Release date:2020/2/19 10:11:25
Polyethylene glycol (PEG) is a polymer of ethylene glycol with a relative molecular weight of 200 to 8000 or above. It is composed of repeated oxyethyl groups, which not only has good water solubility but is also soluble in organic solvents such as benzene, acetonitrile, and ethanol. The characteristics of PEG molecules are as follows:

① Low dispersion: The dispersion with a relative molecular mass (Mr) of less than 5000 is 1.01, and the dispersion with a molecular weight (Mr) of more than 5000 is 1.1, with a wide distribution and greater selectivity;
② Unique amphiphilic: unique structure makes it soluble in both organic solvents and water;
③ Non-toxic: Studies show that polyethylene glycols greater than 1000 are non-toxic and have been used in various foods, cosmetics and drugs;
④ Biodegradable: Polyethylene glycol is directly eliminated in the body without any structural changes. Metabolites with a molecular weight of less than 20,000 can be metabolized by the kidneys, and larger molecules can be metabolized by the digestive system.

Characteristics of PEGylated Drugs

Most protein drugs, peptide drugs, and chemical drugs are accompanied by some problems that cannot be overcome by themselves, such as short period of action, large immunogenicity, and side effects. PEG is neutral, non-toxic, has unique physical and chemical properties and good biocompatibility, and is one of the few chemicals approved by the FDA for injectable drugs in vivo. Therefore, by connecting the activated polyethylene glycol to proteins, peptides, small molecule drugs and liposomes by chemical methods, that is, by performing PEGylation of the drug molecule, the biological half-life of the drug molecule can be effectively improved and its toxic side effects can be reduced. Among them, the most studied is the PEG modification of proteins. Compared with unmodified protein drugs,
PEGylated protein drugs have the following advantages:

(1) Stronger biological activity;
(2) Liposomes have a stronger passive targeting effect on tumors;
(3) longer half-life;
(4) lower maximum blood concentration;
(5) minor fluctuations in plasma concentration;
(6) less enzymatic degradation;
(7) Less immunogenicity and antigenicity;
(8) less toxic;
(9) Better solubility;
(10) reduce the frequency of medication;
(11) Improve patient compliance, improve quality of life, and reduce treatment costs.
PEGylation advantage
Main advantages of PEGylated proteins. (Image Source: Drug discovery today 2005, 10 (21), 1451-1458)
PEGylation Method
In view of the huge impact of PEGylation on drug properties, PEGylation has become an important way for drug development and improvement of the efficacy of already marketed drugs. So, how to carry out PEGylation becomes the top priority. 
First, the appropriate PEG needs to be selected for molecular modification. The selection of modifiers mainly considers the following 5 aspects:

(1) The
selection of the relative molecular weight (Mr) of PEG should consider both biological activity and pharmacokinetic factors. Applying too large a PEGylated protein drug will cause the drug to lose most of its biological activity. When using low-Mr (<20000) PEGylated protein drugs, the modified protein drugs have no essential changes in biological activity and pharmacokinetic properties compared to the prototype drugs. Therefore, PEG in the range of 40,000-60000 is generally selected as the modification.
(2) The selection of modification sites should be based on the analysis of the structure-activity relationship of the protein. The surface residues of the proteins that do not bind to the receptor are selected as modification sites, so that the modified protein can retain high biological activity. Common modification sites are amino modification, carboxy modification and thiol modification;
(3) The specificity of the reaction between PEG modifier and amino acid depends on the chemical properties of the modifier and the choice of modification site.
(4) The hydrolytic stability and reactivity of the PEG modifier depend on the stability of the activating group and the control of the modification reaction conditions, especially the pH. In general, PEG modifiers have high reactivity, so their stability is poor and they are easy to hydrolyze;
(5) The activity, toxicity, and antigenicity of PEGylated protein are related to the size and type of PEG modification. Generally, as the relative molecular weight of PEG increases, the loss of protein activity gradually increases. In addition, different PEG modifiers have different effects on the biological activity of proteins.
Secondly, activate PEG. PEGylated proteins are mainly achieved by the reaction of PEG terminal hydroxyl groups with protein amino acid residues. PEG terminal hydroxyl groups have poor activity and must be activated with an activator to covalently modify proteins under mild conditions in vivo. Common PEG activation methods are:

(1) carbonyl diimidazole method: This method was first used in the synthesis of polypeptides and has proven to be a good reagent for forming amide bonds.

carbonyl diimidazole
Carbonyl diimidazole activation of PEG
(2) N-hydroxysuccinimide method: (a) Activate N, N-succinimide carbonate. This reaction needs to be performed under anhydrous conditions. (B) Activate succinic anhydride and N-hydroxysuccinimide. The polyethylene glycol obtained by this method has higher activity. It is best to perform protein coupling in a non-aqueous environment.
N, N-succinimide carbonate activated PEG
N, N-succinimide carbonate activated PEG

Succinic anhydride and N-hydroxysuccinimide activated PEG
Succinic anhydride and N-hydroxysuccinimide activated PEG

(3) Cyanuryl chloride method: Cyanuryl chloride, also known as trichlorazine (TST), is a symmetric heterocyclic compound. David uses TST to react with the hydroxyl group on polyethylene glycol. Only one chlorine atom is substituted, and the other chlorine atoms Reacts with protein amino groups.
Cyanuryl chloride activated PEG
Cyanuryl chloride activated PEG
(4) Activation method involving phosgene: Kurfuerst mentioned in his patent some methods to prepare activated polyethylene glycol by reacting N-hydroxysuccinimide potassium salt, nitrophenol and trichlorophenol with phosgene. The activation is divided into two steps as shown in the figure below.
Phosgene activation of PEG
Phosgene activation of PEG
(5) Chemical modification of cysteine ​​residues of proteins by polyethylene glycol. The common PEG activation methods for specific modification of sulfo groups are shown in the figure below.
PEG activation method for specific modification of thiol
Specific modification of thiol-activated PEG
(6) Polyethylene glycol linked to enzyme sites: In addition to traditional chemical modification methods, modification can also be achieved by other means such as enzyme catalysis, taking G-TGase as an example.
Enzyme-linked PEG
Enzyme-linked PEG

Finally, select the appropriate protein amino acid residue site or small molecule drug site for site-specific modification. The site-specific modification of suitable protein amino acid residues with activated PEG can improve the efficacy of natural proteins. The biggest problem in PEG modification technology of protein drugs is the inability to achieve site-specific modification, the modification products are not uniform, which brings great difficulty to isolation and purification, and also greatly hinders clinical application. According to the amino acid properties of proteins and the characteristics of PEG derivatives, when scientists use PEG to modify, they choose protein surface residues that do not bind to the receptor as modification sites. In this way, in addition to the excellent properties brought about by PEGylation, the modified protein drugs also have high biological activity. At present, the common modification sites in marketed drugs include amino, carboxyl, sulfo, disulfide, glycosyl, and some specific positions of non-polar amino acids.
After decades of research, many PEGylated small molecule drugs have entered the state of clinical research. PEGylated small molecule drugs are widely used, the most representative of which is paclitaxel and camptothecin. More researches also include various types of commonly used anti-tumor drugs with relatively simple structures and a few non-anti-tumor drugs. In 2014, AstraZeneca's Movantik received US FDA approval as the world's first approved PEGylated small molecule drug, with annual sales expected to exceed US$1 billion at maturity.

The Future of PEGylation

At present, PEGylated drugs are mainly irreversible first-generation PEG-drug complexes and second-generation reversible PEG-drug complexes that are connected to the drug through a cleavable bond to exert a sustained release effect. Due to their non-biodegradability and limitation on molecular mass, the first-generation small molecule drug conjugates are not very satisfactory in improving their pharmacokinetics. Therefore, in addition to the peptide small molecule conjugate drugs that have undergone more research, Current research efforts have focused on the use of new biodegradable and spinal degradable water-soluble polymers. Degradable high-molecular-weight N- (2-hydroxymethyl) methacrylamide (HPMA) polymer small molecule drug conjugates show improved pharmacokinetics and pharmacodynamics, while showing ultimate renal clearance . Similarly, new water-soluble, biocompatible, and biodegradable polymers, such as polycarbonate, are also being synthesized and evaluated, expanding the range of polymers available for drug coupling. The future of PEGylation is geared towards the third generation of PEGylation, which aims to achieve the highest potency and extend the half-life of drug molecules, site-specificity, and low doses. This can be achieved by electrostatically linked non-covalent PEG modification. If successful, with the help of further research, PEGylation has the potential to become the main method of protein therapy.

Biochempeg can provide a variety of PEG linkers, which is convenient for pharmacists to carry out the next drug modification.

Related Article:
Things About PEGylation Technology and Biopharmaceuticals You Should Know 
The Applications of PEGylation Reagents
Potential Limitations of PEGylated Therapeutic Proteins
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