Making Site-specific PEGylation Work

Purification and analysis of PEGylated protein pharmaceuticals presents many challenges.
Mar 01, 2005
By BioPharm International Editors
Volume 18, Issue 3

James E. Seely
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 of proteins.2,3

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.

Figure 1. Structures of Methoxy Polyethylene Glycol (mPEG) and Polyethylene Glycol (PEG diol)
PEGYLATION REAGENTS 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).

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.

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