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Release date:2022/6/24 17:45:18

Protein-polymer conjugates are widely used as therapeutic agents for diseases with advantages such as high target specificity. Currently, FDA-approved protein conjugates are covalently linked with polyethylene glycol (PEG). These pegylated drugs have a longer half-life in the blood and are administered with a reduced frequency, which is a significant advantage for patients. However, PEG has some potential disadvantages that require the development of alternatives, so the development of polymers with enhanced pharmacokinetic properties as well as other advantages such as improved stability or degradability is important to advance the field of protein therapy.

Protein-polymer conjugates play an important role as therapeutic agents for diseases due to their high specificity and low off-target. However, there are two main ways to enhance protein properties (such as pharmacokinetics) by rapidly removing or inactivation of proteins through metabolism, excretion and other pathways. One is to use recombinant DNA technology to replace amino acids or create fusion proteins. The other one is conjugates of synthetic polymers, the most common example of which is polyethylene glycol (PEG). However, synthesis of protein-polymer conjugates is not always straightforward, and multiple factors need to be considered: choice of polymer, choice of protein target, choice of conjugation chemistry, and characterization of conjugation in vitro and in vivo.

 Development history of protein-polymer conjugates

Development history of protein-polymer conjugates

Choice of polymers 

PEG and PEG analogues 

Currently, the most widely used polymer is PEG, which not only increases the hydrodynamic radius of binding proteins but also helps immunogenic proteins evade the immune system. The PEG group is biocompatible. However, some people have been shown to develop anti-PEG antibodies, so the PEGylated proteins in these patients can be cleared more quickly. PEG brush polymers not only enhance circulating half-life, but also do not induce anti-PEG antibody responses.

Stimuli-responsive polymers 

In addition to improving pharmacokinetics, polymers can confer new properties on proteins. Polyacrylamides such as poly (N-isopropylacrylamide)(p (NIPAAm)) have a lower critical solution temperature (LCST), allowing the polymer to precipitate with increasing temperature. There are also conjugations of pH-responsive ionic polymers such as polyacrylic acid and p(DMAEMA). The use of stimuli-responsive polymers in disease therapy may enable interesting applications, such as hyperthermia-mediated cancer therapy, but potential toxicity should be considered.

 PEG and PEG analogues

PEG and PEG analogues

Biomimetic polymers 

Polymers inspired by natural molecules have been shown to effectively combine the desirable properties of natural molecules with the advantages of synthetic polymers. Natural disaccharide trehalose polymers can stabilize biomacromolecules to extreme conditions, another example of biomimetic polymers is heparin mimics polymers, which use polystyrene sulfonate and polyethylene sulfonate as synthetic heparin mimics polymers to stabilize the very unstable fibroblast growth factor 2 (FGF2) with therapeutic wound-healing potential.

 Biomimetic polymers

Biomimetic polymers

Degradable polymers 

Non-degradable polymers can accumulate in the body, potentially causing toxic side effects to the human body, so it is necessary to develop degradable polymers for biomedical applications. Cyclic ketene acetal (CKA) is a monomer that can generate hydrolytically degradable backbone ester linkages through free radical ring opening, and CKAs can be copolymerized with vinyl monomers to produce degradable polymers.

There are other natural polymer analogues, such as polypeptides and hydroxyethyl starch (HES). Various proteins have been conjugated to synthetic polypeptides, although these polypeptides are stable, they may be degraded in vivo by proteases. Similarly, HES is degraded by alpha-amylase in plasma, and its conjugates have been extensively studied for for therapeutic use.

 Degradable polymers

Degradable polymers

Other matters related to polymers

Toxicological tests for polymers in vitro and in vivo are necessary, and toxicity assessments should be carried out as early as possible, as safety issues arising during development can be costly.  The main purpose of a polymer to bind to a protein is to increase its cyclic half-life.  The size of the polymer also has an effect on bioactivity, with larger polymers often producing conjugates with lower bioactivity, most likely due to nonspecific steric hindrances.

The process of polymer selection is roughly as follows. First, the molecular weight of the polymer should be determined.  And then determine whether the conjugate only needs to extend its half-life.  A variety of PEGs or PEG analogues are available in the market for this purpose.  For long-term use, polymers that reduce immunogenicity should be considered because patients develop antibodies to PEG after repeated injections throughout their life.

 Polymer selection process

Polymer selection process

Choose the right coupling chemistry 

In general, polymer-protein coupling faces more steric and entropic barriers than small-molecule coupling reactions. Therefore, the commonly used coupling methods need to be very efficient, and the choice of coupling chemistry also needs to consider site selectivity and the availability of residues on the protein.

Reactive groups on proteins 

Site-selective conjugation is essential to maintain sufficient biological activity, and generally, the conjugation site should be kept away from the active site or binding motif to maximize protein activity. Lysine is the most abundant amino acid on the surface of proteins and is often the first residue attempted for non-selective conjugation. Bernardes and colleagues discovered that sulfonyl acrylates can selectively modify single lysines in five different proteins. The high nucleophilicity of cysteine makes it easy to modify, but free cysteines are rare and usually located in hydrophobic pockets. Disulfide bonds are present in most proteins and they can be used as residue-specific binding sites. Although tyrosine is less utilized, it is an amino acid with moderate surface abundance (4.8%), and several coupling methods have been developed, such as diazo coupling, PTAD coupling, etc.

Reactive groups on polymers 

Reactive groups on polymers are electrophiles that react with nucleophilic groups such as lysine, cysteine or tyrosine residues, usually these functional groups are located at one end of the polymer chain. These common reactive groups are activated esters and carbonates, aldehydes, amine reactive thiolation reagents, Michael acceptors, disulfide exchange, N-terminal coupling, tyrosine coupling. Maleimide is the most commonly used reagent for cysteine coupling.  They have very fast reaction kinetics (10 3-10 4 m-1s-1 at pH 7.5), and the coupling is generally stable. Vinyl sulfones are also commonly used Michael receptors, and although they are slightly less reactive than maleimides, hydrolysis is more stable.

Characterization of protein-polymer conjugates 

Purification of Conjugates 

Purification of protein-polymer conjugates is generally relatively difficult because polymers are almost always used in excess, and coupling efficiencies are often less than 100%, so mixtures typically contain three types of macromolecules—protein, polymer, and conjugate. For very small and stable proteins, high performance liquid chromatography (HPLC) can be used for purification, however the use of organic solvents and high pressure within the column can denature the tertiary structure of most proteins. On the other hand, size exclusion chromatography (SEC) is only suitable for a small subset of conjugates because it is best for separating two species that differ by more than a factor of two in size. Other chromatographic methods, such as ion exchange and hydrophobic interaction chromatography, are often better choices and are commonly used in the clinical production of protein drugs.

Characterization of Conjugates

Common characterization methods of conjugated compounds include gel electrophoresis, mass spectrometry, SEC and dynamic light scattering (DLS), none of which is deterministic, and multiple methods should be used for characterization.

Biological Evaluation of Conjugates 

The activity of the conjugate should be tested after purification and characterization. In vitro assays of protein activity range from growth factor-related cell proliferation assays to enzyme-linked immunosorbent assays (ELISA) of antibodies. The structural integrity of polymer-attached proteins can also be examined by circular dichroism (CD), DLS (for potential aggregation), and ELISA. After polymer coupling, protein activity is typically reduced to about 20% to 80%, with activity usually decreasing with increasing molecular weight and associated with the conjugation site. If the protein is not very active, its activity can be increased by site selective binding.

The Heather D. Maynard group synthesized an insulin-trehalose polymer conjugate by nonspecific reductive amination of two of the three amine sites on insulin that significantly improved the cyclic half-life. However, it may be due to non-specific conjugation that the conjugate requires five times the dose required to achieve the hypoglycemic effect in mice compared to free insulin.  In order to improve the conjugation activity, LysB29 was selected for site-selective conjugation. LysB29 was selected among the three possible conjugation sites (GlyAl, PheB1, and LysB29) because the bioactivity of GlyAl conjugate is significantly lower than that of other conjugates. And LysB29 is more reactive than PheB1 [2]. This site-specific conjugation requires only three times as much dose and therefore has a higher activity than previous methods.

 Biological Evaluation of Conjugates

Biological Evaluation of Conjugates

Toxicity and immunological evaluation of conjugation is important and should be carried out as early as possible in the development of protein-polymer conjugation as a therapeutic agent. In addition, the in vivo biological distribution of conjugation has a significant impact on its in vivo biological activity.

Summary and prospect

In the past few years, significant progress has been made in protein-PEG conjugated drugs for medicine. At present, a variety of PEGylated protein drugs have been clinically used to treat a series of diseases. However, there is still room for improvement, with many conjugates increasing the circulating half-life but significantly reducing the biological activity. Studies have shown that by rationally designing coupling sites and utilizing site-selective coupling reactions, the activity of proteins can be fully retained.

In addition, the development of genetic engineering, high-efficient coupling chemistry, and new methods to synthesize end-functional polymers for conjugation have led to degradable PEG substitutes and new biomimetic strategies to improve the stability and activity of natural proteins. Protein-polymer coupling will be an exciting field that requires the joint efforts of scientists with expertise from different disciplines. It is believed that in the future scientists can improve the highly evolved mechanisms of nature to promote human health.

PEGylation is being used as a universal therapeutic technique to provide diverse conjugation with peptides, proteins, antibody fragments, aptamers, enzymes, and small molecules. Dozens of PEGylated drugs are currently on the market for the treatment of cancer, chronic kidney diseases, hepatitis, multiple sclerosis, hemophilia and so on. As a lead PEG supplier, Biopharma PEG provides thousands of PEG linkers (PEGylation reagents) - maleimide,  pyridyl disulfide, amine, carboxylic acids and NHS esters functionalized PEG to support customers' research. We can also provide custom PEG synthesis services to meet our customer's different PEGylation needs.
 

 

References:
[1] 
Pegloticase immunogenicity: the relationship between efficacy and antibody development in patients treated for refractory chronic gout
[2]
Site-Specific Insulin-Trehalose Glycopolymer Conjugate by Grafting from Strategy Improves Bioactivity
[3] Therapeutic Protein–Polymer Conjugates: Advancing Beyond PEGylation

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