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 such as N-hydroxysuccinimide (NHS)-PEG to attach PEG to free amines, typically at lysine residues or at the N-terminal amino acid. A major limitation of this approach is that proteins typically contain several lysines, in addition to the N-terminal amino acid. The PEG moiety attaches to the protein randomly 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. The different PEGylated species often possess different intrinsic biological activities. This is disadvantageous when developing a PEGylated therapeutic protein product where predictability of biological activity is crucial. For regulatory approval, composition and biological activity of the modified protein must be consistent between manufacturing lots. From a producer's point of view, regulatory approval for sale of a heterogeneously PEGylated therapeutic protein may have added complexities and hurdles. 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.       top

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