VEGF ANTAGONIST FORMULATIONS
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 polynucleotide encoding a VEGF antagonist fusion protein, wherein said VEGF antagonist fusion protein comprises an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor, an Ig domain 3 of a second VEGF receptor, and a multimerizing component; and wherein said fusion protein does not comprise a signal peptide or a C-terminal lysine.
2 . The polynucleotide of claim 1 , wherein said signal peptide comprises amino acids 1-26 of SEQ ID NO:2.
3 . The polynucleotide of claim 1 , wherein said fusion protein comprises amino acids 27-457 of SEQ ID NO:2 or amino acids 27-457 of SEQ ID NO:4.
4 . The polynucleotide of claim 1 comprising nucleic acids 146-1439 of SEQ ID NO:1.
5 . The polynucleotide of claim 1 comprising nucleic acids 78-1371 of SEQ ID NO:3.
6 . A method of manufacturing a VEGF antagonist fusion protein that comprises in order from the N-terminus to the C-terminus an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor, an Ig domain 3 of a second VEGF receptor, and a multimerizing component, and does not comprise a signal peptide or a C-terminal lysine, said method comprising:
a. expressing said VEGF antagonist fusion protein in a mammalian cell, wherein said mammalian cell comprises a polynucleotide that encodes said VEGF antagonist fusion protein; and
b. purifying said VEGF antagonist fusion protein.
7 . The method of claim 6 , wherein said mammalian cell is a CHO cell.
8 . The method of claim 6 , wherein said fusion protein is substantially free of protein contaminants.
9 . The method of claim 8 , wherein at least 90% of the weight of said fusion protein is not present as an aggregate.
10 . The method of claim 6 , wherein said VEGF antagonist fusion protein is glycosylated at one or more asparagine residues.
11 . The method of claim 10 , wherein said VEGF antagonist fusion protein comprises amino acids 27-457 of SEQ ID NO:4.
12 . The method of claim 11 , wherein said VEGF antagonist fusion protein is glycosylated at asparagine residues 62, 94, 149, 222 and 308.
13 . The method of claim 10 , wherein said VEGF antagonist fusion protein comprises amino acids 27-457 of SEQ ID NO:2.
14 . The method of claim 13 , wherein said VEGF antagonist fusion protein is glycosylated at asparagine residues 59, 91, 146, 219 and 308.
15 . A VEGF antagonist fusion protein comprising an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor, an Ig domain 3 of a second VEGF receptor, and a multimerizing component; and not comprising a signal peptide or a C-terminal lysine.
16 . The VEGF antagonist fusion protein of claim 15 , wherein said VEGF antagonist fusion protein forms a dimer.
17 . The VEGF antagonist fusion protein of claim 15 , wherein said signal peptide comprises amino acids 1-26 of SEQ ID NO:2.
18 . The VEGF antagonist fusion protein of claim 15 , wherein said fusion protein comprises amino acids 27-457 of SEQ ID NO:2.
19 . The VEGF antagonist fusion protein of claim 18 , wherein said fusion protein is glycosylated at asparagine residues 59, 91, 146, 219, and 308.
20 . The VEGF antagonist fusion protein of claim 15 , wherein said fusion protein comprises amino acids 27-457 of SEQ ID NO:4.
21 . The VEGF antagonist fusion protein of claim 20 , wherein said fusion protein is glycosylated at asparagine residues 62, 94, 149, 222 and 308.
22 . A mammalian cell comprising a polynucleotide that encodes a fusion protein, wherein said fusion protein comprises an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor, an Ig domain 3 of a second VEGF receptor, and a multimerizing component; wherein said fusion protein does not comprise a signal peptide or a C-terminal lysine; and wherein said fusion protein binds vascular endothelial growth factor (VEGF).
23 . The mammalian cell of claim 22 , wherein said mammalian cell is a Chinese hamster ovary (CHO) cell.
24 . The mammalian cell of claim 22 , wherein said signal peptide comprises amino acids 1-26 of SEQ ID NO:2.
25 . The mammalian cell of claim 22 , wherein said fusion protein comprises amino acids 27-457 of SEQ ID NO:2.
26 . The mammalian cell of claim 25 , wherein said fusion protein is glycosylated at asparagine residues 59, 91, 146, 219, and 308.
27 . The mammalian cell of claim 25 , wherein said polynucleotide comprises nucleotides 146-1439 of SEQ ID NO:1.
28 . The mammalian cell of claim 22 , wherein said fusion protein comprises amino acids 27-457 of SEQ ID NO:4.
29 . The mammalian cell of claim 28 , wherein said fusion protein is glycosylated at asparagine residues 62, 94, 149, 222 and 308.
30 . The mammalian cell of claim 28 , wherein said polynucleotide comprises nucleotides 78-1371 of SEQ ID NO:3.