Peptide PEGylation: The Next Generation - Pharmaceutical Technology

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PharmTech Europe

Peptide PEGylation: The Next Generation
Linking peptides to polyethylene glycol, or PEGylation, has helped improve pharmaceutical therapeutics in several ways. A wave of new techniques is now ushering in further advances.


Pharmaceutical Technology
Volume 35, pp. a26-s28

Second-generation PEGylation techniques

Since the first wave of PEGylated peptide drugs entered the market, many more methods for PEGylating peptides have been developed to confer PEG's advantages on molecules with varying properties and to overcome some of the problems associated with first-generation PEGylation.

Site-specific conjugation. One new technique is to modify the peptide, rather than the PEG (7). This may be necessary if the peptide lacks lysines, or if the lysine is located in an active site. A cysteine can be added, where desired, to generate site-specific PEGylation at places chosen to minimize interference with the peptide's biological function, while maximally decreasing the peptide's immunogenicity. PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, and PEG orthopyridyl disulfide are thiol reactive PEGs that have been created to PEGylate free cysteine residues. This approach has been used in a number of ways including: to make monoPEGylated human growth hormone analog, which could potentially be used to treat growth hormone deficiency and wasting in AIDS patients with an approximately 8-fold longer half-life compared with unmodified growth hormone (8); a long-acting highly potent interferon a-2 conjugate for certain cancers and viral conditions (not PEGIntron) with a 20-to 40-fold longer half-life than unmodified interferonalpha2 (IFN-a2) (9); and PEGylated recombinant human granulocyte macrophagecolony stimulating factor (GM-CSF) for neutropenia and other myeloid disorders.

Another means of achieving site-specific conjugation is to use an N-terminal serine or threonine if it exists. These residues can be converted to glyoxylyl derivatives by periodate oxidation. The N-terminal-introduced reactive carbonyl group specifically reacts, under mild acidic conditions, with an aminooxy-functionalized poly(ethylene glycol) to form a stable oxime bond. Unlike most previous methods, this approach places a single PEG chain at a defined site on the protein and should, therefore, be more likely to conserve biological activity. PEGylated interleukin8 (IL-8), G-CSF, and interleukin1 receptor agonist (IL-1ra) were made this way (10).

HIPEG. HiPEG (PolyTherics, UK) was developed to specifically attach PEG to histidine sequences expressed on the N or C terminal of proteins. After PEGylation, the histidine tag remains available for affinity purification of the protein or peptide. This technology has been shown to be scalable, robust and reproducible (9), and has been successfully used to PEGylate an anti-TNFa domain antibody, which can be used to treat autoimmune disorders, such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, and refractory asthma (9).

Branched and forked PEG. Branched PEGs (PEG2) can be used in lieu of linear PEG molecules, and enable a larger and purer PEG to be linked with only one reactive group. This is particularly useful in cases where the incorporation of many PEG molecules could block a protein–protein interaction. The bulkier nature of branched PEGs helps to repel approaching macromolecules from a peptide's active site, thereby preserving biological activity. They are also more effective at protecting peptides from proteolysis, and reducing immunogenicity and antigenicity. Branched PEG was used to PEGylate lysostaphin, which is used to fight multidrugresistant strains of Staphylococcus aureus.

Forked PEG is the opposite of branched PEG. Rather than one functional group attached to two PEG chains, it has two reactive groups at the end of one PEG chain. Forked PEG can be used to bring two moieties into proximity.

Releasable PEGs. Releasable PEGs (rPEGs) may be desired if the PEG interferes with biological activity. With rPEGs, the PEG conjugation achieves its goal by protecting the peptide in circulation, but it is then discarded to release an active protein. PEG-Intron (originally made by Ezon, but now marketed by Merck), which is currently used to treat Hepatitis C, was made this way: Enzon conjugated PEG-SC to His34 of interferon a-2b using a carbamate linkage. Over time, this linkage degrades to yield native interferon a-2b. Enzymatic degradation, hydrolytic cleavage, or reduction can also be used to release PEG for rPEGs. To date, many rPEGs generated use a linker with an ester bond to control the rate of protein release.

Heterobifunctional PEG. Heterobifunctional PEGs have two different terminal groups. Because they are hydrophilic, flexible, and biocompatible, they can be used as a spacer to link two groups, or to target drugs, or even in peptide synthesis. Preferred end groups include NHS esters, maleimide, vinyl sulfone, pyridyl disulfide, amines, and carboxylic acids.


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