A common and variable type of post-translational modification in glycoproteins is that of O-glycosylation. In this case, sugar groups are linked to the hydroxyls of serine or threonine residues.
"Historically, the industry has placed more emphasis on characterizing N-glycosylation of bioproducts rather than O-glycosylation," says Mark R. Hardy, Ph.D., senior scientist, characterization and analytical development, Wyeth BioPharma (Andover, MA). "This may give the erroneous impression that N-glycosylation is more important than O-glycosylation."
Dr. Hardy feels that O-glycosylation needs to play a more prominent role in the minds of biotech scientists. "Part of the difficulty in analyzing O-glycans is that they are less tractable (than N-linked glycans). There are no generic enzymes to release O-linked oligosaccharides. This post-translational modification occurs in the Golgi apparatus where sugar attachment is catalyzed by an array of specific glycosyltransferases.
"As a result, the type of sugars added varies depending on the cell expressing the glycoprotein, the state of the cell, and competition between enzymes, substrates, and other factors. So, biosynthesis isn't straightforward, it's a rather chaotic pathway."
Although a number of traditional tools are available (such as reductive alkaline cleavage or "beta elimination"), a more holistic approach can be taken using peptide mapping, liquid chromatography, and mass spectrometry.
"These exploit the selectivity of reverse-phase HPLC proteolytic fragment separation and resolution. Site occupancy and forms of glycosylation can be determined. You can also do a combination of these assays. Although this is not necessarily a quantitative technique, it is valuable for characterization and comparisons."
Other reasons for characterizing O-glycosylation include verifying the consistency of manufactured bioproducts and validating function. "Alterations in O-glycosylation can potentially affect potency, half-life, clearance, and immunogenicity, etc.," notes Dr. Hardy.