PEGYLATED G-CSF POLYPEPTIDES AND METHODS OF PRODUCING SAME
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 .- 39 . (canceled)
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 produced by a method 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 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, and
subjecting the PEGylated G-CSF polypeptide intermediate 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 a reduced pH of about 8.0 or lower,
thereby producing the PEGylated G-CSF polypeptide.
41 . The method of claim 40 , wherein the insufficient neutrophil level is a result of chemotherapy or radiation therapy.
42 .- 47 . (canceled)
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 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.
49 . (canceled)
50 . The method of claim 40 , 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 α-methylbutanoate (mPEG-SMB).
51 . The method of claim 50 , wherein the amine-specific activated PEG is mPEG-SPA with a molecular weight of about 5 kDa.
52 . The method of claim 40 , wherein the G-CSF polypeptide is a G-CSF variant comprising the substitutions K16R, K34R, K40R, T105K and S159K relative to wild-type human G-CSF.
53 . 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 comprising a mixture of positional PEG isomers of a PEGylated G-CSF polypeptide, wherein the G-CSF 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.
54 . The method of claim 53 , wherein the insufficient neutrophil level is a result of chemotherapy or radiation therapy.
55 . The method of claim 48 , wherein the insufficient neutrophil level is a result of chemotherapy or radiation therapy.