Preparation of g-csf stoichiometrically conjugated with biocompatible polymers at cystein residue

• US 20050143563A1
• Filed: 03/20/2003
• Published: 06/30/2005
• Est. Priority Date: 03/20/2002
• Status: Active Grant

• Current Assignee: BiopolyMed, Inc.
• Original Assignee: BiopolyMed, Inc.
• Inventors: Park, Myung-Ok
• Granted Patent: US 7,557,195 B2
• Time in Patent Office: Days
• US Class Current: 530/351
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF

Abstract

The present invention relates to a conjugate of a biocompatible polymer and a G-CSF bonded through a thiol group of a dysteine residue in G-CSF at a 1:1 molar ratio, and methods of preparation thereof.

Claims

1. Conjugates of biocompatible polymer and wild type G-CSF, wherein activated biocompatible polymer is bonded to G-CSF at a 1:

1 molar ratio through a thiol group of a cysteine residue in G-CSF.

2. The conjugates according to claim 1, wherein the biocompatible polymers are selected from the group consisting of polyethylene glycol (PEG) and derivatives thereof, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and derivatives thereof, polyacrylic acid and derivatives thereof, polyamino acid, poly(vinyl alcohol), polyurethane, polyphosphazene, poly(L-lysine), polyalkylene oxide (PAO), polysaccharide, dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacryl amide and similar non-immunogenic polymers.

3. The conjugates according to claim 1, wherein G-CSF originates and is purified from a natural or recombinant protein source.

4. The conjugates according to claim 1, wherein the reactive functional group in biocompatible polymers for activation is selected from the group consisting of maleimide, acetamide, pentenoic amide, butenoic amide, isocyanate, isothiocyanate, cyanuric chloride, 1,4-benzoquinone, and disulfide.

5. A method of preparation of conjugates of biocompatible polymer and wild type G-CSF at a 1:

1 molar ratio, wherein the method comprises the steps of activating the biocompatible polymer, reacting the activated biocompatible polymer with G-CSF to form a conjugate of biocompatible polymer and G-CSF bonded through a thiol group of a cysteine residue in G-CSF, and adding SDS solution to prevent the conjugate from aggregating.

6. The method according to claim 5, wherein the biocompatible polymers are selected from the group consisting of polyethylene glycol (PEG) and derivatives thereof, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and derivatives thereof, polyacrylic acid and derivatives thereof, polyamino acid, poly(vinyl alcohol), polyurethane, polyphosphazene, poly(L-lysine), polyalkylene oxide (PAO), polysaccharide, dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacrylamide and similar non-immunogenic polymers.

7. The method according claim 5, wherein G-CSF originates and is purified from a natural or recombinant protein source.

8. The method according claim 5, wherein the reactive functional group in biocompatible polymers for activation is selected from the group consisting of maleimide, acetamide, pentenoic amide, butenoic amide, isocyanate, isothiocyanate, cyanuric chloride, 1,4-benzoquinone, and disulfide.

9. The method according to claim 5, wherein the concentration of SDS solution is from 0.005% to 0.1%.