The biosynthesis of N-glycans in glycoproteins can be described as a four-step process:

The consensus amino acid sequence for N-glycosylation is Asn–Xaa–Thr/Ser (where Xaa is any amino acid other than proline). Overall, only about two-thirds of all potential sequences, termed sequons, are glycosylated, and currently there is no method to predict which sequon will be glycosylated. The role of protein N-glycosylation is usually protein trafficking and secretion.

N-glycans can be categorized as high-mannose, hybrid, or complex, depending on the extent of processing (Figure 1). High-mannose structures (Appendix 1) lack galactose or N-acetylglucosamine (GlcNAc) residues in the antennae, branches at the distal end of the chain. In hybrid structures, both substituted GlcNAc residues and terminal mannose residues are present in the antennae, whereas complex structures have both 1,6- and 1,3-mannose residues substituted with GlcNAc moieties. Hybrid and complex glycans can exist with two or more branches, frequently termed antennae; such glycans are therefore often termed, for example, biantennary, triantennary, or tetraantennary. Both monoantennary and pentaantennary N-glycans are also known to exist. In complex glycans, antennae frequently carry terminal sialic acid (neuraminic acid) residues. Sialylation has been shown to have a great effect on both the pharmacokinetics and the pharmacodynamics of many therapeutic glycoproteins.

O-Glycan chains are built up sequentially via an initial GalNAc residue linked to serine, threonine, and tyrosine, as well as to the less common amino acids hydroxyproline and hydroxylysine. Multiple glycan core structures are known. The sequence and isomeric linkage of monosaccharides show greater variety than that in N-glycans, and at least eight different types have been identified (Figure 2). Although no consensus amino acid sequence for O-glycosylation has been determined, glycosylation is usually favored by the presence of proline one residue before or three residues after the glycosylation site and the absence of charged amino acid residues proximal to serine or threonine. The disaccharide unit N-acetyllactosamine, Gal1,4GlcNAc, is the most common chain extension. Additional modifications, including terminal capping of Gal with sialic acid and fucosylation along the chain, are also frequent.

O-Glycosylation can occur in cluster form, the mucin type, which usually forms part of the cell surface extracellular matrix or secreted glycoproteins. Other O-glycosylation, such as O-GlcNAc, is found on many nucleocytoplasmic proteins; and O-Man–linked glycosylation is found in some muscular and neural glycoproteins and in yeast. O-Fuc– and O-Glc–linked glycosylation types are found on many epidermal growth factor-like proteins that are associated with the Notch signaling pathway.

Not only the type of glycosylation (N- or O-linked), site occupancy, and the site of glycosylation can vary from glycoprotein to glycoprotein, but also the actual oligosaccharide structures (branching and linkages) can differ, even on the same site. This structural variation arises because glycosylation is a process that is not driven by a template. The glycosylation pattern at a given site depends on many factors, including cell-specific and growth-dependent availability of glycosyltransferases and exo-glycosidases found in the Golgi bodies and ER. Heterogeneity leads to different physical and biochemical properties and, therefore, also to functional diversity.

The most direct mode of analysis is direct study of the intact molecule. This mode provides information about the glycosylation profile of the glycoprotein. However, this approach provides limited information when the molecule is large and contains multiple glycosylation sites. One of the most important glycosylation factors defining biological activity is the degree of sialylation, which often determines the half-life of glycoproteins in circulation. This makes ionic-charge–based electrophoresis and ion-exchange chromatography obvious choices of technique. Nearly all types of gel electrophoresis have been used to probe protein glycosylation, including polyacrylamide gel electrophoresis (PAGE), (see Biotechnology-Derived Articles—Polyacrylamide Gel Electrophoresis 1056) and isoelectric focusing (IEF), (see Biotechnology-Derived Articles—Isoelectric Focusing 1054). Similarly, capillary electrophoresis (CE), (see Biotechnology-Derived Articles—Capillary Electrophoresis 1053) has also been found suitable. Strong anion-exchange chromatography has been used for the same purpose, but the resolution is often inferior to that of IEF and CE. Direct mass spectrometry (MS) is another option for the analysis of posttranslational modification. Along with ongoing improvements in the resolution of MS, more and more complex glycoproteins become accessible for direct characterization by this method.

Analysis of glycopeptides provides information about site-specific glycosylation properties, the degree of occupancy, and oligosaccharide structures. Site-specific glycosylation can be affected by cell culture process conditions. Therefore, if a known glycosylation site is critical, manufacturers must monitor site-specific glycan structures. The typical approach is first to generate glycopeptides by protease digestion and to separate them by, for example, RP (reversed-phase) HPLC (see Biotechnology-Derived Articles—Peptide Mapping 1055). Subsequently the separated glycopeptides can be further characterized individually by, e.g., direct analysis using MS, or deglycosylation and subsequent glycan profiling, as described below in the section Profiling of Cleaved Oligosaccharides.

Direct identification of the mixture of glycopeptides and nonglycosylated peptides by MS is limited by masking effects (ion suppression) of peptide signals on glycopeptide signals. One approach to overcoming this effect is to separate peptides and glycopeptides before analysis by MS, e.g., by offline coupling (matrix-assisted laser desorption ionization [MALDI]) or online coupling (electrospray ionization [ESI]). MS analysis of glycopeptides plays an important role in the characterization of O-glycans because these glycans are not always released quantitatively and because, as a result of their smaller size, they are more amenable to characterization by MS as glycopeptides.

The use of CE for high-resolution separation may also be appropriate, especially for the analysis of sialylation.

Profiling of total glycans cleaved from glycoprotein is the most common approach for the characterization of glycoproteins. It provides a way to obtain information about the various populations of glycans present on the protein. The degree of sialylation can also be addressed at this stage. Depending on the chosen method, prior derivatization/labeling may be needed to allow the detection of the glycans. Many protocols are available, and most of the steps in the analysis are well established. The possible drawback with such flexibility is the lack of consensus about which methods to choose under which circumstances; because of the variety of analytical techniques, comparison of resultsobtained by different platforms may not always be possible. So far, the majority of the work has been done on N-glycosylation, because of the following factors:

Different quantitative monosaccharide assays are carried out for a number of purposes. In the glycoprotein field, they provide information about the relative amounts of saccharide in a glycoprotein and about the degree of sialylation of a glycoprotein; and by the measurement of monosaccharide composition, they provide some information about the structure of the glycan chains present.

The simplest assays used are colorimetric tests to demonstrate that the product is glycosylated and to quantify the total amount of saccharide present in the product. These have poor specificity between different types of sugar residues.

Assays of monosaccharide composition are generally simpler to perform than is oligosaccharide profiling, but they provide less information. The most widely used assay is quantification of sialic acid content, because loss of sialylation and exposure of terminal Gal residues may lead to faster clearance of the glycoprotein from the circulation.

The assays can be divided into two types: (1) those that provide compositional information about the intact sample without prior degradation; and (2) others, principally chromatographic, that require hydrolysis of the saccharide chains before analysis and generate quantitative information about several different monosaccharide species simultaneously. In general, the former are colorimetric and the latter are chromatographic. The hydrolysis step is a significant source of assay variability and may require careful optimization for specific samples.

The presence of certain monosaccharides is diagnostic of specific glycan structures. For example, observation of GalNAc is usually a marker for the presence of O-linked glycan chains, and fucose denotes the presence of specific types of chains. As a consequence of the limited diversity of monosaccharide residues present in glycoprotein glycans, accurate quantification of Man, Gal, or GlcNAc residues is required in order to distinguish between large numbers of structurally diverse glycans. The monosaccharide N-glycolylneuraminic acid (Neu5Gc) is not produced in humans and is generally regarded as an unwelcome and potentially immunogenic component of biopharmaceutical products.

High mannose—Glycan chains containing two core GlcNAc residues and between five and nine Man residues, and lacking Gal, GlcNAc, or Neu5Ac residues in the antennae. Such chains are typically found in mammalian glycans.

Hypermannosylation—(i) Addition of Man residues to high mannose chains creating chains with large numbers of Man residues, and (ii) O-Man linked glycan chains with multiple Man residues synthesized by yeast.

Paucimannose—Glycan chains containing two core GlcNAc residues between two and four Man residues. Core-linked Fuc1,3 and/or Fuc1,6 residues may be present.

Oligomannose—Used here as a generic term to include high mannose, paucimannose, and N-linked hyper-mannosylated chains.