title: Site-specific pegylation of G-CSF by reversible denaturation.
abstract: A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.
authors.affiliation: Department of Pharmaceutical Science, University of Padua, via Marzolo 5, 35100, Padua, Italy. francesco.veronese@unipd.it
authors.forename: Francesco M
authors.initials: FM
authors.lastname: Veronese
authors.forename: Anna
authors.initials: A
authors.lastname: Mero
authors.forename: Francesca
authors.initials: F
authors.lastname: Caboi
authors.forename: Mauro
authors.initials: M
authors.lastname: Sergi
authors.forename: Christian
authors.initials: C
authors.lastname: Marongiu
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Pasut
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D011092:Polyethylene Glycols / Q000737:chemistry*; D011489:Protein Denaturation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols