PEGylation is a process through which polyethylene glycol (PEG) chains are covalently attached to proteins, antibodies, peptides, organic molecules, or nanoparticle drug delivery system. PEGylation is routinely achieved by the incubation of an activated PEG with the target molecule.
PEGylation of biomolecules offers several advantages such as favorable pharmacokinetics (DMPK), increased stability, and enhanced therapeutic activity. Specifically, the use of PEG is seen as an advantage since it is highly water-soluble, non-toxic, and non-immunogenic. PEGylation of a molecule can cause changes in physicochemical properties such as increase in hydrophilicity, molecular weight & size change, conformation changes, and steric hindrance.
PEG compounds, either monodispersed or polydispersed, can be attached to a wide variety of compounds or biomolecules through the use of different chemistry. Functional groups on the PEG molecule, located at the ends of the PEG chains, can be an homo- or hetero-functional depending on the application requirements. Some popular functional groups for condensation include amine, carboxylic acid, NHS ester, thiol, maleimide, or biotin groups. When click chemistry reactions are desired azide, tetrazine, alkyne, BCN, DBCO, or TCO.
Figure 1 highlights the conjugation of a PEG NHS ester to the amine group on the protein. The NHS ester can react with primary amines very readily at pH 7-9 to form a stable, irreversible amide bond.
Figure 1. A nucleophilic reaction between an amine and a PEG NHS ester.
Figure 2 shows the thiol group on an antibody reacting with an maleimide PEG compound. Maleimide group reacts specifically with sulfhydryl groups to form a stable thioether linkage when the pH is between 6.5 and 7.5. The reaction does not produce side products and the PEG chain improves water-solubility of the antibody.
Figure 2. A coupling reaction between an antibody and a PEG maleimide.
Figure 3 depicts a click chemistry reaction occurring between a fluorescent dye molecule with an azide handle and a BCN PEG compound. BCN functional group is very reactive toward azides and does not require a Cu catalyst. The reaction can be run either in organic solvents or in aqueous solution depending on the solubility and property of substrate molecules.
Figure 3. A click chemistry reaction between an azide on a fluorescent dye and a BCN PEG molecule.
PEGylation has been used in a variety of therapeutic proteins, peptides, and antibody, as well as small molecule drugs, over the past 50 years. The following examples highlight the application of PEG in protein drugs, peptide-base compounds, and liposomes.
The PEGylated drugs currently are used in the treatment of chronic kidney diseases, hemophilia, hepatitis, gastrointestinal disorders, multiple sclerosis, and a multitude of different cancers. Two of the earliest examples of PEGylated drugs are Pegaspargase for leukemia and Pegademase for severe combined immunodeficiency disorder (Figure 4).
Pegademase is a conjugate of numerous strands, typically listed as 11-17, of monomethoxy polyethylene glycol (mPEG), average molecular weight 5,000, covalently attached to the enzyme ADA. Pegademase works by eliminating the toxic metabolites of ADA deficiency which results in improved immune function.
Pegaspargase is produced through the PEGylation of the enzyme asparaginase. Approximately 69 to 82 molecules of mPEG are linked to L-asparaginase; each mPEG has an average molecular weight of 5,000. The medication works by breaking down asparagine, which effects the leukemia cell’s ability to make protein and leads those cells to die.
Figure 4. Adagen (left side) and Oncaspar (right side) both utilize mPEG polymers to increase the effectiveness of use, with Adagen typically having 11-17 PEG attachments and Oncaspar utilizing 69-82 covalently bonded PEG polymers.
Synthetic peptides are used as potent and selective drugs. The in vivo bioavailability of a typical peptide drug is limited by its low circulating half-life, which could measure a few hours or, even more likely, a few minutes. This is due to enzymatic digestion and other endogenous factors, such as renal clearance. Coupling even small PEG chains to a peptide can dramatically improve the drugs performance by overcoming these issues.
One example of a PEGylated peptide compound is Semaglutide, under the trade name Ozempic, which was approved by the FDA in 2017 for treatment of Diabetes. Semaglutide is pegylated at the GLP-1 peptide (31 aa in the chain) with hexadecanedioic acid via γ-Glu and monodispersed PEG molecule at Lys. Semaglutide works as an agonist of glucagon-like peptide-1 (GLP-1) receptor, which reduces blood sugar via increasing the production of insulin (Figure 5).
Figure 5. Semaglutide has an amino acid substitution at position 8 prevents chemical breakdown by an enzyme dipeptidyl peptidase-4. In addition, the lysine at position 26 is in its derivative form (acylated with stearic diacid), which enables longer presence in the blood circulation.
PEGylated liposome (PEGLip) technology is a new approach to improving the pharmacodynamic properties of therapeutic proteins. One example is of PEGylated liposomes used as carriers with the protein bound non-covalently but with high specificity to the outer surface. This strategy can be very useful when developing drug delivery systems that need to have very long half-life values in vivo.
For example, one of the most problematic complications of hemophilia treatment is the induction of inhibitory antibodies. PEGLip formulation does not change the structure of FVIII and FVIIa, so it avoids changes in structure that induce antibody formation. Figure 6 shows how PEGLip was used by researchers to bind coagulation factor VIII, which significantly reduce bleeding times in animal tests.
Figure 6. The PEGylated liposome shown above is composed of a 97:3 molar ratio of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to 1,2 distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG 2000). The DSPE-PEG 2000 is shown having a non-covalent interaction with a factor VIII protein.
BroadPharm is a leading supplier of PEGylation reagents, including high purity monodisperse PEG and Polymer PEG with M.W. from 2,000 to 20,000 with all the popular PEGylation functional groups. Please review our product page for all our in-stock PEGylation reagents.