PEGylation Technology in Nanomedicine - Polymers in Nanomedicine - page 117

clinical use by the FDA. However, nonselective conjugations of the amino and/or
thiol groups in protein molecules results in product heterogeneity, which often
causes significant deactivation of the product. For example, 20-70% of native
interferon-beta-1b (IFN- b -1b) antiviral activity was retained in mono-PEGylated
IFN- b -1b, but the activity was greatly reduced or disappeared almost completely in
multi-PEGylated IFN- b -1b [ 4 ]. Therefore, the development of site-specific
PEGylation technology is quite important for developing more active and safer
drugs.
2.1 Site-Specific PEGylation
PEGylation of drugs with an amine- or thiol-specific reagent is effective and thus
the most popular method in current use. The thiol group of cysteine is often used for
PEGylation of protein because PEGylation at cysteine is more specific than that of
the amino group of lysine. Generally, cysteine forms dithiol linkages and the free
thiol group is not available. To utilize the free thiol group for conjugation, it is
necessary to engineer a new and free cysteine into the protein via recombination
techniques. Although this approach works well, the genetic engineering involved in
the process requires high skill, and protein misfolding and aggregation often occur
during the purification process. To overcome this problem, Brocchini et al. developed
site-specific PEGylation technology [ 5 , 6 ]. An outline of their technology is shown
in Fig. 3 . Briefly, this technique involves the synthesis of a novel bis-thiol-specific
PEG reagent (PEG monosulfone) containing a thiol-specific bis-alkylating group,
which comprises an a , b -unsaturated carbonyl group possessing a sulfomethyl
group at the a -position of the unsaturated double bond. After the reduction of the
disulfide bond in the protein, both free reactive thiol groups react with the PEG
reagent to produce disulfide-bridging PEGylation with a three-carbon bridge.
Fig. 3 Chemistry of site-specific PEGylation developed by Brocchini et al. [ 5 , 6 ]. After cleavage
of the native disulfide bond between two cysteine thiols by reduction, the free cysteines are reacted
with an a , b -unsaturated PEG derivative to produce a PEG conjugate via a three-carbon bridge