Glycosylation is the process by which a protein or lipid is attached to a sugar under the control of an enzyme, starting at the endoplasmic reticulum and ending at the Golgi apparatus. The sugar is transferred to the protein by the action of a glycosyltransferase, and the amino acid residue on the protein forms a glycosidic bond. The protein undergoes glycosylation to form glycoproteins. Glycosylation is an important modification of proteins and regulates protein function.
Process
N-linked sugar chain synthesis begins in the endoplasmic reticulum and is completed in the Golgi apparatus. The glycoprotein formed in the endoplasmic reticulum has a similar sugar chain. After the Cis surface enters the Golgi apparatus, a series of ordered processing and modification occurs during the transport between the membrane capsules. The original sugar chain is large. Part of the mannose is excised, but a variety of glycosyltransferases are sequentially added with different types of sugar molecules to form oligosaccharide chains with different structures. The spatial structure of the glycoprotein determines that it can bind to that glycosyltransferase and undergo specific glycosylation modifications.
Many glycoproteins have both N-linked sugar chains and O-linked sugar chains. O-linked glycosylation is carried out in the Golgi apparatus. Usually, the first attached sugar unit is N-acetylgalactose, and the linked sites are the hydroxyl groups of Ser, Thr and Hyp, and then the glycosyl groups are successively transferred to form. Oligosaccharide chains, sugar donors are also nucleoside sugars, such as UDP-galactose. The result of glycosylation allows different proteins to be labeled differently, altering the conformation of the polypeptide and increasing the stability of the protein.
One or more aminoglycan chains can also be attached to the serine residue of the core protein via xylose on the Golgi to form proteoglycans. Some of these proteins are secreted outside the cell to form an extracellular matrix or mucus layer, some anchored to the membrane.
Classification
According to the type of glycoside chain, protein glycosylation can be divided into four types, that is, the hydroxyl group of serine, threonine, hydroxylysine and hydroxyproline is used as a point of connection to form an -O-glycosidic bond. Using an amide group of asparagine, an α-amino group of an N-terminal amino acid, and an ω-amino group of lysine or arginine as a point of attachment to form an -N-glycosidic bond; aspartic acid or glutamic acid The free carboxyl group is a point of attachment, forming a lipotypic bond type and a glycopeptide bond with cysteine as a point of attachment.
Future prospects
Drug glycosylation is a unique post-translational modification, and their synthesis is not a model-driven process, so obtaining a consistent glycoform is a daunting task. It is known that glycoforms have a regulatory effect on a range of complex functions, and the strategy of the invention to easily monitor glycoforms in real time is pertinent. In addition to significant advances in process analysis, glycosylation level control remains an unrealized goal. The biopharmaceutical industry and academic researchers have worked hard to bridge this gap, and have made great progress in understanding the complexity of post-translational modifications, the effects of various process variables on glycoforms, and the effects of various glycan types on the efficacy of biopharmaceuticals. Have some understanding. This also clearly implies that in the development of biosimilar drugs, similar drug manufacturers cannot obtain research and development information related to the original drug.
Risk assessment based on biopharmaceutical CQA helps manufacturers make wise decisions early in drug development. In mAbs, the glycoform range as a mass attribute can be estimated by the effector function, half-life, immunogenicity, and pharmacokinetic/potency of the drug. For example, a fucose-free modification enhances ADCC activity, while sialylation affects the anti-inflammatory properties of mAb molecules. Therefore, based on the mechanism of action of the drug and our understanding of the biological role of different glycan species, biosimilar drug manufacturers can make decisions based on data early in the development process. This will significantly reduce the risk of late clinical trial failure and increase our trust in comparability analysis.
Therefore, the use of powerful analytical instruments allows real-time analysis and monitoring of glycosylation modifications, such as NMR using a single amino acid resolution combined with advanced statistical tools to analyze protein characteristics. In addition, mathematical models will be a powerful tool for predicting real-time glycosylation levels in the near future, and can be used to adjust the ongoing culture process in situ based on culture parameters. With the advent of system biology in cell culture, factors such as kinetic parameters, metabolic constants, and cell viability parameters can be easily established. Biosimilars are a valuable and affordable alternative to existing biologics, and powerful instruments and tools have a significant impact on the emergence of biosimilars.
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