PEGylation (also often styled pegylation) is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG, in pharmacy called macrogol) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated (pegylated). PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. 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.
Around 1970, Frank F. Davis, a professor of biochemistry at Rutgers University, became interested in developing a process to render usable bioactive proteins of potential medical value. After considerable study, he concluded that the attachment of an inert and hydrophilic polymer might extend blood life and control immunogenicity of the proteins. Polyethylene glycol was chosen as the polymer. A team of Davis, Theodorus Van Es and Nicholas C. Palczuk conducted animal studies and found that PEG attachment greatly extended blood life and controlled immunogenicity of the proteins. A patent application was filed in 1973 and patent issued in 1979. The inventors and Abraham Abuchowski conducted extensive additional PEGylation studies on various enzymes. In 1981 Davis and Abuchowski founded Enzon, Inc., which brought three PEGylated drugs to market. Abuchowski later founded and is CEO of Prolong Pharmaceuticals.
PEGylation is the process of attaching the strands of the polymer PEG to molecules, most typically peptides, proteins, and antibody fragments, that can improve 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 also have the following commercial advantages:
- Opportunities for new delivery formats and dosing regimens
- Extended patent life of previously approved drugs
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 low immunogenicity
- Ready clearance from the body
- Altered distribution in the body
PEGylated pharmaceuticals on the market
The clinical value of PEGylation is now well established. ADAGEN (pegademase bovine) 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. Sales of the two most successful products, Pegasys and Neulasta, exceeded $5 billion in 2011. All commercially available PEGylated pharmaceuticals contain methoxypoly(ethylene glycol) or mPEG. PEGylated pharmaceuticals currently on the market (in reverse chronology by FDA approval year) include:
- Adynovate — PEGylated Antihemophilic Factor VIII for the treatment of patients with Hemophilia A. (Baxalta, 2015) 
- Plegridy — PEGylated Interferon Beta-1a for the treatment of patients with relapsing forms of multiple sclerosis. (Biogen, 2014)
- Naloxegol (Movantik) — PEGylated naloxol for the treatment of opioid-induced constipation in adults patients with chronic non-cancer pain (un-pegylated methadone can cause adverse gastrointestinal reactions). (AstraZeneca, 2014)
- Peginesatide (Omontys) — once-monthly medication to treat anemia associated with chronic kidney disease in adult patients on dialysis (Affymax/Takeda Pharmaceuticals, 2012)
- Pegloticase (Krystexxa) — PEGylated uricase for the treatment of gout (Savient, 2010)
- Certolizumab pegol (Cimzia) — monoclonal antibody for treatment of moderate to severe rheumatoid arthritis and Crohn's disease, an inflammatory gastrointestinal disorder (Nektar/UCB Pharma, 2008)
- Methoxy polyethylene glycol-epoetin beta (Mircera) — PEGylated form of erythropoetin to combat anemia associated with chronic kidney disease (Roche, 2007)
- Pegaptanib (Macugen) — used to treat neovascular age-related macular degeneration (Pfizer, 2004)
- Pegfilgrastim (Neulasta) — PEGylated recombinant methionyl human granulocyte colony-stimulating factor for severe cancer chemotherapy-induced neutropenia (Amgen, 2002)
- Pegvisomant (Somavert) — PEG-human growth hormone mutein antagonist for treatment of Acromegaly (Pfizer, 2002)
- Peginterferon alfa-2a (Pegasys) — PEGylated interferon alpha for use in the treatment of chronic hepatitis C and hepatitis B (Hoffmann-La Roche, 2001)
- Doxorubicin HCl liposome (Doxil/Caelyx) — PEGylated liposome containing doxorubicin for the treatment of cancer (Ortho Biotech/Schering-Plough, 2001)
- Peginterferon alfa-2b (PegIntron) — PEGylated interferon alpha for use in the treatment of chronic hepatitis C and hepatitis B (Schering-Plough/Enzon, 2000)
- Pegaspargase (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, 1994). This drug was recently approved for front line use.
- Pegademase bovine (Adagen) — PEG-adenosine deaminase for the treatment of severe combined immunodeficiency disease (SCID) (Enzon, 1990)
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.
Limitations of PEGylation
Unpredictability in clearance times for PEGylated compounds may lead to the accumulation of large molecular weight compounds in the liver leading to inclusion bodies with no known toxicologic consequences. Furthermore, alteration in the chain length may lead to unexpected clearance times in vivo.
Four decades of development in PEGylation technology have proven its pharmacological advantages and acceptability. As a multibillion-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.
- Polyethylene glycol
- Size exclusion chromatography
- Matrix-assisted laser desorption/ionization
- Granulocyte colony-stimulating factor
- Cytochrome c
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