PEGylation

PEGylation is the process of covalent attachment of polyethylene 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), and 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 Dr. Frank Davis, Dr. Abraham Abuchowski and colleagues foresaw the potential of the conjugation of Polyethylene glycol (PEG) to proteins. Dr. Abuchowski founded Enzon, Inc, which brought three PEGylated drugs to market. He is the founder and president of Prolong Pharmaceuticals.

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

PEGylated drugs have 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 the U.S. Food and Drug Administration (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 interferon alpha for use in the treatment of chronic hepatitis C and hepatitis B (Hoffmann-La Roche)


 * Pegintron: PEGylated interferon alpha for use in the treatment of chronic hepatitis C and hepatitis B (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). This drug was recently approved for front line use.


 * Neulasta: PEGylated recombinant methionyl human granulocyte colony-stimulating factor for severe cancer chemotherapy-induced neutropenia (Amgen)


 * Doxil/Caelyx: PEGylated liposome containing doxorubicin for the treatment of cancer (Ortho Biotech / Schering-Plough)


 * Pegloticase (Krystexxa) - PEGylated Uricase for the treatment of gout (Savient)

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 are known as “homobifunctional”, whereas 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 overall PEGylation processes used to date for protein conjugation can be broadly classified into two types, namely a solution phase batch process and an on-column fed-batch process. The simple and commonly adopted batch process involves the mixing of reagents together in a suitable buffer solution, preferably at a temperature between 4 and 6°C, followed by the separation and purification of the desired product using a suitable technique based on its physicochemical properties, including size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and membranes or aqueous two phase systems.

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 as a site specific site by conjugation with aldehyde functional polymers.

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 useful in linking two entities, where a hydrophilic, flexible and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and NHS esters.

Third generation pegylation agents, where the shape of the polymer has been branched, Y shaped or comb shaped are available which show reduced viscosity and lack of organ accumulation.

Future perspectives
Even though nearly four decades of development in PEGylation technology has proven its pharmacological advantages and acceptability, the technology still lags in providing a commercially attractive, generic process to produce highly specific PEGylated therapeutic products at high yield. As a multi-million dollar annual business with growing interest from both emerging biotechnology and established multinational pharmaceutical companies, there is great scientific and commercial interest in improving present methodologies and in introducing innovative process variations.