Igf-i poly (ethylene glycol) conjugates

Abstract

Herein is reported a method for producing of a polypeptide conjugated to one poly (ethylene glycol) comprising a) providing a nucleic acid encoding an expression construct comprising in 5′ to 3′ direction a nucleic acid encoding a polypeptide, and a nucleic acid encoding a trypsin site of SEQ ID NO: 01, b) expressing the nucleic acid of a) in a cell and recovering the expression construct from the cell and/or the cultivation medium, c) providing a target peptide with man amino acid sequence of SEQ ID NO: 02 covalently conjugated to a poly (ethylene glycol) at the C-terminal lysine residue, d) incubating the expression construct and the target peptide with the trypsin mutant D189K, K60E, N143H, E151H, and e) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) from the incubation mixture.

1 . A method for the production of a polypeptide conjugated to a poly (ethylene glycol) residue comprising the following steps
 a) incubating a fusion polypeptide, which is encoded by a nucleic acid comprising in 5′ to 3′ direction i) a nucleic acid encoding the polypeptide, and ii) a nucleic acid encoding an amino acid sequence of SEQ ID NO: 01, and a target polypeptide, which has the amino acid sequence of SEQ ID NO: 02 and which is covalently conjugated to a poly (ethylene glycol) residue at the C-terminal lysine residue, with the trypsin mutant D189K, K60E, N143H, E151H, and   b) recovering and thereby producing the polypeptide conjugated to one poly (ethylene glycol) residue.   
     
     
         2 . The method according to  claim 1 , characterized in that the incubating comprises
 i) incubating the fusion polypeptide and the target peptide with guanidinium hydrochloride in a buffered solution,   ii) incubating the trypsin mutant D189K, K60E, N 143H, E 151 H with a Zn(II)-salt, and   iii) combining and incubating the incubation mixtures of i) and ii).   
     
     
         3 . The method according to  claim 1 , characterized in that the fusion polypeptide is encoded by a nucleic acid comprising in 5′ to 3′ direction:
 i) a nucleic acid encoding the polypeptide, 
 ii) a nucleic acid encoding an amino acid sequence of SEQ ID NO: 01, and 
 iii) a nucleic acid encoding an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 27. 
 
     
     
         4 . The method according to  claim 1 , characterized in that the incubating is for 150 minutes to 180 minutes. 
     
     
         5 . The method according to  claim 1 , characterized in that the nucleic acid in step a) comprises in addition iii) a nucleic acid encoding an amino acid sequence of SEQ ID NO: 13. 
     
     
         6 . The method according to  claim 1 , characterized in that the incubating is in HEPES buffered solution. 
     
     
         7 . The method according to  claim 1 , characterized in that the polypeptide is human IGF-I. 
     
     
         8 . The method according to  claim 7 , characterized in that the fusion polypeptide has an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. 
     
     
         9 . The method according to  claim 1 , characterized in that the nucleic encoding a fusion polypeptide is a nucleic acid encoding a polypeptide with the amino acid sequence of SEQ ID NO: 10. 
     
     
         10 . A polypeptide that has the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29 and that is conjugated to one poly (ethylene glycol) residue at the C-terminal lysine residue. 
     
     
         11 . A pharmaceutical composition comprising a polypeptide according to  claim 10 . 
     
     
         12 . Use of a polypeptide according to  claim 10  for the manufacture of a medicament for the treatment of Alzheimer's disease. 
     
     
         13 . A polypeptide according to  claim 10  for use as a medicament. 
     
     
         14 . A polypeptide according to  claim 10  for the treatment or prevention of Alzheimer's disease. 
     
     
         15 . A method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide according to  claim 10 .