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.
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:
1) analysis of intact molecules and inference of glycan chain composition
2) proteolytic cleavage and determination of glycopeptides
3) 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.