This article is about PEGylation in a pharmaceutical context. For the bulk industrial process, see
Ethoxylation.
PEGylation (or 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.[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 (reducing
immunogenicity and
antigenicity), and increase its hydrodynamic size (size in solution), which prolongs its circulatory time by reducing
renal clearance. PEGylation can also provide water solubility to
hydrophobic drugs and proteins. Having proven its
pharmacological advantages and acceptability, PEGylation technology is the foundation of a growing multibillion-dollar industry.[7]
Methodology
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.[9][10] It produces alterations in the physiochemical properties including changes in
conformation,
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 a
molecule, can impart several significant pharmacological advantages over the unmodified form, such as improved
drug solubility, reduced dosage frequency with potentially reduced
toxicity and without diminished efficacy, extended circulating life, increased
drug stability, and enhanced protection from proteolytic degradation; PEGylated forms may also be eligible for patent protection.[11]
PEGylated drugs
The attachment of an inert and
hydrophilic polymer was first reported around 1970 to extend blood life and control
immunogenicity of
proteins.[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 the
U.S. Food and Drug Administration (FDA) in March 1990, to enter the market. It is used to treat a form of
severe combined immunodeficiency syndrome (ADA-SCID), 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.[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 the
Moderna vaccine and the
Pfizer–BioNTech COVID-19 vaccine. Both
RNA 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, 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 as
DMG-PEG 2000.
PEGylation has practical uses in biotechnology for protein delivery,[28] cell
transfection, and
gene editing in non-human cells.[29]
Process
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 include
lysine,
cysteine,
histidine,
arginine,
aspartic acid,
glutamic acid,
serine,
threonine and
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.[34]
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. 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 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.[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 of
organ accumulation.[38] Recently also
enzymatic 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.
Limitations
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.[41] Furthermore, alteration in the chain length may lead to unexpected clearance times in 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 biodegradable
polysaccharides, which is a promising alternative to PEGylation, is another way to solve the
biodegradability issue of PEG.[46]
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