Analytical Tools for Glycosylated Drug Characterization: A Guide for R&D Teams

Why Glycan Characterization is so Challenging?

(1) Heterogeneity of Glycoforms

Glycans are structurally diverse and variable because of the relatively large number of monosaccharide building blocks. The anomeric carbon of one monosaccharide attaches to the hydroxyl group of another monosaccharide to create glycosidic bonds. The presence of multiple hydroxyl groups in monosaccharides enables their combination to form various disaccharide molecules which differ in regiochemistry and stereochemistry and exhibit different biological activities. As the number of monomers conjugated increases, the number of possible disaccharides and oligosaccharides increase exponentially. For example, if one type of monosaccharide is used, 1792 different structures could be generated for a tetrasaccharide. Glycans also show structural heterogeneity due to the heterogeneity of glycosylation, the biosynthetic process by which glycans are generated. In contrast to protein biosynthesis, glycosylation is not template driven. The structure of glycans depends on several elements including sugar donor substrates presence, specific biosynthetic enzymes availability and biosynthetic pathway competition. A particular glycoprotein may be present in several glycoforms which can differ in both number and site of glycosylation on the peptide backbone (macro-heterogeneity) as well as monosaccharide composition, sequence and branching of the carbohydrate chain at each site.

Fig. 1 Major sources of complexity in the structural analysis of glycans.^1,2

(2) Isobaric Species and Structural Isomers

Isobaric species and structural isomers add complexity to glycan analysis methods. Isobaric species may be distinguished based on structural differences despite having equal molecular weight. Structural isomers have the same molecular formula but their atoms are arranged differently. Isobaric species may be problematic in MS analysis, because they are expected to have the same mass to charge ratio. Structural isomers can be problematic in MS analysis because they often give rise to overlapping fragmentation patterns. Different fragmentation techniques can produce distinct fragment ions that serve as unique signatures for each specific structural isomer. Two other analytical methods that may be used to separate and characterize a mixture of glycans are HILIC and HPAEC. In HILIC, glycans are separated on the basis of their hydrophilicity. In HPAEC, glycans are separated on the basis of their charge. Glycan profiles can be complex and methods for their analysis should be carefully developed to be as robust and reliable as possible.

(3) Sensitivity and high throughput requirements

A related issue that further complicates the structural analysis of glycans is that, with a few exceptions, they exist in most biological systems in small amounts. Carbohydrates isolated from biological mixtures (cells, tissues, body fluids) often need to be subjected to a number of additional steps of purification and concentration to reach the level of sensitivity necessary to detect the glycan constituents. Methods that can simplify and expedite the current time-consuming, costly and laborious process of isolating glycans to a level of concentration and purity that can be studied are highly sought. A final point that has contributed to the challenges in the study of glycans is the desire to be able to analyze glycosylation at the level of the whole cell, tissue and even organism. Changes in glycosylation at the whole cell level are known to occur with a variety of cellular events, particularly in disease. For example, many malignant tumor cells express truncated, branched and hyper-sialylated cell surface glycans and this fact has spurred development of glycans as biomarkers for cancer. Analyzing individual cells and tissue glycomes stands out as an effective strategy to discover new disease biomarkers and molecular drug targets. Due to the large numbers of glycans that need to be surveyed to define a glycome, many of which are conjugated to membrane-associated molecules and thus require additional steps to recover glycans for analysis. Analytical technologies with high throughput capabilities have, therefore, received increasing interest due to the diversity of glycan structures that need to be characterized.

Analytical Methods for Glycopeptide and Glycoprotein Characterization

(1) Mass Spectrometry: LC-MS/MS, MALDI-TOF

The superior analytical technique for examining glycopeptides and glycoproteins is MS. LC-MS/MS and MALDI-TOF are two MS techniques used for analyzing glycopeptides and glycoproteins, the former is a powerful analytical technique that is used for the analysis of complex biological samples. It is highly sensitive and can provide high-resolution mass measurements. LC-MS/MS combines the separation capabilities of LC with the detection power of MS/MS to allow for the identification of glycan structures and attachment sites. Additionally, LC-MS/MS can be used to quantify glycopeptides in complex samples, making it a valuable tool for the study of glycosylation patterns in various biological systems. MALDI-TOF is involves the use of a laser to ionize the sample, which is then analyzed by measuring the time it takes for the ions to reach the detector, with the rapid and accurate advantages, and can be used for high-throughput screening and analysis of glycoproteins.

(2) HILIC, RP-HPLC, and CE for Separation

Researchers can modify carbohydrate analysis separation techniques to identify glycan structures indirectly. LC and capillary electrophoresis(CE) stand out as the two primary techniques among such separation methods. High-performance liquid chromatography (HPLC) is the most robust separation technique for carbohydrates. Experiments with HPLC propel solutions containing mixed glycans through columns filled with stationary phase material using pressure-driven flow. Components of the mobile phase have different affinities for the stationary phase, and so they are eluted from the column at different rates. The resulting retention data can be compared to annotated chromatographic data obtained under similar conditions to make educated guesses about glycan structure. Many types of HPLC methods are used for carbohydrate analysis, with the most commonly used being HILIC and reverse-phase LC (RPLC). Major drawbacks for the use of HPLC are low throughput, as run times are usually in the 20 to 45 min/experiment range and the high cost of conducting separation experiments. CE is an electrophoretic separation method also commonly used in carbohydrate analysis. During CE, glycans are placed in an electric field, causing them to migrate differentially through a separation medium depending on the charge of the molecule. CE is attractive for use as a separation method because separations proceed rapidly compared to liquid chromatography. The major limitation to the use of CE is poor coupling to MS, as CE has flow rates that are orders of magnitude lower than are used for MS (<20 nL/min).

(3) Lectin-Based and Antibody-Based Binding Assays

Carbohydrate analytical approaches can be classified by examining the structural information they reveal. Basic methods yield qualitative results indicating whether monosaccharides or oligosaccharides motifs exist. The most prevalent of these assays are lectin profiling assays. Lectins are a family of proteins that recognize specific monosaccharide or oligosaccharides motifs, and by monitoring the presence of the corresponding carbohydrate binding partner can provide general information on glycan composition. Fluorescently tagged lectins are used to visualize and quantitate the abundance of the corresponding monosaccharide(s). There are >60 commercially available lectins, which allow for a wide variety of structural motifs to be assayed in a high-throughput manner. However, since many lectins are promiscuous binders, these assays are vulnerable to false structural assignments. In a similar strategy, carbohydrate-specific antibodies can be used to obviate non-specific binding. However, the higher specificity and cost of antibodies mean that antibody-based profiling is less affordable and versatile. Antibody-Based Binding Assays (ABBAs) specifically target glycopeptides or glycoproteins that present unique glycan structures within complex biological samples. ABBAs, such as ELISA and Western blotting, are commonly used to study glycosylation patterns and their biological functions.

How Our Glycopeptide QC Services Support Your Research?

(1) Standard Reference Glycopeptides for Method Calibration

Our quality control services deliver standard reference glycopeptides as part of the portfolio. Standard reference glycopeptides are highly pure and well-characterized glycopeptides that can be used as standards for analytical method development and calibration. MS can be used to determine the glycan structure and site of attachment. NMR spectroscopy can be used to determine the three-dimensional structure of a glycoprotein. Three-dimensional structures of glycoproteins can be revealed through the technique of X-ray crystallography.

(2) Batch Consistency Reports and Structural Validation

Batch-to-batch consistency is important for the research and development of glycopeptides. QC services offer batch consistency report and structural validation for each batch of glycopeptide synthesized. The report for batch consistency contains information on analytical data, such as MS, HPLC, nuclear magnetic resonance, to make sure the batch is right on the glycan structure and purity specifications. The structural validation of glycopeptides is conducted by an analysis using state-of-the-art techniques to assure identity and integrity of the glycopeptides. The process includes identification of the exact mass and glycan composition by MS, the purity and homogeneity of the product by HPLC, and if needed, NMR can be used for detailed structural information of glycopeptide, including the configuration and linkage of glycan residues.

Glycoamino acids we can provide

References

1. Image retrieved from Figure 1 " Major sources of complexity in the structural analysis of glycans," Pinnock F.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Pinnock F.; et al. " More small tools for sweet challenges: advances in microfluidic technologies for glycan analysis." Frontiers in Lab on a Chip Technologies, 2024, 3: 1359183.
3. Mastrangeli R, Satwekar A, Bierau H. Innovative Metrics for Reporting and Comparing the Glycan Structural Profile in Biotherapeutics[J]. Molecules, 2023, 28(8): 3304. https://doi.org/10.3390/molecules28083304.
4. Delafield D G, Li L. Recent advances in analytical approaches for glycan and glycopeptide quantitation[J]. Molecular & Cellular Proteomics, 2021, 20: 100054. https://doi.org/10.1074/mcp.R120.002095.
5. Niazi S K, Omarsdottir S. Lectin-Based Fluorescent Comparison of Glycan Profile—FDA Validation to Expedite Approval of Biosimilars[J]. International Journal of Molecular Sciences, 2024, 25(17): 9240. https://doi.org/10.3390/ijms25179240.