Polyethylene glycol modification, or PEGylation, is to chemically couple activated PEG to a protein or polypeptide molecule by a covalent bond. Since Davies modified bovine serum albumin with PEG in 1977, PEG modification technology has been widely used in chemical modification of various proteins and polypeptides, and many PEG modified drugs have been listed or in clinical research. PEG modification has the advantages of prolongation of half-life, reduction or disappearance of immunogenicity, reduction of toxic and side effects, and enhancement of physical, chemical and biological stability.
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, antibodies and small molecule drugs.
1 PEG chemical structure and properties
1.1 Chemical structure of PEG
Structural formula CH2(OH)-(CH2CH2O)n-CH2OH
1.2 Properties of PEG
Polyethylene glycol series products are usually soluble in water and various organic solvents, insoluble in aliphatic hydrocarbons, benzene, ethylene glycol, etc., and will not be hydrolyzed and deteriorated. They have a wide range of dissolution and excellent compatibility. Stability, lubricity, film formation, plasticity, dispersibility, etc. Department of low toxic substances, and non-irritating, is a non-ionic polymer.
2. Polyethylene glycol modifier
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. According to the different molecular weights, molecular structures and physicochemical properties of the compounds to be modified, including proteins, polypeptides, monoclonal antibody fragments, and small molecular compounds, different PEGylation techniques are used to modify these compounds.
2.1 Random modification
Randomly modified proteins mostly target ε-NH2 or α-NH2 of lysine. Since lysine is usually in large quantity in the protein, this modification causes multiple lysines in the protein to be modified, and the resulting product is The mixture of PEGylated isomers, most of the new PEGylated drugs approved by the FDA are currently randomly modified products.
2.2 Fixed-point modification
PEGylation site-directed modification is a second-generation polyethylene glycol modification technology developed on the basis of random modification. By optimizing the selection of modification methods, PEG modifiers and reaction pH, site-directed modification is realized. This modification The obtained product is a homogeneous product with less isomers, better activity retention and greatly reduced immunogenicity.
2.2.1 N-terminal amino group site-directed modification
Due to the existence of multiple α-NH2 and ε-NH2 groups in proteins and polypeptides, random modification of amino groups may lead to a significant decrease in drug activity, which brings obstacles to the modification of proteins and polypeptides. At present, we adopt the site-directed modification of the N-terminal amino group, especially for the N-terminal site-directed modification far from the active center, which can effectively maintain the original biological activity of the drug.
2.2.2 Thiol site-directed modification
Combined with genetic engineering technology or Mutagenesis and other methods, thiol groups or some specific groups are introduced into the preset sites of proteins and polypeptides (such as glycosylation sites that do not affect activity, antigenic determinants, etc.) In this way, site-directed modification products with higher activity retention and reduced immunogenicity can be obtained.
2.2.3 Carboxyl site-directed modification
Using polyethylene glycol derivatives with hydrazide activating groups to modify the carboxyl groups of proteins and polypeptides, a site-specific modification product can also be obtained.
2.2.4 Enzymatic modification
Enzyme catalysis is used to induce targeted modification of polyethylene glycol molecules and specific sites on proteins, polypeptides and other molecules. This method has the following characteristics:
(1). No structural modification of the original protein and polypeptide is required, and the physical and chemical properties of the original compound are kept unchanged;
(2). Fixed-point modification;
(3). The process is simple, and it is easy for large-scale production and quality control.