Abstract
Glycomics has a growing interest in the biopharmaceutical industry and biomedical research requiring new high-performance and high-sensitivity bioanalytical tools. Analysis of N-glycosylation is very important during the development of protein therapeutics and it also plays a key role in biomarker discovery. The most frequently used glycoanalytical methods are capillary electrophoresis, liquid chromatography, and mass spectrometry. In this chapter, the capillary electrophoresis-based N-linked carbohydrate analysis methods are conferred with emphasis on its use in the biopharmaceutical and biomedical fields.
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1 Introduction
Glycosylation of proteins is one of the most important post-translation modifications, which impacts their function and their lifespan, also taking part in important biochemical and physiological processes as well as in cell-cell interactions. The buildup of the glycan structure may therefore modify the cell functions and can serve as indicators of various diseases. In the course of N-glycosylation, the carbohydrate structures bind to the polypeptide chains of the proteins being synthesized (co-translational) and then modified post-translationally [1, 2]. The glycan structures are usually made up of several glycoforms, which significantly increase the structural diversity of glycans. This so-called microheterogeneity depends on the expression, concentration, and kinetic features of glycosyltransferases and glycosidases. The glycoforms might have various binding sites within a protein, which is referred to as glycosylation macroheterogeneity (site specificity) [3,4,5,6].
The structural diversity of recombinant glycoproteins is very important for the pharmaceutical industry to avoid unwanted side effects and allergic reactions. During the development of these new-generation medicines, the producing microorganism must be chosen carefully to avoid immunogenic effects caused by nonhuman glycan epitopes. The most immunogenic nonhuman sugar residues are alpha-1,3-galactosylation and N-glycolylneuraminic acid (Neu5Gc) [7,8,9].
2 The Biochemical Background of Glycosylation
Asparagine (N)-linked glycans have three main structural subtypes. If it contains only mannose in addition to the core structure (Fig. 1, left panel), it is called “high mannose” type (Fig. 1, right panel). Hybrid glycans consist of both mannose and other sugar units in addition to the core. Complex-type glycans have other (non-mannose) sugar units added to the core structure (Fig. 1, right panel) [3, 10].
Another major type of protein glycosylation (not discussed in this chapter) is O-glycosylation via Thr or Ser residues. O-Glycans are synthetized in the Golgi apparatus and they predominantly appear on the surface of cells synthetizing mucins and on epithelial cell surfaces rich in serine and threonine [2, 11,12,13,14,15].
3 Glycobiomarker Discovery
Modifications in N-glycan structures significantly influence the half-life of proteins, their maturity state, cellular adhesion characteristics, migration, tumor invasion, and the formation of metastases. Various serological assays are available to identify organ-specific tumor glycobiomarkers, and they provide information on the prognosis of the disease [8, 16,17,18]. Biomarker assays recognize the glycan structures on the surface of the cells, for example, carbohydrate antigen (CA) 19-9 (pancreas/colorectal/gastro-carbohydrate antigen), CA 72-4 (colorectal/gastric), CA 125 (ovary), CA 15-3 (breast), AFP-L3 (hepatic cells), and PSA (prostate-specific antigen). Carcinoembryonic antigen (CEA) is a general and diagnostically widespread tumor marker, rich in N-glycan structures. The serum level of these biomarkers can be specific for determination of tumor genesis; therefore, the mapping of new, more specific glycobiomarkers is desirable in the future (Fig. 2) [16,17,18,19,20,21,22,23,24,25].
4 Glycosylated Biopharmaceuticals
The asparagine-linked carbohydrate moieties also have high significance in the pharmaceutical industry, because most of the new-generation biotherapeutics are glycosylated protein-based medicines, manufactured by recombinant techniques. In addition to monoclonal antibody (mAb)-type drugs, a number of hormones, coagulation factors, and lecithin-type compounds are continuously entering the market these days. Modifications of their linked glycan structures highly influence their efficiency, stability, safety, and half-life (Fig. 3) [6, 7, 26,27,28,29].
The glycosylation decoration of biological medicines greatly depends on the type of the producing microorganism/mammalian cell lines and the production environment. Nonhuman cell lines and microorganisms may synthesize immunogenic glycan residues such as N-glycolylneuraminic acid (Neu5Gc) (CHO cell line), alpha1,3-galactose epitope (BHK, NS0 cells), core alpha1,3-fucose (insects), beta1,2-xylose/core and alpha1,3-fucose (plants), and hyper-mannosylation (yeast) [3, 7, 10, 30]. The abovementioned sugar monomers are immunogenic, so minimizing their presence by better optimization of the production conditions (glycoengineering) is very important. The decrease and avoidance of extreme microheterogeneities in production cells facilitated more reproducible manufacturing of both innovative and biosimilar medicines [3, 5, 7].
5 Glycan Analysis Options
Analysis of the asparagine-linked carbohydrate moieties of glycoproteins is very important for the pharmaceutical industry and in the area of biomarker research requiring high sensitivity and high-resolution separation and detection methods. These bioanalytical techniques should provide detailed N-glycan profile information, including data about linkages and positional isomers. High-sensitivity glycoanalytical tools are readily available today on a wide scale to map protein glycosylations, help to identify smaller structural dissimilarities in the carbohydrate structures, and discover new glycan epitopes [6, 31,32,33]. The most frequently utilized methods are capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography (HILIC), affinity chromatography, mass spectrometry (MS), and their combinations. Most of these methods require removal of the N-linked oligosaccharides from the glycoproteins by endoglycosidase enzymes, such as peptide-N-glycosidase F (PNGase F), followed by labeling, e.g., with fluorescent dyes. Glycans can later be digested with exoglycosidase enzymes for sequence (residue, linkage, and positional) analysis (Fig. 4) [33,34,35,36].
HPLC and UPLC are widely used methods in the profile analysis of released glycans. Combined with mass spectrometry, it provides some structural information about the glycans [37, 38]. An excellent method for the N-glycosylation analysis of intact glycoproteins is MS combined with RP-HPLC, providing detailed information about the heterogeneity of carbohydrate binding sites, most frequently combined with ESI-TOF analyzer. MALDI is another efficient way for glycan analysis, but its accuracy is not as good as with HPLC. However, it is but appropriate for O-glycan analysis/characterization [39,40,41,42,43]. The advent of HILIC chromatography in carbohydrate analysis was an important step forward, as this method is capable of efficiently separating N-glycans [34, 44].
Capillary electrophoresis (CE) has proven to be one of the most excellent separation methods for the analysis of complex N-glycan structures (Fig. 5). Coupled with laser-induced fluorescence detection (LIF), it is possible to reach very high sensitivity and resolution. In the case of carbohydrate sequencing, serial exoglycosidase digestion is required, similar to that of LC. The fluorescent labeling of glycans for CE-LIF is performed using APTS (8-aminopyrene-1,3,6-trisulfonate). Various subsets of CE such as micellar electrokinetic chromatography or isoelectric focusing increase the efficiency and resolution of the separation at the glycopeptide and/or glycoprotein level with lower sample requirement than that of MS or HPLC [45,46,47,48].
These commonly used analytical methods can give valuable structural information about the main carbohydrate groups on glycopeptides, intact proteins in addition to released glycans and monosaccharides. Intact protein glycan mapping is possible with lectin microarrays and CE or MS [43, 46, 49, 50]. MS coupled with ESI or MALDI can determine various glycoforms. CE with MS can reveal sialylation heterogeneity of intact proteins and can give detailed site identification of glycopeptides as well [41, 51, 52]. Lectin microarrays can detect glycan-lectin interactions of intact proteins and give useful information about glycoconjugates, but cannot provide structural information [53]. MALDI-MS offers detailed structural information about intact glycoproteins including linkage and branches and can provide information about glycosylation site specificity, but with lower mass accuracy [54]. Charge-based electrophoresis such as capillary isoelectrofocusing (cIEF) is useful for quality control testing for sialylated species [55]. HPLC with ESI-MS or with MALDI-MS is widely used to gain information about protein glycosylation sites and occupancy also suitable for rapid glycopeptide profiling [56, 57]. RPLC-MS could provide complete sequence analysis of glycoproteins but needs higher sample amounts. On the other hand, CE in combination with MS offers high-resolution analysis of glycoproteins providing useful information about the entire glycan structures while only needing very low sample amounts [58, 59].
6 Summary
The growing interest in glycomics in the pharmaceutical industry and in biomedical research demanded the development of high-resolution and high-sensitivity glycoanalytical techniques. N-Glycosylation analysis is very important for the development of monoclonal antibody-based and new modality medicines, showing promising results in the field of biomarker research. Due to the complex structures of N-linked carbohydrates, their analysis is challenging that requires new-generation, high-resolution/high-sensitivity methods such as CE-LIF and various liquid chromatography-based methods preferably connected to mass spectrometry. Indeed, the additional mass spectrometry data provides deeper structural information, but mostly requires coupling to liquid phase separation methods as described in Fig. 6.
Abbreviations
- AFP:
-
alpha-fetoprotein
- APTS:
-
8-aminopyrene-1,3,6-trisulfonate
- BHK:
-
baby hamster kidney
- CA:
-
carbohydrate antigen
- CEA:
-
carcinoembryonic antigen
- CE-LIF:
-
capillary electrophoresis with laser-induced fluorescence detection
- CHO:
-
Chinese hamster ovary
- HILIC:
-
hydrophilic interaction liquid chromatography
- HPLC:
-
high-performance liquid chromatography
- mAB:
-
monoclonal antibody
- MS:
-
mass spectrometry
- NS0:
-
murine myeloma cell line
- PNGase F:
-
peptide-N-glycosidase F
- PSA:
-
prostate-specific antigen
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Acknowledgments
The authors gratefully acknowledge the grants from the National Research, Development and Innovation Office (K116263, NN127062, 2018-2.1.17-TÉT-KR-2018-00010). This work was also sponsored by the BIONANO_GINOP-2.3.2-15-2016-00017 project of the Hungarian Government. This is contribution #161 from the Horváth Csaba Memorial Laboratory of Bioseparation Sciences.
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Kun, R., Jóna, E., Guttman, A. (2021). Capillary Electrophoresis-Based N-Glycosylation Analysis in the Biomedical and Biopharmaceutical Fields. In: Colnaghi Simionato, A.V. (eds) Separation Techniques Applied to Omics Sciences. Advances in Experimental Medicine and Biology(), vol 1336. Springer, Cham. https://doi.org/10.1007/978-3-030-77252-9_7
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