Applications

Clinical Biomarker Discovery

Changes in glycosylation are a hallmark of many cancers and inflammatory diseases and show great potential as clinical disease markers.

The interaction of cell surface glycans with complementary glycan binding proteins (lectins) located on neighboring cells, other cell types, or pathogens like virus, bacteria or parasitesmediates is crucial in biologically and biomedically important processes like cell–cell adhesion, cell migration, development, pathogen recognition and infection.

Their implication in nearly every pathological condition, consequently suggests an increasing role for glycans as disease markers. The majority of mammalian proteins are N-glycosylated, a common post-translational modification of the asparagine residue within the consensus sequence Asn-X-Ser/ Thr. N-glycans are large complex and branched glycans that share a common pentasaccharide core and present considerable microheterogeneity due to variations in the number of antennae, terminal glycan residues and core modifications.

Glycan Biomarker Research

Although previous glycan biomarker research has also included analysis of glycosphingolipid and glycosaminoglycan levels by mass spectrometry (MS), immunostaining or lectin arrays the focus of most studies has been on the plasma N-glycome followed by the analysis of O-glycans on mucins which are highly over-expressed in carcinomas. This preference can be explained by the high abundance of N-glycans in serumand other body fluids, a manageable number of structures and more mature techniques for sample preparation and analysis of N-glycans compared to those available for other glycan classes. Human N-glycans, that are present on the majority of secreted proteins are easily obtained from a larger number of body fluids including serum/plasma, urine, saliva, tears, milk, semen or amniotic fluid by enzymatic or chemical removal.

Although estimates for the human N-glycan repertoire go as far as 2000 different structures, the currently experimentally accessible human N-glycome is far smaller and very dependent on the employed analytical method. The plasma N-glycome has been reported to contain more than 100 different glycan structures but depending on the analytical method used only a fraction is routinely quantified due to either intense peak overlap during the chromatographic separation or a lack of sensitivity for detecting less abundant species by direct injection methods or MALDI-TOF (matrix assisted laser desorption/ionization coupled to time-of-flight detection) analysis. By nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC MS/MS) recently over 170 distinct N-glycan structures where registered in the plasma glycome and partially assigned by exoglycosidase digestion and diagnostic fragment ions. The 20 most abundant plasma Nglycans that account for over half of the plasma glycome are mono and bis-sialylated or neutral core-fucosylated bi-antennary structures.

These are major structures present on the most abundant plasma glycoproteins like the immunoglobulin isotypes IgG, IgMand IgA, transferrin, alpha-2-macroglobin, C3-complement or haptoglobin. Alpha-2-macroglobin, C3-complement and haptoglobin are acute phase proteins (APPs) which are part of the innate immune system and that show expression levels that are sensitive to inflammatory processes. The high basal concentration of some APPs in plasma together with their significant changes in expression levels during inflammation ranging from 50% of ceruloplasmin to over 1000-fold for the C-reactive protein, can have an impact even on the total serum glycan levels which is measurable as an increase of mobile hexosamine N-acetyl methyl groups by nuclear magnetic resonance (NMR).

Clinical Disease markers

Only absolute glycan quantification can provide a complete picture of the disease related changes and will provide the method robustness required by clinical applications.

The entry into the clinical practice of glycan markers is delayed in large part due to a lack of adequate methodology for the precise and robust quantification of protein glycosylation. Probably the most important area for absolute quantification of specific glycan levels is clinical diagnostics, which requires robust, reliable and precise methods for the quantification of disease markers. A large number of glycan biomarker studies have revealed the perturbation of glycan profiles, but methods for quantifying singlemarkers in the context of a complexmatrix are still underdeveloped. As a result, the decade-long exploration of glycans as biomarkers has not been matched by their introduction as disease markers into clinical practice. An important reason for this could be the lack of robust, transferable and quantitative methods for themeasurement of single glycans in the clinical practice.

Glycosylation not only changes qualitatively but is also increased in absolute terms as several pioneering studies suggest, allowing the definition of clinically relevant cut-off values for individual serumglycan levels for the clinical practice. Elevated serum glycan levels for a large number of structures have been shown in a pancreatic cancer study by Nishimura et al. [1] which show little or no overlap with 95% confidence intervals for healthy controls over a broad age range. As the majority of methods employ mass spectrometry either as a standalone technique or coupled with chromatography for glycan analysis, improvements in reproducibility and robustness of MS facilitating method transfer between labs are urgently needed.

The use of stable isotope labeled glycans as internal standards and heavy-isotope labeling methods will provide the necessary method precision and robustness acceptable for clinical use.

Internal stable isotope labeled standards for glycan quantification.

The use of internal stable isotope labeled standards for glycan quantification by mass spectrometry has the potential to solve many of the current challenges in MS glycan analyses and to aid in the transfer and acceptance of glycan markers into the clinical practice.

An isotopically enriched and quantified internal standard allows the absolute quantification of individual glycan levels in complex mixtures independent of changes in a glycan profile. A recent roadmap report from the US National Research Council highlighted the need for developing isotopically labeled glycan standards formass spectrometry to overcome “the current practice of relative quantification”.

In analogy to existing clinical methods for quantifying metabolites or drug levels we anticipate that a single or a limited number of glycan disease markers will be most efficiently quantified by isotopic dilution e.g. by employing stable isotope labeled glycans. Glycan synthesis has come a long way and current chemo-enzymatic approaches are now capable of producing well defined pure and strategically labeled glycans for this purpose.

The use of internal standards for absolute glycan quantification accelerates glycan analysis rather than slowing it down as any external calibration is unnecessary. This will be particularly the case for the rapid assessment of individual glycan levels as surrogate metabolites, which reflect impact of hereditary and environmental factors on patient physiology, in a platform independent and robust manner.

Biosimilar Glycosylation

Comparability studies of biosimilars reveal that products differ widely in composition and not always meet self-declared specifications. Glycosylation profile can exhibit batch-to-batch variation affecting the activity of a recombinant protein directly, and products that are similar from a qualitative perspective often differ quantitatively in the glycosylation profile, i.e. the clones are undergalactosylated.
Customized mAbsolute kits allow the absolute quantification of  the glycans attached to a given specific biosimilar in a few hours.

BioPharmaceutical N-Glycan Profiling

Most eukaryotic proteins, both membrane bound and soluble, and the large majority of commercial recombinant therapeutic proteins are modified with N-glycans that can have a major impact on protein solubility, structure, immunogenicity, circulatory half-life, and consequently drug efficacy.

Changes in protein glycosylation are also a hallmark of many cancers, infectious and autoimmune diseases, and the growing number of congenital disorders of glycosylation (CDG)4 suggesting an increasingly important role of glycans as biomarkers.

Consequently, robust and quantitative methods for the analysis of glycans are not only required for mapping glycan structure to function but also highly relevant in biopharmaceutical quality control and in the development of glycans as selective and complementary disease markers.

Relative Quantification methods

Many profiling methods require the enzymatic or chemical release of the glycans from the peptide backbone. The resulting mixture of glycans can then be chemo-selectively derivatized with a fluorescent label8 like 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), or 9-aminopyrene-1,4,6-trisulfonic acid (APTS), separated by HPLC or capillary electrophoresis and analyzed by fluorescence and/or mass spectrometry detection. Alternatively, glycans can be profiled, often after permethylation, directly by mass spectrometry although isobaric structures remain unresolved, unless diagnostic fragment ions can be produced by tandem MS. While the chromatographic methods are sensitive and provide relative quantification of glycans via uniform labeling,10 they are more time-consuming, expensive, and in general more prone to error due to additional sample preparation steps. More importantly, current methods only provide relative but no absolute quantification of individual glycans, e.g., for diagnostic applications or quantification of immunogenic glycan levels. While the relative quantification of glycans is thought to be sufficient to track changes in glycosylation between samples in many biopharmaceutical applications, a clinical use of glycans as disease markers that goes beyond glycan ratios would require methods that measure absolute concentrations of individual glycans.

A new Absolute Quantification method

Isotopic dilution mass spectrometry has been the golden standard for absolute metabolite quantification in newborn screening, quantification of immunosuppressor levels, in screening for illicit drugs, and in various approaches for quantitative proteomics. An application of isotopic dilution to glycan analysis, however, has been hampered largely by a lack of heavy isotope labeled glycan standards.

Monoclonal Antibody N-glycan Characterization

Human Serum IgG High-Throughput N-glycan Absolute Quantification

Glycobiology

Several applications in the field of glycobiology research and proteomics:

• Rapid Absolute glycan quantification
• Increase reproducibility in lab-to-lab method transfer (internal calibration standard)
• De-convolute and quantify co-eluting peaks in LC-MS
• Quantify glycan recovery after sample preparation
• Protein characterization: Quick determination of the glycosylation profile of a expressed recombinant protein depending on the heterologous host. Correlation of the differences in glycosylation and protein function