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Site-Specific PEGylation

Polyethylene Glycol (PEG) Background
Covalent modification of proteins with polyethylene glycol (PEG) has proven to be a very useful method to extend the circulating half-lives of proteins and several PEGylated proteins are now approved for use in humans. Covalent attachment of PEG to a protein increases the protein's effective size and reduces its rate of clearance from the body. PEGs are commercially available in several sizes, allowing the circulating half-lives of PEG-modified proteins to be tailored for individual clinical indications through use of different size PEGs. In addition to improving protein half-life, PEG modification can increase protein solubility and stability and decrease protein immunogenicity.

Disadvantages of Amine-Reactive PEGs for Protein PEGylation
The most commonly employed method for PEGylating proteins uses compounds that attach PEG to free amines, typically at lysine residues or at the N-terminal amino acid. A critical limitation of this approach is that proteins typically contain several lysines, in addition to the N-terminal amino acid. The PEG moiety can attach to the protein at any of the available free amines, resulting in a heterogeneous product mixture consisting of mono-, di-, tri-, etc, PEGylated species modified at different lysine residues and the N-terminus. The different PEGylated species often possess different intrinsic biological activities. This can present problems when developing a PEGylated protein therapeutic because predictability of biological activity and manufacturing reproducibility are crucial for regulatory approval. Many amine-PEGylated proteins are unsuitable for commercial use because of low specific activities. Biological activities of amine-PEGylated proteins often are reduced 10-100-fold relative to the non-modified protein. Inactivation results from covalent modification of one or more amino acids required for biological activity or from covalent attachment of the PEG moiety near the active site or ligand binding site of the protein.

Advantages of Site-Specific PEGylation
Site-Specific PEGylation overcomes the problems of product heterogeneity and loss of biological activity characteristic of amine-PEGylation. Site-Specific PEGylation allows a protein to be selectively modified with PEG at a single, unique, pre-determined site. The site of PEGylation potentially can be any amino acid position in the protein and can be varied depending upon the protein. By targeting the PEG molecule to an optimal site in a protein, it is possible to create PEGylated proteins that are homogeneously modified and have no significant loss of biological activity. Site-Specific PEGylation is achieved by introducing a "free" cysteine residue, i.e., a cysteine residue not involved in a disulfide bond, into a target protein using site-directed mutagenesis. The free cysteine residue serves as the attachment point for covalent modification of the protein with a cysteine-reactive PEG molecule. Attachment of the PEG molecule to the free cysteine residue is highly specific because most native cysteine residues in proteins participate in disulfide bonds and are not available for PEGylation using cysteine-reactive PEGs. PEGylation of the cysteine muteins yield a single monoPEGylated species modified at the free cysteine residue. Because the PEGylated protein is homogeneously modified, it is readily purified and retains high biological activity. Our PEGylated proteins undergo a rigorous screening process to identify those with the best biological activities and PEGylation efficiencies. We use commercially available, off-the-shelf PEGs in our products, which allows us to take advantage of the latest developments in PEG technology.       top

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