PEGylation

Overview
PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

History
In 1970s, pioneering research by Davis, Abuchowski and colleagues foresaw the potential of the conjugation of Polyethylene glycol (PEG) to Proteins. This technique is now well established and is known as PEGylation.

PEGylation, is a process of attaching the strands of the polymer PEG to molecules most typically peptides, proteins, and antibody fragments, that can help to meet the challenges of improving the safety and efficiency of many therapeutics. It produces alterations in the physiochemical properties including changes in conformation, electrostatic binding, hydrophobicity etc. These physical and chemical changes increase systemic retention of the therapeutic agent. Also, it can influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.

PEGylation, by increasing the molecular weight of a molecule, can impart several significant pharmacological advantages over the unmodified form, such as:

•	Improved drug solubility

•	Reduced dosage frequency, without diminished efficacy with potentially reduced toxicity

•	Extended circulating life

•	Increased drug stability

•	Enhanced protection from proteolytic degradation

The PEGylated drugs are having the following commercial advantages also:

•	Opportunities for new delivery formats and dosing regimens

•	Extended patent life of previously approved drugs

PEGylated Pharmaceuticals on the Market
The clinical value of PEGylation is now well established. ADAGEN (PEG- bovine adenosine deaminase) manufactured by Enzon Pharmaceuticals, Inc., US was the first PEGylated protein approved by FDA in March 1990, to enter the market. It is used to treat X-linked severe combined immunogenicity syndrome, as an alternative to bone marrow transplantation and enzyme replacement by gene therapy. Since the introduction of ADAGEN, a large number of PEGylated protein and peptide pharmaceuticals have followed and many others are under clinical trial or under development stages. Some of the successful examples are:

• PEGASYS: PEGylated alpha-interferons for use in the treatment of hepatis C (Hoffman-La Roche)

• PEG-Intron: PEGylated alpha -interferons for chronic hepatis C (Schering-Plough / Enzon)

• Oncaspar:	PEGylated L-asparaginase for the treatment of acute lymphoblastic leukemia in patients who are hypersensitive to the native unmodified form of L-asparaginase (Enzon)

• Neulasta: PEGylated recombinant methionyl human granulocyte colony stimulating factor for severe cancer chemotherapy induced neutropenia (Amgen) • Doxil: PEGylated liposome containing doxorubicin for the treatment of Cancer (Sequus)

PEG Moiety Properties
PEG is a particularly attractive polymer for conjugation. The specific characteristics of PEG moieties relevant to pharmaceutical applications are:

•	Water solubility

•	High mobility in solution

•	Lack of toxicity and immunogenicity

•	Ready clearance from the body

•	Altered distribution in the body

PEGylation Process
The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety is known as “homobifunctional”, where as if the functional groups present are different, then the PEG derivative is referred as “heterobifunctional” or “heterofunctional.” The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates and carbonates. In the second generation PEGylation chemistry more efficient functional groups such as aldehyde, esters, amides etc made available for conjugation.

As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very much useful in linking two entities, where a hydrophilic, flexible and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulphones, pyridyl disulphide, amine, carboxylic acids and NHS esters.

Allergic Responses
In general the immune reaction to PEGylated products is no different than that of the underlying molecule that the agent is conjugated to. The risk of complement activation is correlated with the antigenicity of the moiety that is being PEGylated. Rarely patients who are repeatedly exposed to PEGylated proteins will develop anti-PEG antibodies (eg. PEG-Uricase or Krystexxa). The Hershfield laboratory at Duke University has experience in measuring anti-PEG antibody responses in humans. It is believed that the Epitope for anti-PEG IgG is the repeating unit of polyethylene glycol. The incidence of anti-PEG IgG in the population is unknown. The PEGylated Echo contrast agent Definity (echo contrast dye) is associated with a risk of allergic reactions in 0.006% to 0.008% of patients.

Suggested Reading
Abuchowski, McCoy, Palczuk, van Es and Davis (1977). "Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase." Journal of Biological Chemistry 252(11): 3582-3586.

Fee (2003). "Size-exclusion reaction chromatography (SERC): A new technique for protein PEGylation." Biotechnology and Bioengineering 82(2): 200-206.

Fee and Alstine (2006). "PEG-proteins: Reaction engineering and separation issues." Chemical Engineering Science 61(3): 924-939.

Kodera, Matsushima, Hiroto, Nishimura, Ishii, Ueno and Inada (1998). "Pegylation of proteins and bioactive substances for medical and technical applications." Progress in Polymer Science 23(7): 1233-1271.

Morar, Jeffrey and Mark (2006). "PEGylation of Proteins: A Structural Approach." Biopharm International 19(4): 34.

Roberts, Bentley and Harris (2002). "Chemistry for peptide and protein PEGylation." Advanced Drug Delivery Reviews 54(4): 459-476.

Veronese (2001). "Peptide and protein PEGylation: a review of problems and solutions." Biomaterials 22(5): 405-417.

Veronese and Harris (2002). "Introduction and overview of peptide and protein pegylation." Advanced Drug Delivery Reviews 54(4): 453-456.

Veronese and Pasut (2005). "PEGylation, successful approach to drug delivery." Drug Discovery Today 10(21): 1451-1458.