Preparation and characterization of monopegylated human granulocyte-macrophage colony-stimulating factor.
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.
Lee
Darin L
DL
Cangene Corporation, Mississauga, ON, Canada M9W 6A9.
Sharif
Isha
I
Kodihalli
Shantha
S
Stewart
Donald I H
DI
Tsvetnitsky
Vadim
V
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
D011994:Recombinant Proteins; C527993:monopegylated granulocyte-macrophage colony-stimulating factor, human; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor