Site-Specific Protein PEGylation Application to Cysteine Analogs of Recombinant Human Granulocyte Colony-Stimulating Factor

APRIL 2005 G ranulocyte colonystimulating factor (G-CSF) is a 19-kDa glycoprotein that stimulates proliferation, maturation, and functional activation of cells in the granulocyte lineage. Recombinant G-CSF is widely used to ameliorate neutropenia resulting from myelosuppressive chemotherapy and bone marrow transplantation as well as to mobilize peripheral blood progenitor cells for transplantation and blood banking (1, 2). G-CSF has a short circulating half-life, which necessitates that the protein be administered to patients through daily subcutaneous injection. Development of modified forms that last longer in vivo — and thus can be administered less frequently — is of significant interest to patients and healthcare providers. Covalent modification of G-CSF with polymers such as polyethylene glycol (PEG) increases its circulating half-life in animals and humans (3– 6). Covalent attachment of PEG to a protein increases that protein’s effective size and reduces its rate of clearance from the body. Previous studies have used amine-reactive PEGs to modify G-CSF at exposed amine groups on lysine residues and the N-terminal amino acids. Such an approach is not optimal for G-CSF, however, because the protein contains four lysine residues in addition to its N-terminal amino acid, and those lysine residues are located in regions implicated in receptor binding (7–10). Not surprisingly, modification of G-CSF with amine-reactive PEG reagents reduces in vitro biological activity of the protein by threeto 50-fold, depending on the number and sizes of attached PEG molecules (3, 4). Loss of in vitro bioactivity is greatest when G-CSF is modified with large PEGs — e.g., 20-kDa PEGs, which are most useful for extending the protein’s half-life. Amine-PEGylated G-CSF also is heterogeneous, occurring as a complex mixture of at least four isoforms and multiple molecular weight species, all of which have different specific activities (3–5). Less heterogeneous PEG–G-CSF conjugates with less severe loss of in vitro bioactivity can be prepared by preferential attachment of PEG to the N-terminus of G-CSF (5, 6, 11). An alternative method for PEGylating proteins covalently attaches PEG to cysteine residues using cysteine-reactive PEGs such as maleimide PEGs. At near-neutral pH values, these PEG reagents selectively attach to the thiol groups of “free” cysteine residues (those not involved in disulfide bonds). Because most proteins contain no surface-exposed free cysteine residues, attachment of a PEG molecule can be targeted to a specific site by introducing a free cysteine residue into a protein using site-directed mutagenesis followed B I O P R O C E S S TECHNICAL