Structure and structural dynamics are important attributes that enable proteins to perform many different activities in vivo. Although most of the unique structural and functional properties of a protein are dictated by its amino-acid sequence, there are many changes, called post–transitional modifications (PTMs) that can alter a protein as it is expressed, folds, and ages (1). These alterations can significantly change and control the specificity and strength of protein interactions, as well as influence the physico–chemical properties of the protein (e.g., stability, solubility, immunogenicity) (2). Probably one of the most common and important of these PTMs is glycosylation—the attachment of oligosaccharides to proteins.
During recent years, it has become increasingly apparent that glycosylation is important in controlling the function and solution behavior of proteins in solution (3, 4). More than half of all eukaryotic proteins are glycoproteins (5). The oligosaccharides, also referred to as glycans or carbohydrates, commonly found on a protein exist as either N–linked or O–linked oligosaccharides and typically consist of 2 to 14 monosaccharides chemically linked in a linear or branched configuration. N–linked oligosaccharides are chemically linked to asparagines (and to a lesser extent arginine) within the consensus sequence –Asn–Xaa–Ser/Thr (6), while O–linked oligosaccharides are chemically linked to any serine or threonine (7).
To date, most of the progress in characterizing the carbohydrates on glycoproteins has focused on studying their structure and composition. These results have shown that a diversity of oligosaccharide structures can exist at any given glycosylation site on a protein. This diversity is one of the major causes of the microheterogeneity observed in proteins and is one of the major challenges biopharmaceutical companies face in demonstrating lot–to–lot comparability in manufacturing a glycosylated protein biopharmaceutical. Differential glycosylation on proteins will also be an important factor in determining similarity between innovator products and their follow–ons.