Biology Reference
In-Depth Information
Pegylated molecules have an increased hydrodynamic size because they create
a “water shell” around the molecule. The attachment of PEG is thought to
improve solubility and possibly reduce immunogenicity due to shielding by
the conjugate. In addition, the increased hydrodynamic size can result in
reduced clearance and thus increase
in vivo
activity.
Mixing activated polyethylene polymers with proteins under appropriate
chemical reaction conditions produces pegylated proteins. The duration of the
drug development process using this strategy is relatively short because exist-
ing starting materials can often be used for the chemical reaction. PEG is
thought to be relatively inert and non-immunogenic by itself,so it is a suitable
starting material for protein-conjugate therapeutics.
One issue with drugs made by solution or solid-phase chemistry can be poor
specificity of conjugation in the chemical reaction or generation of undesirable
by-products. Many pegylation chemistries have been tried to reduce undesir-
able by-products, improve the specificity and efficacy of PEG attachments,
and minimize immunogenicity risk of the protein conjugate while maximizing
the
in vitro
and
in vivo
activity of the resultant molecule [41]. The current
chemistries typically target the reactive amino groups on Lys or the amino ter-
minal amine. rHuEPO has eight Lys, some of which are part of the active site
[17]. Therefore, some pegylated EPO molecules conjugated to Lys may have
low activity because PEG may interfere with receptor binding and activation.
Other pegylated EPO molecules may have low activity because attached poly-
mer results in structural alterations that interfere with receptor interaction.
Analogs of proteins can be made to increase specificity. For example, Cys
substitutions at targeted regions can allow addition of the conjugate with high
specificity to the sulfhydryl group [42, 43]. Another strategy is to make pegy-
lated EPO synthetically: During synthesis, a PEG-conjugated amino acid
could be introduced instead of the unconjugated amino acid. This approach
allows specific targeting of particular amino acid positions for PEG attach-
ment, such as the glycosylation sites, and reduces heterogeneity and the poten-
tial for loss of
in vitro
activity. It is not clear that these molecules will retain
the same stability,
in vivo
activity, and lack of immunogenicity as their glyco-
sylated counterparts, however.
Fusion proteins
Several EPO gene fusion proteins have been reported, including EPO/inter-
leukin-3 (IL-3) [44], and EPO/granulocyte-macrophage colony-stimulating
factor (GM-CSF) [45]. The theory behind creation of such molecules is that
they can impart to the fusion protein biologic properties of both molecules.
One can imagine that co-administration of an early-acting growth factor such
as rHuIL-3 with rHuEPO can increase efficacy of rHuEPO. The fusion protein
being larger may impart increased activity for both partners because of
reduced clearance. The fusion protein also ensures that both molecular entities
