Effects of Glycosylation on the Stability and Half-life of Antibody Drugs


Among various post-translational modifications of proteins, glycosylation is one of the most important and complex modifications, and it is also one of the key quality attributes to evaluate antibodies. The realization of the function of monoclonal antibody is closely related to its glycosylation, which will affect the performance of protein, such as conformation, stability, solubility, pharmacokinetics, activity and immunogenicity.

Glycosylation is one of the important post-translational modifications of proteins. According to the modified sites of glycosylation, it can be divided into N-glycosylation and O-glycosylation. N-glycosylation is located in asn-297. N-acetylglucosamine in oligosaccharide links itself with amide nitrogen on asparagine residue to modify protein,which starts from endoplasmic reticulum and completes in Golgi body. O-glycosylation is completed in Golgi body by n-acetylgalactose in oligosaccharide linking with hydroxyl on serine or threonine residue to modify protein.

N-glycosylation is the most common glycosylation modification in immunoglobulins secreted by animal cells, and it is also the most studied glycosylation modification. Taking IgG1 as an example, its important glycosylation modification site is located at the FC end, and it can be divided into complex type, heterozygous type and high mannose type according to its terminal fine structure (length, branch and monosaccharide arrangement), as shown in Figure 1.

Fig 1. The position (left) and three main types (right) of N-glycosylation in McAbs

In Fig. 1, we can see that the glycosylation of IgG1 is complex type, and its glycosylation takes fucose as the core, and then n-glcnac divides into two equal branches, which are accompanied by mannose (man), galactose (Gal) and sialic acid (SIA), forming the N-glycosylation of FC end of IgG1. According to the different monosaccharides in the two branches, the glycosylation modification types of IgG1 can be divided into different sugar types in Fig. 1. It is because of the different sugar types that the immunogenicity, biological activity and pharmacokinetics of monoclonal antibodies are different.

Different glycosylation modifications have different effects on stability, half-life, safety and biological activity of the antibody.

Stability and Half-life

The most obvious function of glycosylation is that it can increase the stability and solubility of protein. Studies have shown that glycosylation can protect proteins by hiding the binding sites between proteins and proteases. Researchers found that the steric resistance of N-glycosylation to adjacent peptides was due to the formation of hydrogen bonds between glycosylation and hydrophilic amino acids. Glycosylation can also block the binding of protease and antibody, thus increasing the stability of antibody.

Sialic acid glycosylation in glycosylation can prolong the existence time of glycoprotein in serum. When the glycan of glycoprotein is terminated by galactose modification or de sialylated, it can be recognized by ASGPR, resulting in a significant reduction of half-life. ASGPR located on the surface of hepatocytes can't recognize the whole sialylated glycoprotein, but it will be de sialyzed by non-specific sialidase in the blood when it circulates with the blood. The galactosylation of naked dew can be recognized by ASGPR, and the glycoprotein can be degraded. Other studies have shown that high mannose type can reduce the half-life of antibodies in serum.

Researchers also found that fucosylation can effect the half-life of IgG1. Compared with the normal trastuzumab, the pharmacokinetics of trastuzumab has changed, resulting in a moderate reduction of its half-life.

In general, the higher the stability and the longer the half-life of the drug in the human body, the more effective it will play. As for anti-tumor drugs, if the stability of the drug is high and the half-life is long, the dosage can be appropriately reduced and the interval of administration can be designed to be longer in clinical treatment, and the effect is the same. However, with the increase of the stability and half-life of the drug in the human body, the safety of the drug will become one of the factors to be considered due to the potential miss.

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