This article is about PEGylation in a pharmaceutical context. For the bulk industrial process, seeEthoxylation.
Polyethylene glycol
PEGylation (orpegylation) is the process of both covalent and non-covalent attachment or amalgamation ofpolyethylene glycol (PEG, in pharmacy calledmacrogol) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described asPEGylated.[1][2][3][4] PEGylation affects the resulting derivatives or aggregates interactions, which typically slows down their coalescence and degradation as well as elimination in vivo.[5][6]
PEGylation is routinely achieved by the 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 (reducingimmunogenicity andantigenicity), and increase its hydrodynamic size (size in solution), which prolongs its circulatory time by reducingrenal clearance. PEGylation can also provide water solubility tohydrophobic drugs and proteins. Having proven itspharmacological advantages and acceptability, PEGylation technology is the foundation of a growing multibillion-dollar industry.[7]
A comparison ofuricase andPEG-uricase; PEG-uricase includes 40 polymers of 10kDa PEG. PEGylation improves its solubility at physiological pH, increases serum half-life and reduces immunogenicity without compromising activity. Upper images show the whole tetramer, lower images show one of the lysines that is PEGylated. (uricase fromPDB:1uox and PEG-uricasemodel from reference;[8] only 36 PEG polymers included)
PEGylation is the process of attaching the strands of the polymer PEG to molecules, most typicallypeptides,proteins, andantibody fragments, that can improve the safety and efficiency of manytherapeutics.[9][10] It produces alterations in the physiochemical properties including changes inconformation,electrostaticbinding,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 amolecule, can impart several significant pharmacological advantages over the unmodified form, such as improveddrug solubility, reduced dosage frequency with potentially reducedtoxicity and without diminished efficacy, extended circulating life, increaseddrug stability, and enhanced protection from proteolytic degradation; PEGylated forms may also be eligible for patent protection.[11]
The attachment of an inert andhydrophilic polymer was first reported around 1970 to extend blood life and controlimmunogenicity ofproteins.[12] Polyethylene glycol was chosen as the polymer.[13][14] In 1981 Davis and Abuchowski founded Enzon, Inc., which brought three PEGylated drugs to market. Abuchowski later founded and is CEO of Prolong Pharmaceuticals.[15]
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 theU.S. Food and Drug Administration (FDA) in March 1990, to enter the market. It is used to treat a form ofsevere combined immunodeficiency syndrome (ADA-SCID), as an alternative tobone marrow transplantation and enzyme replacement bygene therapy. Since the introduction of ADAGEN, a large number of PEGylated protein and peptidepharmaceuticals 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.[16][17] All commercially available PEGylated pharmaceuticals contain methoxypoly(ethylene glycol) or mPEG. PEGylated pharmaceuticals on the market (in reverse chronology by FDA approval year) have included:[18]
A PEGylated lipid is used as an excipient in both theModerna vaccine and thePfizer–BioNTech COVID-19 vaccine. BothRNA vaccines consist of Messenger RNA, or mRNA, encased in a bubble of oily molecules called lipids. Proprietary lipid technology is used for each. In both vaccines, the bubbles are coated with a stabilizing molecule of polyethylene glycol. As of December 2020, there is some concern that PEG could trigger an allergic reaction,[19][20] as appears to have occurred by 19 December,[needs update] in at least three "Alaska health care worker" people who were administered the Pfizer–BioNTech COVID-19 vaccine.[21] The particular PEGylated molecule in the Moderna vaccine is known asDMG-PEG 2000.
The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both ends. PEGs that are activated at each end 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.[30]
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 includelysine,cysteine,histidine,arginine,aspartic acid,glutamic acid,serine,threonine andtyrosine. The N-terminal amino group and the C-terminalcarboxylic acid can also be used as a site specific site by conjugation withaldehyde functionalpolymers.[34]
The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typicallyanhydrides,acid chlorides,chloroformates andcarbonates. In the second generation PEGylation chemistry more efficient functional groups such as aldehyde,esters,amides etc. are 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 ahydrophilic, flexible andbiocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide,vinyl sulfones,pyridyl disulfide,amine,carboxylic acids andNHS esters.[35][36][37]
Third-generation pegylation agents, where the polymer has been branched, Y-shaped or comb-shaped are available and show reduced viscosity and lack oforgan accumulation.[38] Recently alsoenzymatic approaches of PEGylation have been developed, thus further expanding the conjugation tools.[39][40] PEG-protein conjugates obtained by enzymatic methods are already in clinical use, for example:Lipegfilgrastim,Rebinyn,Esperoct.
Unpredictability in clearance times for PEGylated compounds may lead to the accumulation of large-molecular-weight compounds in the liver leading toinclusion bodies with no known toxicologic consequences.[41] Furthermore, alteration in the chain length may lead to unexpected clearance timesin vivo.[42]Moreover, the experimental conditions of PEGylation reaction (i.e. pH, temperature, reaction time, overall cost of the process and molar ratio between PEG derivative and peptide) also have an impact on the stability of the final PEGylated products.[43]To overcome the above-mentioned limitations different strategies such as changing the size (Mw), the number, the location and the type of linkage of PEG molecule were offered by several researchers.[44][45] Conjugation to biodegradablepolysaccharides, which is a promising alternative to PEGylation, is another way to solve thebiodegradability issue of PEG.[46]
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