Three-branched polyethylene glycol-G-CSF conjugate

1. A three-branched PEG-G-CSF conjugate of the following general formula (1):

in which a bonding ratio of three-branched polyethylene glycol(PEG) and G-CSF is 1:1 (mol/mol), and PEG has an average molecular weight of about 23,000 daltons,

wherein,

n is an integer of 1 to 1,000;

m is an integer of 10 to 1,000;

Z is (CH 2 )s or (CH 2 ) s NHCO(CH 2 )s as a linker of G-CSF and PEG wherein S is an integer of 1 to 6;

Y is an amide bond formed by combining NH 2 functional group in G-CSF and a functional group of PEG derivative.

2. A method of preparing a three-branched PEG-G-CSF conjugate of the general formula (1) of claim 1 comprising:

preparing a mixed solution including a three-branched PEG derivative of the following general formula (2) and G-CSF;

reacting the three-branched PEG derivative and G-CSF by stirring the mixed solution at a temperature of about 4° C. for about 90 minutes to forma covalent bond between the three-branched PEG derivative of the following general formula (2) and G-CSF;

then adding an acid to the mixed solution to complete the reaction of the three-branched PEG derivative and G-CSF; and

then identifying the three-branched PEG-G-CSF conjugate by subjecting said mixed solution to chromatography,

wherein PEG (polyethylene glycol) has an average molecular weight about 23,000 daltons;

wherein,

n is an integer of 1 to 1,000;

m is an integer of 10 to 1,000;

Z is (CH 2 ) s or (CH 2 ) s NHCO(CH 2 ) s as a linker of G-CSF and PEG wherein S is an integer of 1 to 6; and

the functional group which can chemically react with proteins and peptides containing G-CSF is N-hydrosuccinimide.

3. The method of claim 2 , wherein the molar ratio of G-CSF to three-branched PEG derivative is from 1:0.5 to 1:50.

4. The method of claim 3 , wherein the molar ratio of G-CSF to three branched PEG derivative in the reaction is from 1:0.5 to 1:5.

5. A pharmaceutical composition comprising a three-branched PEG-G-CSF conjugate of the following general formula (1):

in which a bonding ratio of three-branched polyethylene glycol (PEG) and G-CSF is 1:1 (mol:mol), and PEG has an average molecular weight of 23,000 daltons,

wherein,

n is an integer of 1 to 1,000;

m is an integer of 10 to 1,000;

Z is (CH 2 ) S or (CH 2 ) S as a linker of G-CSF and PEG wherein S is an integer of 1 to 6;

Y is an amide bond formed b combining NH 2 functional group in G-CSF and a functional group of PEG derivative, and

at least one pharmaceutically acceptable diluent, antiseptic, solubilizer, emulsifier, adjuvant, or combination thereof.

6. A method of treating neutropenia caused by treatment by cyclophosphamide in a patient in need thereof, comprising administering to said patient pharmaceutical composition of claim 5 .

7. A composition comprising the three-branched PEG-G-CSF conjugate of claim 1 , in further combination with at least one diluent, antiseptic, solubilizer, emulsifier, adjuvant, or combination thereof.

8. A composition comprising the conjugate of claim 1 as effective ingredient and at least one diluent, antiseptic, solubilizer, emulsifier, adjuvant, or combination thereof.