Site-Specifically Modified Recombinant Human Granulocyte Colony- Stimulating Factor with Polyethylene Glycol

To prepare PEGylated recombinant human granulocyte colony-stimulating factor (rHuG-CSF) with enhanced pharmacokinetic properties, we prepared a cysteine-substituted mutant of rHuG-CSF (mrHuG-CSF). For site-specific PEGylation of mrHuG-CSF, Threonine 134 residue that is one of the glycosylation sites and thought not to be critical in structure and function of rHuG-CSF was substituted by Cysteine. Also, Cysteine 17 residue at N-terminus that does not make di-sulfide bonds and is partially buried was substituted by Serine. This mrHuG-CSF was then site-specifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide and the correct molecular weight of the conjugates was confirmed by MALDI-TOF mass spectrometry analysis. Compared with unmodified rHuG-CSF, both PEGylated mrHuG-CSF showed similar biological activities in vitro and the plasma half-life of the 20 kDa PEGylated mrHuG-CSF was about 5-folds increased. Taken together, site-specific PEGylation of mrHuG-CSF may increase their therapeutic potency in humans.