1 research outputs found
Cysteine-Specific PEGylation of rhG-CSF via Selenylsulfide Bond
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) <i>N</i>-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 <i>ortho</i>-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 <i>in vivo</i> 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 <i>in vitro</i> biological activity and aggregate formation of
the maleimide conjugate. The conjugate prepared with PEG-Se had the
highest <i>in vitro</i> biological activity, while the conjugate
prepared with PEG-OPSS had the best <i>in vivo</i> performance