulation comprises 40 mg/ml of said VEGF antagonist fusion protein.
104. The method of claim 103 , wherein said formulation further comprises a tonicity agent.
105. The method of claim 104 , wherein said tonicity agent is sodium chloride.
106. The method of claim 103 , wherein said organic co-solvent comprises 0.1-0.5% polysorbate.
107. The method of claim 103 , wherein said organic co-solvent comprises 0.05-0.15% polysorbate 20.
108. The method of claim 103 , wherein said organic co-solvent comprises 0.003-0.15% polysorbate.
109. The method of claim 103 , wherein said stabilizing agent comprises a sugar.
110. The method of claim 109 , wherein said sugar is selected from group consisting of dextrose, ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
111. The method of claim 109 , wherein said sugar is 5-20% sucrose.
112. The method of claim 109 , wherein said sugar is 0.5-6.0% sucrose.
113. The method of claim 103 , wherein said stabilizing agent comprises an amino acid.
114. The method of claim 113 , wherein said amino acid is glycine.