PEGylation of Peptides and Proteins

Because peptides/protein-based drugs have strong immunogenicity, when they are used in the body, they will trigger the production of antibodies, which will reduce the efficacy and even cause allergic reactions. Therefore, researchers try to use soluble inert polymers, such as polyethylene glycol (PEG), combined with peptides/proteins to solve the dilemma, which can not only reduce its immunogenicity, but also can extend its half-life and retain its biological activity. PEGylation is a popular technology gradually developed in the late 1970s. At present, there are more than ten kinds of PEGylated protein drugs on the market with excellent clinical medical effects.

BOC Sciences' PEGylation services are designed to optimize the properties of peptides and proteins for a variety of applications, including drug delivery, diagnostics and biotechnology. Our team of experienced scientists and experts in the field of PEGylation can work with customers to develop custom PEGylation strategies that meet their specific requirements. We also offer a range of analytical services to characterize and validate PEGylated peptides and proteins, including techniques such as mass spectrometry, chromatography and spectroscopy to ensure the purity, identity and stability of the modified molecules.

Why is PEG Added to Drugs?

PEG contains a large number of ethoxy groups, which can form hydrogen bonds with water, has a high degree of hydrophilicity, and has a large hydrodynamic volume in an aqueous solution. It can change the biological distribution behavior of drugs in the aqueous solution and create a space barrier around the modified drugs to protect drugs from enzymatic hydrolysis, avoiding rapid elimination in the metabolism of the kidney. PEG is immunologically inert, even if its molecular weight (Mw) is as high as 5.9×106 Da, its immunogenicity is also very low. When using PEGylated protein in clinical treatment, no anti-PEG antibody was found. PEG is one of the few synthetic polymers approved by the Food and Drug Administration (FDA) that can be used for injectable medicine in the body.

PEGylation of Proteins

Research on PEGylation modification of proteins began in the 1970s. The original purpose was mainly to reduce the immunogenicity of protein drugs. Further studies have shown that PEGylation of proteins can also improve many other properties, such as increasing the stability of proteins to enzymes, extending the half-life of proteins in plasma, increasing solubility in water, etc. PEGylated modifications of some proteins have achieved good results in their application as drugs. In 1991, the first PEG-modified protein drug, PEG-adenosine deaminase, was approved by the FDA for the treatment of a severe childhood immunodeficiency disorder. In 2001, interferon modified with polyethylene glycol (PEG-INTRON) was approved by the FDA for the treatment of chronic hepatitis C.

Fig. 1. Strategies of site-selective PEGylation (Journal of controlled release. 2014, 192: 67-81).

PEGylation of Peptides

Peptides and proteins have many similarities in physical and chemical properties, and the research on PEGylation modification of peptide compounds was later than the related research on proteins. In recent years, research on the pegylation modification of peptide compounds has also made some progress. In particular, the site-directed pegylation modification of peptide compounds is easier to achieve than that of proteins. Therefore, it is also of great significance to carry out research on PEGylation modification of peptide compounds. The PEG modification sites of the peptide are at the N-terminal and C-terminal of the peptide, the Lys side chain and the thiol group of Cys. The molecular weight range of PEG single molecules used for modification is between PEG2 and PEG24; the molecular weight range of PEG macromolecules is between PEG 500 and PEG 40K.

Our PEGylation Services

The key points of design and synthesis of PEG modifiers for PEGylation need to be considered comprehensively, such as the types and modification sites of proteins and peptides; the hydrolysis stability, reactivity and selectivity of the modifier; the stability, toxicity and antigenicity of the linker between the modifier and the protein or peptide; whether the synthesis of modifier is simple and economical. Based on this, BOC Sciences has developed a one-stop service process for peptides and proteins.

Screening of PEG Raw Materials

Since the alcoholic hydroxyl group at the end of PEG is chemically inactive, in order to ensure the appropriate reaction speed with the amino group, the alcoholic hydroxyl group at the end of PEG needs to be activated. In order to avoid cross-linking and agglomeration during the modification process, we usually use mPEG as the synthetic raw material of the modifier. Furthermore, different polymerization processes provide significantly different levels of dispersion of mPEG. The dispersion of mPEG is high, and the dispersion of the prepared modifier is also high. Thus, the dispersion of the conjugate formed between the modifying agent and the protein or peptide also increases accordingly, and it is difficult to ensure the uniformity of the conjugate. Therefore, we generally first determine the distribution width index (MW/Mn) value of mPEG polymers from different sources through mass spectrometry or gel filtration chromatography analysis, and select appropriate mPEG raw materials accordingly.

Protection of Active Sites

In order to prevent the active site of a protein or peptide from reacting with the modifying agent during the PEGylation process and thus reducing or destroying its efficacy, it is often necessary to include the active site. Generally, we can avoid this by introducing active site protecting agents, using modifiers with different structures, adjusting modification reaction conditions, and controlling the reaction ratio between modifiers and proteins or polypeptides. For example, the reactivity of non-essential amino acid residues on protein and peptide molecules can be adjusted through changes in solvent type, reaction temperature, buffer medium and its pH value.

Identification of Modification Sites

Identification of modification sites is based on analysis of amino acids released during the stepwise degradation of the conjugate. For short peptides, the exact location of the linkage between the modifier and the protein or peptide can be relatively easily identified. However, it is very difficult to identify the modification sites of conjugates with larger molecular weights. In proteins, cleavage of the protein into short peptides can only occur. By using state-of-the-art techniques such as mass spectrometry, chromatography, and nuclear magnetic resonance spectroscopy, BOC Sciences can accurately identify modification sites within a peptide or protein, providing valuable information for optimizing the PEGylation process.

Analytical Qualification of Conjugates

For the analysis and characterization of the conjugate formed between the modifier and the protein or peptide, it is necessary to consider the average degree of modification of the protein or peptide and the homogeneity of the conjugate. The average degree of modification of a protein or peptide is the average number of modifier molecules coupled to each conjugate. The average degree of modification is often determined photometrically. The homogeneity of a conjugate is the respective relative content of the conjugate coupled with different numbers of modifiers. Currently, we can determine the homogeneity of conjugates through gel filtration chromatography, mass spectrometry, capillary electrophoresis and other methods. Conjugates with different degrees of modification have different residence times in gel filtration chromatography. Through this approach, the distribution of the conjugates can be determined.

Our PEGylation Technology

The most commonly used method in the study of PEGylation modification of peptides or protein is mPEG. First introduce carboxyl, amino or other reactive groups at the end of mPEG, or prepare mPEG-modified amino acid derivatives. Then use solid phase or liquid phase methods to couple it to the peptide sequence to achieve PEGylation modification of the N-terminal, C-terminal and certain amino acid side chains of the peptide. The following mainly introduces our methods for PEGylation modification of peptides and proteins.

Site-selective PEGylation

Enzymatic PEGylation

In the field of PEGylation, enzymes due to their highly specificity and selectivity have also been considered as tools for PEGylation of peptides/proteins. Transglutaminase (TGase)-mediated PEGylation is the most common used method, which catalyzes addition of acyl residues to primary amines. Moreover, glycoPEGylation, involving two enzymes to mimic O-glycosylation, aims to solve the problem that threonine and serine in the case of O-glycosylation and aspargine cannot be PEGylated directly without affecting other traditional PEGylation sites.

Releasable and Non-covalent PEGylation

Releasable and non-covalent PEGylation strives to overcome the loss of bioactivity resulting from PEG coupling at receptor binding site. For the releasable PEGylation, tailor made PEGylating agents, containing hydrolysable linkers, are exploited to allow a slow and controlled protein release in vivo. As regards to the non-covalent PEGylation, the polymer coupling does not involve a covalent bond but the polymer interacts with the protein through hydrophobic interactions or coordination bonds.

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PEGylation Service Process

Reference

1. Kolate, A. et al. PEG - a versatile conjugating ligand for drugs and drug delivery systems. Journal of controlled release: official journal of the Controlled Release Society. 2014, 192: 67-81.