Pegylated g-csf polypeptides and methods of producing same

Abstract

A method for increasing the stability and uniformity of a PEGylated G-CSF polypeptide having at least one PEG moiety attached to the epsilon amino group of a lysine residue or the N-terminal amino group and at least one PEG moiety attached to a hydroxyl group, comprising subjecting the polypeptide to an elevated pH of above 8.0 for a period of time suitable to remove PEG moieties attached to a hydroxyl group, and reducing the pH to about 8.0 or lower; as well as PEGylated G-CSF polypeptides and compositions produced according to the method and methods for increasing neutrophil levels in a patient using the PEGylated G-CSF polypeptides and compositions.

1 . A method for increasing the stability and uniformity of a PEGylated G-CSF polypeptide having at least one PEG moiety attached to the epsilon amino group of a lysine residue or the N-terminal amino group and at least one PEG moiety attached to a hydroxyl group, comprising subjecting the polypeptide to an elevated pH of above 8.0 for a period of time suitable to remove PEG moieties attached to a hydroxyl group, and reducing the pH to about 8.0 or lower.  
     
     
         2 . The method of  claim 1 , wherein the elevated pH is in the range of from about 8.5 to about 10.5.  
     
     
         3 . The method of  claim 2 , wherein the elevated pH is in the range of from about 9.0 to about 10.0.  
     
     
         4 . The method of  claim 3 , wherein the elevated pH is in the range of from about 9.2 to about 9.8, e.g. about 9.5.  
     
     
         5 . The method of  claim 1 , wherein the polypeptide is subjected to the elevated pH for a period of from about 2 hours to about 100 hours.  
     
     
         6 . The method of  claim 5 , wherein the polypeptide is subjected to the elevated pH for a period of from about 4 hours to about 72 hours.  
     
     
         7 . The method of  claim 6 , wherein the polypeptide is subjected to the elevated pH for a period of from about 8 hours to about 48 hours.  
     
     
         8 . The method of  claim 7 , wherein the polypeptide is subjected to the elevated pH for a period of from about 12 hours to about 30 hours.  
     
     
         9 . The method of  claim 1 , further comprising subjecting the polypeptide at a reduced pH to at least one chromatographic purification step.  
     
     
         10 . The method of  claim 9 , wherein the chromatographic purification step is ion exchange chromatography.  
     
     
         11 . The method of  claim 10 , wherein the pH is reduced to below about 7.0 and the chromatographic purification step is cation exchange chromatography.  
     
     
         12 . The method of  claim 9 , wherein the chromatographic purification step is gel filtration chromatography.  
     
     
         13 . The method of  claim 1 , wherein the pH is reduced to a pH in the range of from about 2.0 to about 5.0.  
     
     
         14 . The method of  claim 13 , wherein the pH is reduced to a pH in the range of from about 2.5 to about 4.5.  
     
     
         15 . A method for producing a PEGylated G-CSF polypeptide, comprising subjecting a G-CSF polypeptide to a PEGylation reaction with an amine-specific activated polyethylene glycol (PEG) to produce a PEGylated G-CSF polypeptide intermediate, and subsequently subjecting the PEGylated polypeptide intermediate to an elevated pH of at least about 9.0 for a period of time suitable to remove PEG moieties attached to a hydroxyl group to produce the PEGylated G-CSG polypeptide.  
     
     
         16 . The method of  claim 15 , wherein the PEGylation reaction is performed at a pH in the range of about 7.0-9.0.  
     
     
         17 . The method of  claim 15 , wherein the PEGylation reaction is performed at a pH above 9.0.  
     
     
         18 . The method of  claim 17 , wherein the PEGylation reaction and subsequent removal of PEG moieties attached to a hydroxyl group are performed as a single step at a single pH value.  
     
     
         19 . The method of  claim 15 , wherein the amine-specific activated PEG is mPEG-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butanoate (mPEG-SBA), or mPEG-succinimidyl α-methylbutanoate (mPEG-SMB).  
     
     
         20 . The method of  claim 15 , wherein the amine-specific activated PEG is mPEG-SPA with a molecular weight of about 5 kDa.  
     
     
         21 . The method of  claim 15 , wherein the elevated pH is in the range of from about 9.2 to 11.0.  
     
     
         22 . The method of  claim 21 , wherein the elevated pH is in the range of from about 9.2 to 10.0.  
     
     
         23 . A PEGylated G-CSF polypeptide produced by the method of  claim 1 .  
     
     
         24 . The PEGylated G-CSF polypeptide of  claim 23 , wherein the polypeptide is a G-CSF variant comprising the substitutions K16R, K34R, K40R, T105K and S159K relative to wild-type human G-CSF.  
     
     
         25 . A composition comprising a mixture of positional PEG isomers of a PEGylated G-CSF polypeptide, wherein the polypeptide comprises the substitutions K16R, K34R, K40R, T105K and S159K relative to wild-type human G-CSF, and wherein at least about 80% of the positional PEG isomers of the polypeptide are lysine/N-terminal positional PEG isomers having three attached PEG moieties.  
     
     
         26 . The composition of  claim 25 , wherein at least about 85% of the positional PEG isomers of the polypeptide are lysine/N-terminal positional PEG isomers having three attached PEG moieties.  
     
     
         27 . The composition of  claim 25 , wherein at least about 90% of the positional PEG isomers of the polypeptide are lysine/N-terminal positional PEG isomers having three attached PEG moieties.  
     
     
         28 . The composition of  claim 25 , wherein the at least about 80% of the positional PEG isomers having three attached PEG moieties are composed of two positional PEG isomers, wherein one of the isomers has PEG moieties attached at the N-terminal, Lys23 and Lys159 and the other isomer has PEG moieties attached at the N-terminal, Lys105 and Lys159.  
     
     
         29 . The composition of  claim 26 , wherein the at least about 85% of the positional PEG isomers having three attached PEG moieties are composed of two positional PEG isomers, wherein one of the isomers has PEG moieties attached at the N-terminal, Lys23 and Lys159 and the other isomer has PEG moieties attached at the N-terminal, Lys105 and Lys159.  
     
     
         30 . The composition of  claim 27 , wherein the at least about 90% of the positional PEG isomers with three attached PEG moieties are composed of two positional PEG isomers, wherein one of the isomers has PEG moieties attached at the N-terminal, Lys23 and Lys159 and the other isomer has PEG moieties attached at the N-terminal, Lys105 and Lys159.  
     
     
         31 . A composition comprising a mixture of positional PEG isomers of a PEGylated G-CSF polypeptide, wherein the polypeptide comprises the substitutions K16R, K34R, K40R, T105K and S159K relative to wild-type human G-CSF, and wherein at least about 80% of the positional PEG isomers of the polypeptide are lysine/N-terminal positional PEG isomers having 3 attached PEG moieties after storage in an aqueous reference composition for 3 months at a temperature of 5° C.  
     
     
         32 . A composition comprising a pharmaceutically acceptable carrier and a PEGylated G-CSF polypeptide according to  claim 23 .  
     
     
         33 . A method of producing a mixture of lysine/N-terminal positional PEG isomers of a recombinant G-CSF polypeptide comprising the substitutions K16R, K34R, K40R, T105K and S159K relative to wild-type human G-CSF, comprising: 
 a) expressing the recombinant G-CSF polypeptide in a host cell;    b) isolating the recombinant G-CSF polypeptide;    c) reacting the isolated recombinant G-CSF polypeptide with an amine-specific activated PEG to produce a plurality of positional PEG isomers of the recombinant G-CSF polypeptide; and    d) reacting the plurality of positional PEG isomers at a pH of 8.5 to 10.5 to produce a plurality of partially dePEGylated lysine/N-terminal positional PEG isomers of the recombinant G-CSF polypeptide.    
     
     
         34 . The method of  claim 33 , wherein the amine-specific activated PEG is selected from the group consisting of mPEG-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butanoate (mPEG-SBA), or mPEG-succinimidyl c-methylbutanoate (mPEG-SMB).  
     
     
         35 . The method of  claim 33 , further comprising subjecting the plurality of lysine/N-terminal positional PEG isomers of the recombinant G-CSF polypeptide to one or more chromatographic purification steps to produce a substantially purified mixture of lysine/N-terminal positional PEG isomers of the G-CSF polypeptide.  
     
     
         36 . The method of  claim 35 , further comprising combining an effective dose of the substantially purified mixture of lysine/N-terminal positional PEG isomers of the recombinant G-CSF polypeptide with at least one pharmaceutically acceptable excipient to produce a pharmaceutical composition.  
     
     
         37 . The plurality of partially dePEGylated lysine/N-terminal positional PEG isomers produced by the method of  claim 33 .  
     
     
         38 . The substantially purified mixture of lysine/N-terminal positional PEG isomers produced by the method of  claim 35 .  
     
     
         39 . The pharmaceutical composition produced by the method of  claim 36 .  
     
     
         40 . A method for increasing the level of neutrophils in a patient suffering from or at risk of an insufficient neutrophil level, comprising administering to said patient an effective dose of a PEGylated G-CSF polypeptide according to  claim 23 .  
     
     
         41 . The method of  claim 40 , wherein the insufficient neutrophil level is a result of chemotherapy or radiation therapy.  
     
     
         42 .- 43 . (canceled)  
     
     
         44 . A composition comprising a homogeneous mixture of positional PEG isomers of a PEGylated G-CSF polypeptide variant, wherein at least about 80% of the positional PEG isomers of the mixture consist of two positional PEG isomers each having PEG moieties consisting of two PEG moieties bound to epsilon amino groups of lysine and one PEG moiety bound to the N-terminal amino group.  
     
     
         45 . The composition of  claim 44  wherein at least about 85% of the positional PEG isomers of the homogenous mixture consist of two positional PEG isomers each having PEG moieties consisting of two PEG moieties bound to epsilon amino groups of lysine and one PEG moiety bound to the N-terminal amino group.  
     
     
         46 . The composition of  claim 44  wherein at least about 90% the positional PEG isomers of the homogeneous mixture consist of two positional PEG isomers each having PEG moieties consisting of two PEG moieties bound to epsilon amino groups of lysine and one PEG moiety bound to the N-terminal amino group.  
     
     
         47 . A composition comprising a pharmaceutically acceptable carrier and a composition according to  claim 44 .  
     
     
         48 . A method for increasing the level of neutrophils in a patient suffering from or at risk of an insufficient neutrophil level, comprising administering to said patient an effective dose of a composition according to  claim 44 .  
     
     
         49 . (canceled)