The need for better Glycoprotein Standards

Glycosylation is one of the most common post-translational protein modifications in eukaryotic systems (12-14). It has been estimated that 60-90% of all mammalian proteins are glycosylated at some point during their existence (12, 14) and virtually all membrane and secreted proteins are glycosylated (13). Glycoprotein glycans often play crucial roles in physiological events such as cell-cell recognition (15-17), signal transduction (18), inflammation (19), and tumorigenesis (20-25).  Given the important physiological roles of protein glycosylation, numerous research groups have devoted significant effort to the characterization of specific glycan structures, the identification of proteins that express each glycan, and the detailed study of how these structures change, e.g., as cells differentiate or as tumor cells progress.  All of these efforts have given rise to the emerging field of glycomics (26).

Small changes in the glycosylation patterns of glycoproteins have also been demonstrated to have profound effects on glycoprotein therapeutics.  As with many other recombinant glycoprotein therapeutics, mAb glycosylation plays a role in maintaining stability and solubility (27), along with cellular transport and clearance (28), but an interesting story revolves around the complex relationship between glycan structure and Ab effector  function.  The antigen binding fragment (FAB) of an IgG binds to the target antigen causing a change in the conformation of the crystallizable fragment (Fc) region, see Figure 1.  This structural change enables the Ab to induce signaling by binding to one or more of the groups of Fc receptors.  Because the glycan chains maintain the confirmation of the Fc region, the glycans play a role in directing the binding of the activated Ab to specific classes of Fc receptors, and thus determine the response of the Ab binding to its epitope. (29)  For instance, Abs without a core fucose activate the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway, i.e., direct natural killer cells, up to 50 times that of Abs with this glycan motif. (30)  Consequently if the intended purpose of the antibody is to kill the cells it binds to which it binds, the Fc glycans should be not fucosylated.  Alternatively, if the purpose of the mAb is simply to bind to cells with the target epitope, then these glycans should be fucosylated.  Similarly, the anti-inflammatory response of intravenous immunoglobulin G (IVIg) treatment has been attributed to that fraction of the IgG that contains Fc glycans with terminal sialic acids, which may account for as little as 0.02% of the total IgG population  (9, 10).  A possible explanation for this behavior is that the sialic acid containing glycan leads to activation of CD22, which in turn leads to B-cell activation. (31)

Beyond Abs, glycans can play a role in the efficacy of many other therapeutic glycoproteins.   Although there are numerous examples of this, probably the best characterized is recombinant EPO (Epogen), which is used for the treatment of anemia.  The extent of sialylation on its N-glycans significantly alters its pharmacokinetics such that removal of the terminal sialic acid residues decreases its plasma half-life in rodents from hours to minutes (32). This observation inspired Amgen to design a hyperglycosylated form of EPO that has a 3-fold longer half-life in humans (33).  The pharmacokinetic properties of several other recombinant glycoprotein therapeutics have similarly been enhanced by increased levels of sialylation (34).  Although space constraints limit this discussion to only a handful of examples, we hope that this discussion has demonstrated that changes in the ratios of N-linked glycans can have profound biological effects and thus the significance of being able to accurately quantitate these changes.