Features of PEGylation



Because of its low toxicity, no antigenicity, good amphiphilicity, and biocompatibility, macromolecular polyethylene glycol (PEG) has an irreplaceable advantage in protein modification. The PEGylation modification technology couples polyethylene glycol with the modified drug through covalent bonds to improve the physicochemical properties and biological activity of the drug. This technology has been widely used in proteins (peptides), enzymes, and antibodies. and small molecule drugs.

Polyethylene glycol has a wide molecular weight distribution, and the properties vary with the average molecular weight. When the molecular weight is less than 1000Da, polyethylene glycol is a colorless, odorless and viscous liquid. It is a waxy white solid. The melting point of solid polyethylene glycol is proportional to the molecular weight and gradually approaches the limit of 67 °C. The toxicity decreases with the increase of molecular weight. PEG less than 400 Da will be degraded into toxic metabolites by alcohol dehydrogenase in vivo, while PEG with molecular weight greater than 1000 Da has been used in the food industry, cosmetics industry and pharmaceutical industry for many years and proved to be non-toxic.

The polyethylene glycol molecule contains a large number of ethoxy groups, which can form hydrogen bonds with water, so it has good water solubility and is soluble in most organic solvents except ether, hexane, and ethylene glycol. After most proteins are modified with polyethylene glycol, in addition to retaining or increasing their water solubility, they can also obtain solubility in some organic solvents. In protein solutions, polyethylene glycol, whether in free or bound form, has no adverse effects on protein molecules even at high concentrations. The general conformation of polyethylene glycol-modified proteins does not change, and the biological activity of the conjugates is mainly produced by the protein part of the conjugates.

Polyethylene glycol is immunologically inert, and its immunogenicity is very low even if its molecular weight is as high as 5.9×106Da. Clinical use of polyethylene glycol-modified protein treatment has not found anti-PEGylated antibodies.

Among the 20 common amino acids that make up proteins, only the side chain groups of polar amino acid residues can be chemically modified. Commonly used reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, N-terminal amino and C-terminal carboxyl . The reactive groups on these amino acid residues are mostly nucleophilic, and their nucleophilic activity usually decreases in the following order: thiol>α-amino>ε-amino>carboxyl (carboxylate)>hydroxyl. According to the different nature of the reaction between chemical modifiers and proteins, the modification reactions are mainly divided into acylation reactions, alkylation reactions, redox reactions, aromatic ring substitution reactions, etc. group for chemical modification. Sulfhydryl groups usually exist on disulfide bonds and active sites of proteins, while carboxyl groups are difficult to activate without intermolecular or intramolecular neutralization reactions with amino groups on proteins. Therefore, the most likely site of a protein or polypeptide molecule to interact with modifiers is the amino group on the lysine residue on the surface of the molecule.

Polyethylene glycol modification, also known as PEGylation of molecules, is a modification method developed in the late 1970s. Coupling of activated polyethylene glycol with protein molecules affects the spatial structure of the protein, ultimately leading to changes in various biochemical properties of the protein: increased chemical stability, improved resistance to proteolytic hydrolysis, reduced immunogenicity and toxicity or disappear, the half-life in vivo is prolonged, and the plasma clearance rate is reduced.