Conclusions

Protein glycosylation is the most common posttranslational modification in eukaryo-tic cells. One-half to one percent of the genome appears to be involved in glycosyla-tion, on the same order of magnitude as protein phosphorylation. There are two main types of glycosylation: N-linked oligosaccharides and O-linked oligosaccharides. All eukaryotic cells glycosylate proteins via common pathways; however, evolution has imparted many variations. The N-glycosylation pathway has three steps: the dolichol cycle and oligosaccharide transfer to the protein, the trimming reactions where glucose and mannose residues are removed, and the branching reactions that construct the oligosaccharide antennae. O-glycosylation, unlike N-glycosylation, is not generated by the transfer of a lipid-sugar onto the protein, but by the direct, sequential transfer of monosaccharides onto the protein. The glycosylation requirements of a recombinant glycoprotein are completely dependent on efficacy in the patient. Glycosylation is now recognized as a necessary component for many therapeutic enzymes, antibodies, and hormones. It is also recognized that many environmental factors affect glycosylation in cell culture. Recombinant glycoproteins can be produced in yeast, fungi, plants, insect cells, transgenic animals, and mammalian cells. However; to date, the only licensed therapeutic glycoproteins are produced by S. cerevisiae and mammalian cell culture.

Early therapeutic glycoprotein development was hindered by the lack of analytical techniques to measure glycosylation and an incomplete understanding of the cellular processes that controlled glycosylation. More ground is now being covered with respect to the cellular processes involved in glycosylation; however, there is still much work to be done. There are now many analytical techniques available to determine and quantify protein glycosylation. These analytical techniques are highly sensitive and able to detect and separate large molecules containing very small differences. The analytical techniques that are commonly used to quantify glycoproteins are electrophoresis, LC, MS, NMR, and exoglycosidase digestion. To fully characterize a glycoprotein, at least two or more orthogonal analytical techniques need to be used.

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