Process for fractionating polyethylene glycol (PEG) - protein adducts and an adduct of PEG and granulocyt-macrophage colony stimulating factor

Abstract

The present invention provides adducts of polyethylene glycol and granulocyte-macrophage colony stimulating factor as well as methods for modifying a protein with pleiotropic activities which involve coupling polyethylene glycol to the protein.

1 . A process for fractionating a mixture of polyethylene glycol (PEG)-protein adducts comprising partitioning the PEG/protein adducts in a PEG-containing aqueous biphasic system.  
     
     
         2 . A process according to  claim 1  comprising the futher step of recovering a PEG-protein adduct of pre-determined degree of PEG substitution from one phase of the biphasic system.  
     
     
         3 . A process according to  claim 1  or  claim 2  wherein the PEG-protein adduct is an adduct of mono methoxyPEG.  
     
     
         4 . A process according to  claim 3  wherein the monomethoxyPEG adduct is formed by reaction of the protein with 2,2,2-trifluoroethanesulphonyl monomethoxy polyethylene glycol (TMPEG).  
     
     
         5 . A process according to  claim 4  wherein unreacted TMPEG is destroyed or the adduct-forming reaction is quenched by addition of lysine or albumin.  
     
     
         6 . A process according to any one of  claims 1  to  5  wherein partitioning is performed batchwise or continuously.  
     
     
         7 . A process according to  claim 6  wherein partitioning is performed by counter-current flow of the two phases.  
     
     
         8 . A process according to any one of  claims 1  to  7  wherein the protein is granulocyte-macrophage colony stimulating factor (gm-CSF).  
     
     
         9 . A PEG-gm-CSF adduct.  
     
     
         10 . A pharmaceutical formulation comprising a PEG-gm-CSF adduct and a pharmaceutically acceptable carrier or diluent therefor.  
     
     
         11 . A formulation according to  claim 10  wherein the diluent of carrier is suitable for injection.  
     
     
         12 . A formulation according to  claim 11  wherein the diluent or carrier is or comprises sterile water for injection.  
     
     
         13 . A composition according to any one of  claims 11  to  13  further comprising at least one buffer, isotonic agent, preservative of antioxidant.  
     
     
         14 . A PEG-gm-CSF adduct according to  claim 10  or a pharmaceutical formulation according to any one of  claims 11  to  13  for use in a method of treatment of the human or animal body or in a diagnostic method practised on the human or animal body.  
     
     
         15 . Use of a PEG-gm-CSF adduct according to  claim 10  or a pharmaceutical formulation according to any one of  claims 11  to  13  in the preparation of a medicament for use in a method of treatment of the human or animal body or a diagnostic method practised on the human or animal body.  
     
     
         16 . Use according to  claim 15  wherein the medicament is administered by intravenous or subcutaneous injection or infusion.  
     
     
         17 . A therapeutic of diagnostic method for the treatment of the human or animal body comprising administering an effective non-toxic amount of a PEG-gm-CSF adduct according to  claim 10  or a pharmaceutical composition according to any one of  claims 11  to  13  to a human or non-human animal in need thereof.