Glycosylation modification of therapeutic antibodies

• Posted by jessica Young December 20, 2021 - Filed in Technology - #bio  - 1,289 views

  The structural and functional heterogeneity of therapeutic antibodies (IgG type) is achieved by post-translational modifications (PTM), among which the glycosylation of antibody Fc is the most heterogeneous one that can affect the stability of the molecule and its interaction with the Fc receptor in vivo. Thus, glycan distribution can influence a drug's mechanism of action, as well as its biological activity, safety, and efficacy.

   

  Polysaccharides can be co-translated with asparagine residues of proteins in the endoplasmic reticulum in eukaryotes. The oligosaccharide transferase complex recognizes the sequence motif of asparagine-X-serine/threonine (N-X-S/T) in unfolded proteins, where X can be any amino acid other than proline. In IgG-type antibodies, such motifs are mainly located in the CH2- structural domain of the heavy chain.

   

  The Fc sugar chain molecule has the core structure of a complicated double-antenna type 'pentasaccharide' molecule consisting of mannose (Man) and N-acetylglucose. The variability of polysaccharide length, branching patterns, and monosaccharide sequences leads to the complexity of the polysaccharide molecule structure. This N-glycan core structure can be further diversified in the presence of other enzymes to form a variety of structures.

   

  Numerous factors influence the conformation of therapeutic antibody glycoconjugates, the most important of which is the cell line. Mammalian cell lines, such as CHO, Baby Hamster Kidney (BHK) cells, human embryonic kidney 293 (HEK 293) cells, human embryonic retinoblast (PER.C6) and leukemia cell lines (NM F-9), and mouse myeloma cell lines such as NS0, SP2/0, and Y0, are the principal cell lines currently generating mAb. The CHO cell line is the most commonly used because it grows quickly and has a glycosylation pattern comparable to that of human cells.

   

  However, CHO cell lines differ from human cells in the sialic acid-galactose linkage because CHO cells lack -2,6 sialyltransferase and produce only -2,3-linked sialylated polysaccharides, whereas human cells produce both -2,3- and -2,6-linked sialylated polysaccharides, preferring the latter. Inadequate sialylation results from a lack of -2,6- sialylation, which affects the circulation lifetime of antibody molecules. Another significant difference is the lack of a functional GnTIII enzyme to prevent the insertion of GlcNAc residues. Other characteristics during fermentation that may alter the final glycan chain pattern of the therapeutic antibody, in addition to the host cell line, are glucose content, dissolved oxygen, bioreactor pH, sodium butyrate, ammonia content, CO2 concentration, and temperature.

   

  The polysaccharide structure can have a direct effect on the conformational space of the Fc structural domain, influencing the interaction with immune receptors and thus the functional properties of antibodies, such as half-life, antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cytophagy (ADCP), and other receptor-mediated immunomodulatory effects. The biological effects of various Fc-polysaccharide forms have been investigated.

   

  For the assessment of therapeutic antibodies, many alternative approaches for the analysis of polysaccharides have been devised. Using separation and mass spectrometry techniques, three primary options are possible:

  • analysis of intact molecules and inference of glycan chain composition
  • proteolytic cleavage and determination of glycopeptides
  • enzymatic (e.g., with PNGase F) or chemical release to identify polysaccharides

   

  Glycosylation of therapeutic antibodies is an unique class of post-translational modifications. Achieving constant levels of polysaccharides is difficult. Given the critical role it plays in controlling a wide range of functions, glycosylation control techniques are required. Glycosylation can be quantified as a quality attribute via its impact on a drug's effector function, half-life, immunogenicity, and pharmacokinetics/pharmacodynamics.