title: Cysteine-specific PEGylation of rhG-CSF via selenylsulfide bond.
abstract: A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.
authors.affiliation: Sandoz Biopharmaceuticals, Mengeš, Lek Pharmaceuticals d.d., Kolodvorska 27, SI-1234 Mengeš, Slovenia. menci.kunstelj@sandoz.com
authors.forename: Menči
authors.initials: M
authors.lastname: Kunstelj
authors.forename: Katarina
authors.initials: K
authors.lastname: Fidler
authors.forename: Spela
authors.initials: S
authors.lastname: Skrajnar
authors.forename: Maja
authors.initials: M
authors.lastname: Kenig
authors.forename: Vanja
authors.initials: V
authors.lastname: Smilović
authors.forename: Mateja
authors.initials: M
authors.lastname: Kusterle
authors.forename: Simon
authors.initials: S
authors.lastname: Caserman
authors.forename: Irena
authors.initials: I
authors.lastname: Zore
authors.forename: Vladka Gaberc
authors.initials: VG
authors.lastname: Porekar
authors.forename: Simona
authors.initials: S
authors.lastname: Jevševar
mesh_terms: D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification / Q000493:pharmacokinetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics; D018036:Selenium Compounds / Q000737:chemistry* / Q000302:isolation & purification / Q000493:pharmacokinetics
chemical_list: D011994:Recombinant Proteins; D018036:Selenium Compounds; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols; D003545:Cysteine; C025698:selenium disulfide