Vegf antagonist formulations

Abstract

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.

We claim: 
     
         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.