Glycosylation and Carbohydrate Structure

Glycosylation is not controlled through a simple template in the way proteins are via the DNA template. Rather, glycosylation of proteins occurs in the endoplasmic reticulum and Golgi apparatus via the action of specifically localized enzymes on the glycoprotein as it passes through the secretory pathway from site of translation to site of release at the plasma membrane of the cell. This is true for both N and O-linked glycosylation of proteins. This means that the N-glycans and O-glycans produced are complex and can exhibit a high degree of heterogeneity, depending on the glycosylation enzymes that have acted and the extent to which that action has occurred.

There are many factors that can affect the profile of glycans on a glycoprotein. These include types of glycan biosynthesis enzymes present in the secretory pathway (which is a function of cell type used in manufacturing), the overall 3D structure of the protein itself, speed of the protein through the secretory pathway, and bioreactor conditions (such as pH or nature of growth media). Furthermore, different glycosylation sites within a molecule can have their own specific sets of glycans.

Protein glycosylation occurs in both plants and animals but the nature of glycosylation varies through the different kingdoms as well as between differ phyla and further down through taxonomic classification. Thus plants, insects and mammals all produce glycans with unique sets of structural features. Likewise, different mammalian cell types are also capable of producing unique glycan profiles that are specific for that cell type. This means that cell lines used to produce glycoproteins must be carefully considered since glycan structures should either match the natural source of the glycoprotein or be engineered in a controlled way to give the designed functionality.

The most important point to consider here is that humans produce a specific range of glycans. If an inappropriate cell line is used in the manufacturing process, glycoproteins could be produced with potentially immunogenic structures, since they would be not found on natural human glycoproteins. An example of this is the Galα1-3Gal epitope which is produced in murine cell lines. Bacterial protein glycosylation has also been observed and again this will not be the same as for human glycosylation and could potentially produce immunogenic glycan epitopes.

The role of protein glycosylation has been shown to be very diverse, with different effects observed depending on the nature of the glycans and protein to which they are attached. A wide variety of specific functions such as targeting of a glycoprotein to a site of action (eg inflammatory response, lysosomal targeting), control of half-life of a glycoprotein in the blood stream, control of function (e.g. decrease in fucosylation of a monoclonal antibody leads to an increase in antibody dependent cellular cytotoxicity) and maintenance of glycoprotein structure have all be attributed to glycosylation.

Many proteins are modified with glycans, indeed the majority of the therapeutic proteins on the market today are glycoproteins. These include monoclonal antibodies, Erythropoietin, Darbepoietin and FSH amongst others. The fact that so many biotherapeutics are glycosylated makes glycosylation one of the most important post-translational modifications to consider during protein characterization. This potentially high degree of heterogeneity may appear to make glycan analysis a daunting undertaking. However, BioPharmaSpec scientists are used to working with this high degree of complexity and will use their product knowledge and many years of technical expertise in glycan analysis to suggest the best glycan characterization assessment approach for your biopharmaceutical.