Polyethylene glycol is a type of polyether polymer. Its properties vary with its average molecular weight. Low-molecular-weight polyethylene glycol is a colorless, odorless, viscous liquid, while high-molecular-weight polyethylene glycol is a white solid. Polyethylene glycol molecules contain numerous ethoxy groups, which can form hydrogen bonds with water, making them highly hydrophilic. Polyethylene glycol is one of the very few synthetic polymers approved by the US Food and Drug Administration (FDA) for injectable pharmaceutical use.
Types of Polyethylene Glycol Modification Reactions
Of the 20 common amino acids that make up proteins, only the side chains of polar amino acid residues can be chemically modified. Activated polyethylene glycol is conjugated to proteins by chemically reacting with amino acid residues on the protein molecule.
Amino Group Modification
Because free amino groups on protein molecules react easily with nucleophiles and usually aren’t in active sites, they’re the go-to place for modifications. Numerous types of PEG are used for amino group modification, with cyanuric chloride, alkylation, and acylation being the most common methods.MPEG-SS, MPEG-SC, MPEG-SPA, MPEG-NHS, MPEG-CHO, and MPEG-ALD are often used to change amino groups.
Thiol Modification
Cysteine thiol groups usually stay inside proteins. There aren’t as many free thiol groups on the protein surface as there are amino groups, but we know exactly where they are. That means we can make site-specific changes to these free thiol groups without messing up how the protein works. And if the protein doesn’t have any cysteine to begin with, we can use genetic engineering to add one or more to specific spots, so we can change the protein exactly where we want. This approach not only achieves highly selective modification but also minimizes loss of biological activity and reduces immunogenicity. The most commonly used modifying agent is MAL-PEG.
Carboxyl Group Modification
Carboxyl group modification sites include aspartic acid, glutamic acid, and terminal carboxyl groups. First, the terminal group of the polyethylene glycol molecule is converted to an amino group, which is then conjugated to the protein’s carboxyl group in the presence of carboxydiimide. This can also easily lead to other cross-linking reactions. Recently, PEG-hydrazide has been shown to specifically bind to carboxyl groups. In the presence of EDC, under acidic conditions (pH = 4.5-5), the amino groups in the protein are protonated to avoid the occurrence of cross-linking reactions.
Selection of PEG modifiers
The selection of modifiers mainly considers the following aspects:
- PEG’s molecular size and weight spread matter. Research shows that how long a modified protein drug works in the body depends on how much PEG is attached. But, its activity in the lab goes down as more PEG is added. The use of PEG with too large Mn to modify protein drugs will cause the drug to lose most of its biological activity. PEG with a high molecular weight (over 20,000) is often used as a modifier these days. Also, a lower PDI for the PEG modifier is better. The wider the molecular weight distribution, the more unfavorable it will be for the separation and purification of the modified protein drug.
- Modification site and functional group of PEG modifier: When picking a spot to attach something to a protein, think about how the protein’s structure relates to what it does. It’s best to choose spots on the protein’s surface that don’t connect to any receptors. This helps make sure the changed protein still works well. How well a PEG modifier reacts with amino acids depends on what the modifier is made of and where you attach it. To achieve the specificity of the modification reaction, it is necessary to select a PEG modifier with an appropriate functional group.
- Molecular chain structure of PEG modifier: In addition to the molecular weight of polyethylene glycol, the branched nature of polyethylene glycol can affect many pharmacokinetic parameters of protein drugs. PEGylated molecules with different branched natures have different biological properties.
