Patent ID: 7655766

Claim:
A plurality of partially dePEGylated lysine/N-terminal positional PEG isomers of a recombinant G-CSF polypeptide, said recombinant G-CSF polypeptide comprising the substitutions K16R, K34R, K40R, T105K and S159K relative to wild-type human G-CSF, produced by a method comprising: a) expressing the recombinant G-CSE 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 selected from the group consisting of mPEG-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butanoate (mPEG-SBA), or mPEG-succinimidyl α-methylbutanoate (mPEG-SMB) under conditions to produce a plurality of positional PEG isomers of the recombinant G-CSF polypeptide. d) reacting the plurality of positional PEG isomers at a pH of 8.5 to 10.5 under conditions which produce a plurality of partially dePEGylated lysine/N-terminal positional PEG isomers of the recombinant G-CSF polypeptide, wherein at least 80% of the plurality of the partially de PEGylated lysine/N-terminal positional PEG isomers are a mixture of two positional PEG isomers each having three PEG groups, wherein one of the two positional PEG isomers has PEG moieties attached the N-terminal, Lys23 and Lys159 and the other of the two positional PEG isomers has PEG moieties attached at the N-terminal, Lys105 and Lys159.