How to Perform Polyethylene Glycol (PEG) Modification?

Polyethylene glycol (PEG) is represented by the chemical structure HO-(CH₂CH₂)_n-OH, denoting a class of ethylene glycol polymers with molecular weights ranging from 200 to over 8000. Widely acknowledged as the most biocompatible polymer to date, PEG boasts colorlessness, odorlessness, low toxicity, minimal immunogenicity, and exceptional water solubility. Its attributes include biocompatibility, lubricity, and moisturizing qualities. PEG finds extensive utility in the pharmaceutical sector, serving as a versatile component. With its specific viscosity, PEG functions as a binder and suspending agent, dissolves readily in water for various applications, acts as a lubricant, enhances transdermal absorption due to its skin affinity, and can modify drugs effectively. The realm of PEG modification stands out as a highly promising and valuable avenue for its application.

What is Polyethylene Glycol (PEG) Modification?

PEG modification represents a sophisticated technique for modifying drug molecules and enhancing drug delivery. By attaching polyethylene glycol derivatives to the drug molecule surface, various properties such as hydrophilicity, volume, molecular weight, spatial conformation, and steric hindrance of molecular interactions are notably enhanced or altered. This modification can enhance the solubility of drug molecules, create a protective barrier, and decrease enzymatic hydrolysis. It offers benefits such as reduced toxicity and immunogenicity, altered tissue distribution, extended half-life, and increased concentration at target sites.

Polyethylene Glycol Modification Process

Before modifying PEG, it is essential to source high-purity PEG raw materials. This is because during the polymerization of polyethylene glycol, impurities such as trace water and incomplete reactions can form, potentially leading to unstable connection bonds and unwanted side reactions during drug modification. These impurities can directly impact the efficacy of the drug. The production process for PEG modification can be outlined as follows:

Step 1: Select the appropriate PEG for molecular modification

In general, the choice of modifiers mainly considers the following five aspects:

(1) Selection of PEG relative molecular mass (Mr)

The selection of PEG relative molecular mass (Mr) should take into account both biological activity and pharmacokinetics. The use of excessively large PEG-modified protein drugs will cause the drug to lose most of its biological activity. When using low Mr (<20000) PEG-modified protein drugs, the modified protein drugs have no essential changes in biological activity and pharmacokinetic properties compared to the prototype drugs. Therefore, PEG with Mr in the range of 40000-60000 is generally selected as modification.

(2) Molecular weight distribution coefficient (PDI) of PEG

The smaller the PDI of the PEG repair reagent, the better. The wider the molecular weight distribution, the more unfavorable it is for the separation and purification of modified protein drugs.

(3) Selection of modification sites

The selection of modification sites should be based on protein structural relationship analysis, and protein surface residues that do not bind to receptors should be selected as modification sites, so that the modified protein can retain higher biological activity. Commonly used modification sites include amino modification, carboxyl modification and sulfhydryl modification.

(4) Functional groups of PEG modifier

The specificity of the reaction between PEG modification reagents and amino acid residues depends on the choice of modification sites and the chemical nature of the modification agent. For the specificity of the repair reagent reaction, it is necessary to select a PEG repair reagent with appropriate functional groups.

(5) Molecular chain structure of PEG modifier

In addition to the molecular weight of PEG, the branched chain characteristics of PEG will affect many pharmacokinetic parameters of protein drugs. PEGylated molecules with different branched chain characteristics have different biological properties. Take PEG IFNα-2β as an example: when PEGylation uses small-molecule linear PEG, the half-life is about 40 h; when PEGylation uses large-molecule branched-chain PEG, the half-life is 80 h.

Step 2: PEG activation

PEG modification of proteins is mainly achieved through the reaction of PEG terminal hydroxyl groups with protein amino acid residues. However, PEG terminal hydroxyl groups have very poor activity, so they must be activated with an activator to covalently modify proteins under mild conditions in the body. Commonly used PEG activation methods are:

• Carbonyldiimidazole method
• N-hydroxysuccinimide method
• Cyanuric chloride method
• Activation method involving phosgene
• Chemical modification of cysteine residues of proteins with polyethylene glycol
• Enzymatic site-linked polyethylene glycol

Step 3: Select appropriate sites for site-directed modification

Use activated PEG to carry out site-specific modification of appropriate protein amino acid residues to improve the efficacy of natural proteins. The biggest problem in PEG modification technology for protein drugs is the inability to achieve site-specific modification and the uneven modification products, which brings great difficulty to separation and purification, and also greatly hinders clinical application. According to the amino acid properties of the protein and the characteristics of PEG derivatives, when modifying with PEG, researchers analyze the structural relationship of the protein and select protein surface residues that do not bind to the receptor as modification sites. In addition to the excellent properties brought by PEG modification, the modified protein drugs still have high biological activity. At present, common modification sites in marketed drugs include modification of amino groups, carboxyl groups, thiol groups, disulfide bonds, glycosyl-groups, and certain specific sites of non-polar amino acids.

(1) Site-specific modification of amino groups

The amino groups on the surface of protein molecules have high nucleophilic reactivity and are therefore the most commonly modified groups in protein chemical modifications, such as PEG modification of human interferon.

(2) Site-specific modification of carboxyl groups

The N-terminus of some proteins plays an important role in their biological activity. If PEG is modified at the N-terminus (amine group), the biological activity of the protein will be lost. Therefore, transferring the PEG modification site to the C-terminus (carboxyl group) is an effective modification strategy, such as polyethylene glycol modified loxenatide.

(3) Site-directed modification of thiol groups

Currently, mPEGs that can be used for thiol modification include mPEG-maleimide, mPEG-o-pyridine-disulfide, mPEG-vinylsulfone, and mPEG-iodoacetamide. For example, PEG modification of interleukin.

(4) Site-specific modification of disulfide bonds

Currently, certolizumab, one of the drugs on the market, uses PEG to modify the disulfide bonds in the antigen-binding region to extend its half-life, thereby exerting a longer-lasting drug effect.

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What is the Degree of Polymerization of Polyethylene Glycol?

As the degree of polymerization increases, the physical appearance and properties of polyethylene glycol gradually change. Those with a relative molecular weight of 200-600 are liquid at room temperature, while those with a relative molecular weight of more than 600 gradually become semi-solid. As the molecular weight increases, it changes from a colorless and odorless viscous liquid to a waxy solid, and its hygroscopic capacity decreases accordingly.

In addition, polyethylene glycol has strong water absorption and can absorb moisture from the air under normal temperature conditions, and the liquid can be miscible with water in any proportion. When the temperature rises, any fraction of solid polyethylene glycol can be miscible with water in any proportion. When the temperature reaches the boiling point of water, the polymer will precipitate. The precipitation temperature depends on the molecular weight and concentration of the polymer. Polyethylene glycol PEG is a non-ionic polymer and is stable under normal conditions. It can oxidize with oxygen in the air at temperatures of 120 °C or higher, and it will not change at 200~240 °C when protected by inert gases such as carbon dioxide or nitrogen. When it rises to about 300 °C, the molecular links break and degrade.

Applications of PEG with Different Degrees of Polymerization

According to the degree of polymerization of polyethylene glycol, its applications can basically be summarized as:

• PEG-200 can be used as a medium for organic synthesis and a heat carrier with higher requirements. It is used as a humectant, inorganic salt solubilizer, and viscosity regulator in the daily chemical industry; it is used as a softener and antistatic agent in the textile industry; it is used as a wetting agent in the papermaking and pesticide industries.
• PEG-400, PEG-600, and PEG-800 are used as bases for medicines and cosmetics, and as lubricants and wetting agents in the rubber and textile industries. PEG-600 is added to the electrolyte in the metal industry to enhance the grinding effect and enhance the luster of the metal surface.
• PEG-1000 and PEG-1500 are used as matrix or lubricant and softener in the pharmaceutical, textile and cosmetics industries. It is used as a dispersant in the coating industry to improve the water dispersibility and flexibility of the resin. It can improve the solubility of dyes and reduce their volatility in inks. It is especially suitable for wax paper and ink pad inks. It is used as a dispersant in the rubber industry to promote vulcanization and as a dispersant for carbon black fillers.
• PEG-2000 and PEG-3000 can be used as metal processing molding agents, lubricants and cutting fluids for metal wire drawing, stamping or forming, grinding, cooling, lubricating and polishing agents, welding agents, etc. It is also used in the paper industry as a lubricant, etc., and as a hot melt adhesive to add rapid rewetting capabilities.
• PEG-4000 and PEG-6000 are used as excipients in the pharmaceutical industry for the preparation of suppositories and ointments. It is used as a coating agent in the paper industry to increase the gloss and smoothness of paper. It is used as lubricant and coolant in rubber and metal processing industries; it is used as dispersant and emulsifier in pesticide and pigment industrial production; it is used as antistatic agent and lubricant in textile industry.
• PEG-8000 is used as a matrix in the production of pharmaceuticals and cosmetics industries to adjust viscosity and melting point; it is used as a lubricant and coolant in the rubber and metal processing industries; it is used as a dispersant in the industrial production of pesticides and pigments and emulsifier; it is used as antistatic agent and lubricant in the textile industry.