The modification of proteins with polyethylene glycol (PEGylation) is an established technology that has many benefits
in the biopharmaceutical industry. For instance, modifying proteins with multiple PEGs masks immunogenic sites and prevents
neutralizing-antibody formation to certain proteins and therapeutic enzymes.1-3 Due to the amphiphilic nature of polyethylene glycol, PEGylation can also improve the solubility and physical-chemical stability
James E. Seely
PEGylation can increase the circulating half-life of proteins, especially smaller peptides and proteins, which normally have
a rapid glomerular filtration rate and are cleared on the basis of size. PEGs have a high Stokes-radius-to-mass ratio. Attachment
of one or two 10-20 kDa PEG molecules can increase the circulating half-life of small proteins and peptides several-fold,
resulting in a much less frequent dosing regimen.4
In site-specific PEGylation, a significant portion of the biological activity of the protein can be maintained while increasing
the half-life. Currently available products include PEGASYS (peginterferon alpha-2a) by Roche, Neulasta (pegfilgrastim) by
Amgen, Somavert (pegvisomant) by Roche and PEG-INTRON (peginterferon alpha-2b) by Schering-Plough. Several other companies
have site-specific PEGylated products in development.
In this article, we present some of the challenges of developing commercially viable, site-specific PEGylated protein and
peptide pharmaceuticals, with particular emphasis on the purification and analysis of PEGylated products and the analysis
of the PEG linkers.
The most common modification agents, or linkers, are based on methoxy PEG (mPEG) molecules (Figure 1). Their activity depends
on adding a protein-modifying group to the alcohol end. In some instances polyethylene glycol (PEG diol, Figure 1) is used
as the precursor molecule. The diol is subsequently modified at both ends in order to make a hetero- or homo-dimeric PEG-linked
molecule (as shown in the example with PEG bis-vinylsulfone).
Figure 1. Structures of Methoxy Polyethylene Glycol (mPEG) and Polyethylene Glycol (PEG diol)
PEGylation at Cysteinyl Residue
Proteins are generally PEGylated at nucleophilic sites such as unprotonated thiols (cysteinyl residues) or amino groups.2,5,6 Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone.2,5,6 All four are strongly cysteinyl-specific under mild conditions and neutral to slightly alkaline pH but each has some drawbacks.
The amide formed with the maleimides can be somewhat unstable under alkaline conditions7 so there may be some limitation to formulation options with this linker. The amide linkage formed with iodo PEGs is more
stable, but free iodine can modify tyrosine residues under some conditions. PEG thiols form disulfide bonds with protein thiols,
but this linkage can also be unstable under alkaline conditions. PEG-vinylsulfone reactivity is relatively slow compared to
maleimide and iodo PEG; however, the thioether linkage formed is quite stable. Its slower reaction rate also can make the
PEG-vinylsulfone reaction easier to control.
Site-specific PEGylation at native cysteinyl residues is seldom carried out, since these residues are usually in the form
of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate
cysteinyl PEGylation sites for thiol-specific linkers. The cysteine mutein must be designed such that it is accessible to
the PEGylation reagent and is still biologically active after PEGylation.