title: Preparation and characterization of monopegylated human granulocyte-macrophage colony-stimulating factor.
abstract: ABSTRACT Conjugates of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) attached to polyethylene glycol (PEG) chains were prepared using amine-reactive chemistry. Molecular masses of the PEGs were 20, 30, and 40 kDa. The monopegylated forms were isolated by anion-exchange chromatography and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography, mass spectrometry, reverse-phase high-performance liquid chromatography (HPLC), peptide mapping, in vitro cell proliferation bioassays, and rat pharmacokinetic studies. The pegylation site of the purified monopegylated products was identified as the N-terminus of the protein. All forms of pegylated GM-CSF were able to stimulate TF-1 cell proliferation in a colorimetric bioassay at concentrations equal to or lower than that of GM-CSF. Pharmacokinetic studies in rats demonstrated 32-fold, 27-fold, and 40-fold extensions in elimination half-lives for 20, 30, and 40 kDa PEG-GM-CSF, respectively, as compared with nonmodified GM-CSF.
authors.affiliation: Cangene Corporation, Mississauga, ON, Canada M9W 6A9.
authors.forename: Darin L
authors.initials: DL
authors.lastname: Lee
authors.forename: Isha
authors.initials: I
authors.lastname: Sharif
authors.forename: Shantha
authors.initials: S
authors.lastname: Kodihalli
authors.forename: Donald I H
authors.initials: DI
authors.lastname: Stewart
authors.forename: Vadim
authors.initials: V
authors.lastname: Tsvetnitsky
mesh_terms: D000818:Animals; D001681:Biological Assay; D049109:Cell Proliferation / Q000187:drug effects; D002845:Chromatography; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D013058:Mass Spectrometry; D010449:Peptide Mapping; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins
chemical_list: D011994:Recombinant Proteins; C527993:monopegylated granulocyte-macrophage colony-stimulating factor, human; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor