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Nihms 1596902
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Expert Rev Proteomics. Author manuscript; available in PMC 2021 May 28.
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Abstract
Introduction—Protein glycosylation influences characteristics such as folding, stability, protein
interactions, and solubility. Therefore, glycan moieties of therapeutic proteins and proteins that are
likely associated with disease pathogenesis should be analyzed in-depth, including glycan
heterogeneity and modification sites. Recent advances in analytical methods and instrumentation
have enabled comprehensive characterization of highly complex glycosylated proteins.
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Correspondence: Jan Novak, Department of Microbiology, University of Alabama at Birmingham, 845 19th Street S, Birmingham,
AL 35294, USA, jannovak@uab.edu, Tel: +1(205)934-4480; Kazuo Takahashi, Department of Biomedical Molecular Sciences, Fujita
Health University School of Medicine. 1-98 Dengakugakubo, Kutsukake, Toyoake, Aichi 470-1192, Japan, kazuot@fujita-hu.ac.jp,
Tel: +81(562)93-2430, Fax: +81(562)93-1830.
Disclosure of interests
The authors have no relevant affiliation or financial involvement with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript. In the interest of full disclosure, we report that M. B.
Renfrow and J. Novak are co-founders of Reliant Glycosciences, LLC and co‑inventors on the US patent application 14/318,082
(assigned to UAB Research Foundation that distributes royalties to the inventors).
Reviewer disclosures
APeer reviewers on this manuscript have no relevant financial or other relationships to disclose.
Ohyama et al. Page 2
Keywords
N-glycosylation; O-glycosylation; Immunoglobulin glycosylation; Mucin 1 (MUC-1); virus
glycoconjugates; intravenous immunoglobulin (IVIG); Fc fusions protein therapeutics;
erythropoietin
1. Introduction
Glycosylation is the most common post-translational modification (PTM) of proteins. The
two major types of glycosylation, N-linked and O-linked, impact many functions, including
protein folding and stability, protein interactions, and protein solubility [1]. Notably, changes
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in the N- and/or O-glycosylation patterns of various proteins have been reported in several
diseases, such as cancer, infection, autoimmune diseases, diabetes, and chronic
inflammatory diseases [1–3]. Glycosylation also influences therapeutic efficacies of protein
drugs through changes of activity, pharmacokinetics, clearance, and immunogenicity [4]. To
date, more than 100 proteins have been approved as therapeutics, and most of them are N-
and/or O-glycosylated [5]. The glycosylation of biological products in the list of the US
Food and Drug Administration’s Center for Drug Evaluation and Research are summarized
in Table 1. With so many glycoproteins used as drugs and many more at different stages of
development, it is critical to develop robust quantitative methods for in-depth analysis of
protein glycosylation in terms of glycan structure and attachment sites. The same is true for
pathogenic proteins to better understand their (patho)biological roles. While mass
spectrometry (MS) approaches for quantitative proteomics have greatly advanced, the
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analytical workflow for highly glycosylated proteins still needs to be optimized for the
evaluation of glycan structure and their site-localization. The aim of this review is to
summarize recent technical developments and advancements in MS analysis toward
understanding of the biological roles for therapeutic or pathogenic glycosylated proteins. We
review general methodological advances for the analysis of glycosylated proteins and also
provide specific examples of analytical methods, especially those using MS, that have
enabled detailed analysis of highly complex glycosylation of therapeutic and pathogenic
glycoproteins. These efforts will lead to a better understanding of the biological roles and
specific function of glycoproteins.
a glycan precursor to the nitrogen atom of an Asn side chain by a β−1 N-linkage. There is a
common core for all eukaryotic N-glycans that includes three mannose (Man) and two
GlcNAc residues attached to Asn, Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ1-
Asn. Based on additional sugar residues that extend from the core glycan, N-glycans are
classified into three types: oligomannose, hybrid, and complex glycans.
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action of GnT-II to yield the precursor for all biantennary complex N-glycans;
GlcNAc2Man3GlcNAc2. Additional branches can be added at C-4 of the core α1–3 Man and
at C-6 of the core α1–6 Man by GnT-IV (MGAT4A, MGAT4B) and GnT-V (MGAT5,
MGAT5B). The “bisecting” GlcNAc residue can be attached to the β-Man of the core by
GnT-III (MGAT3). Hybrid N-glycans are formed if the GlcNAcMan5GlcNAc2 glycan
produced by GnT-I is not digested by α-Man II (MAN2A1,MAN2A2) [6]. Further
modifications are performed by α1–6-fucosyltransferase (FUT8), β1,4-
galactosyltransferases (Gal-T), α2,3-sialyltransferase (α2,3 Sialyl-T) and α2,6-
sialyltransferase (α2,6 Sialyl-T), which is distributed in the medial to trans face of Golgi
apparatus [3].
1.2 O-glycosylation
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O-glycosylation most often occurs on Ser and Thr residues, which are hydroxyl functional
amino acids. The most common sugars attached to Ser/Thr are GlcNAc and N-
acetylgalactosamine (GalNAc) in humans. Attachment of O-GlcNAc occurs in proteins in
the nucleus, mitochondria, and cytoplasm; these proteins play important roles in regulating
cellular processes, such as epigenetics, gene expression, translation, protein degradation,
signal transduction, mitochondrial bioenergetics, the cell cycle, and protein localization [7].
O-GlcNAc is dynamically added and removed from proteins by O-GlcNAc transferase and
O-GlcNAcase, respectively. O-GlcNAc modification is reported to act in a reciprocal
manner to O-phosphate modification, and their respective addition and removal can affect
protein structure, activity, and function [8].
Protein modifications with O-GalNAc are often represented by mucins as the prototype.
Mucins are the class of glycoproteins carrying the greatest number of O-GalNAc glycans
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(mucin-type O-glycans). O-GalNAc glycan is usually extended to form one of four common
structures (core 1 to 4). In tumor-associated mucin glycoforms, shorter glycans are enriched,
such as a single unextended GalNAc linked to Ser/Thr (the Tn antigen), T antigen, and
sialyl-Tn antigen. These glycoforms are thought to be involved in tumorigenesis and/or
progression [9] (Figure 1b).
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for analysis by intact methods. Furthermore, native MS can be applied for the
characterization of plasma glycoprotein microheterogeneity toward understanding of
protein-drug interactions, pharmacokinetics of proteins, and glycosylation status changes of
proteins in various diseases[12–14].
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enriched prior to MS analysis, to addres the issues related to their ionization efficiency that
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is lower than that of other peptides. Lectin affinity chromatography is a commonly used tool
to enrich for glycopeptides [15, 16]. Typically, broadly selective lectins such as Con A are
used [15]. Alternatively, sequential or multi-lectin columns are used for the selective
enrichment of glycopeptides [17]. Hydrophilic interaction chromatography (HILIC) is also
available for glycopeptide enrichment. HILIC resins, such as cellulose, Sepharose CL4B,
silica, and zwitterionic types have been used [18]. Recently, HILIC/ reversed-phase (RP)
stop-and-go-extraction tip (Stage Tip) has been developed for enrichment in small sample
amounts [19]. The MS analysis of enriched glycopeptides is commonly carried out with
matrix assisted laser dissociation (MALDI) MS or liquid chromatography (LC)-tandem MS
(MS/MS) [20, 21]. In particular, the development of high-resolution hybrid mass
spectrometers has improved glycopeptide characterization [22] due to their ability to
unambiguously distinguish between isobaric glycopeptide mixtures via accurate mass. Also,
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the use of several fragmentation methods that enable tandem MS sequencing (MS/MS) to
provide glycan structural information as well as site specific information about glycosylation
heterogeneity. These fragmentation techniques include as higher-energy collision
dissociation (HCD) / collision-induced dissociation (CID), and electron transfer dissociation
(ETD) [23, 24]. Recently, the combination of these fragmentation methods, e.g., ETD/HCD
(EThcD) has been used for their superior fragmentation efficacy [25].
The last strategy is based on the analysis of released N- or O-glycans from glycoproteins
(Figure 2). It is currently the most recognized approach for quantitative analysis of the
complete, detailed glycan structure, including monosaccharide composition, sequence,
branching, and linkages. However, in the case of glycoproteins with multiple sites of
glycosylation, the context of site-specific heterogeneity is lost. So often these last two
strategies are combined for complete glycoprotein analysis. For released glycan analysis,
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glycans are typically released through enzymatic or chemical methods and analyzed either
by high performance LC (HPLC) and exoglycosidase digestion, or by MS using various
MS/MS techniques [5] and more recently ion mobility technology [26]. For N-glycan
analysis, first, peptide N-glycosidase F (PNGase-F) digestion is frequently conducted for
their release. PNGase-F releases all asparagine-linked oligosaccharides, but it is not effective
if they are α1–3-fucosylated, which is a common core-modification in plants [27]. In
contrast, for the release of O-glycans, a general O-endoglycosidase is not available. The
available endoglycosidase, O-glycanase, only cleaves the core 1 type O-glycan, without any
other modification. Thus, releasing of O-glycans is widely conducted by chemical methods
known as reductive β-elimination. β-elimination cleaves the glycosidic bond using a strong
alkaline reaction, followed by reduction of the reducing terminus with sodium borohydride
(NaBH4). An alternative method is oxidative release of O- and N-glycans [28]. Finally, the
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released glycans with a reducing terminal are analyzed, most of which are derived by
permethylation or fluorescent derivatization prior to analysis. Permethylation of released
glycans has become more standardized in recent years. Permethylation neutralizes the
negative charge of sialylated glycans and improves glycan tandem mass spectra with high
structural information [29–31]. In contrast, the labeling of released glycans with fluorescent
reagents is most useful for their specific and highly sensitive detection. 2-aminopridine (PA)
and 2-aminobenzamide (2-AB) are commonly used fluorescent dyes for HPLC approaches
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[32, 33]. Recently, procainamide was shown to enhance electrospray ionization (ESI)
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efficiency and fluorescence intensity [34]. Because 2-aminobenzoic acid (2-AA) carries one
negative charge, it is suitable for capillary electrophoresis (CE) and MS analysis. 8-
aminopyrene-1,3,6-trisulfonic acid (APTS), or 8-amino-nephtalene-1,3,6-trisulfonate
(ANTS) are also used for CE methods [35, 36]. Additionally, underivatized glycan alditols
after β-elimination treatment are often analyzed by LC-MS on a porous graphite carbon
column [37, 38]. As an alternative approach, the ion mobility technique has gained attention
for its ability to distinguish between different isobaric glycans. This technology enables gas-
phase separation of ions with identical m/z values but different compositional structure,
linkage, or branching [39, 40]. Further improvements are needed in the future for facilitating
glycosylation analysis and quantitative data output.
The final step of protein glycosylation analysis involves MS-output data processing. Due to
the complexity and time needed for MS-output data interpretation, specialized bioinformatic
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tolls have been developed. The acquired MS data need to be compared with the knowledge
databases for the assignment of glycan/glycopeptide structures. Several automated programs
were developed for analysis of released glycan, such as GlycoWorkbench, SysBioWare,
MultiGlycan, Glycan Builder, and GRITS Toolbox [41, 42]. For in-depth structural analysis
including sites of glycan attachment, several groups have developed algorithms that have
been summarized by Cao WQ et al [43]. For the glycopeptide analysis, commercially
available software solutions, such as Byonic and SimGlycan are powerful tools for the rapid
detailed analysis of complex N-and O-linked glycan structures from the LC-MS/MS data of
glycopeptides [42]. As manual data validation is still needed due to high false-discovery
rates of automated tools, several types of bioinformatics softwares have newly been
developed, as summarized by Yu A et al [44]. However, each software should be validated in
the specific samples and most of software can be applied to the analysis of either of N-
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glycoform or O-glycoform.
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the Fc region include Fc receptors (FcγRI, FcγRII, FcγRIII), the C1q component of
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complement, and the neonatal Fc receptor. Other receptors may also bind IgG, either free, in
an immune complex, or in aggregates. N-linked glycosylation of Asn297 of the CH2 domain
in the Fc region impacts binding to and activation of Fcγ receptors and of the C1q
component of complement and IgG retention in the circulation [51]. N-linked glycosylation
of the Fab region is relevant to the affinity and avidity of antibodies for antigens [52, 53] and
also to antibody half-life [54].
The oligosaccharides present in the IgG Fc region are of the complex biantennary type and
are comprised of a core heptasaccharide, i.e., GlcNAc2Man3-GlcNAc2, forming a
biantennary N-glycan with variable content of fucose, bisecting GlcNAc, Gal, and sialic
acid. Due to the variable modifications of the core heptasaccharide, at least 36 different
structurally unique oligosaccharide chains may be found at Asn297 [55]. Furthermore, due to
the asymmetric glycosylation to each heavy chain, there can be more than 400 IgG
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glycoforms [56]. In healthy subjects, more than 90% of serum IgG contains core fucose and
50 to 70% are galactosylated. Furthermore, 10 to 20% of IgG is sialylated and a small
proportion of IgG glycans (about 10–15%) contains a bisecting GlcNAc [49, 57, 58]. The
composition of glycans tends to change with age [59–62], sex [61], and pregnancy [27, 63].
Alteration of IgG glycoforms have also been reported in diseases. Rheumatoid arthritis is
among the diseases in which reduced galactosylation and sialylation of IgG has been
observed [64–66]. Similar detects of IgG galactosylation have also been found in patients
with systemic lupus erythematosus, Crohn’s disease, and other chronic and inflammatory
diseases [3, 56, 67–70]. To assess IgG glycoforms, various analytical methods have been
developed. The standard N-glycan analysis begins with IgG purification followed by
analysis of released N-glycans and analysis of N-glycopeptides.
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With regard to purification of IgG, the most popular technique is affinity-purification using
immobilized Protein A [71, 72], Protein G [73], and anti-human IgG Fcγ antibodies [54].
For isolation of antigen-specific antibodies, affinity chromatography can be used with the
specific antigen immobilized on a column [74]. IgG separation using SDS-PAGE, followed
by in-gel digestion and N-glycan release has also been reported [75–77]. To analyze N-
glycans in the Fab and Fc portions individually, several approaches have been reported to
separate the two regions of IgG. Pepsin digests on the C-terminal side of disulfide bonding
in IgG-heavy chains [78] (Figure 3a). By dialysis using a size-selective membrane, F(ab’)2
fragments and Fc fragments are separated in the retained and flow-through fluids,
respectively [73, 78]. Furthermore, papain which digests on the N-terminal side of disulfide
bonding in IgG-heavy chains has been used to cleave IgG to Fc and Fab moieties [54, 71,
79] (Figure 3a). After digestion, Protein A columns binds the Fc fragments while Fab
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fragments are in flow-through [80]. Bondt et al. introduced a high-throughput workflow for
separation of the Fc and F(ab’)2 regions of IgG [49] (Figure 3a). Briefly, IgG is captured
from serum or plasma using anti-IgG-Fc coupled beads, followed by digestion using IdeS or
IdeZ proteases, which are cysteine proteases from Streptococcus pyogenes or Streptococcus
equi subspecies zooepidemicus that digest antibodies at a specific site between Gly-Gly of
the IgG hinge region. After digestion by IdeS protease, the F(ab’)2 fragment is collected in
the flow-through and the Fc portion in the eluant. To analyze N-glycopeptides, tryptic
digests are applied to LC-MS/MS [49]. To analyze N-glycans released by PNGase-F, N-
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glycans can be enriched by various solid-phase extraction (SPE) methods, such as HILIC
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[49, 81], RP, and porous graphitic carbon (PGC) chromatography [82]. Permethylation of
sialylated glycans can be applied before MALDI to avoid ionization biases and desialylation
during ionization [83]. However, permethylation or derivatization of carboxyl groups,
including methyl amidation [84] and methyl esterification [85], which enable differentiation
between α2,6- and α2,3-sialylated glycans, require long derivatization in harsh conditions
for a purified glycan sample. Recently, a rapid derivatization protocol with 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole
monohydrate (HOBt) in ethanol has been developed [86]. This ethyl esterification reaction
can be applied to a complex sample mixture before glycan purification, which enables high-
throughput analysis. Furthermore, ethyl esterification of sialic acids enables mass-based
differentiation between α2,6- and α2,3-sialylated glycans [87]. Ethyl esterification is a
technique for the chemical modification of sialylated glycans for enhancing stability and
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[95]. The elevated serum levels of Gd-IgA1 have been confirmed by lectin-based enzyme-
linked immunosorbent assay (ELISA) using Helix aspersa agglutinin, Vicia villosa, Helix
pomatia, and Caragana arborescens which bind terminal, i.e., Gal-deficient, GalNAc [96–
98]. Recently, mAbs specific for IgA1 with Gd O-glycans of IgA1 (Gd-IgA1) were
developed, and their high reactivity with serum IgA1 from IgAN patients was demonstrated
[99, 100]. However, lectin and antibody-based analysis are indirect approaches, and the
strategy for identifying what kind of glycoform increases in whole IgA1 can be
demonstrated only by detailed glycoform analysis using MS-based analytical strategies.
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glycopeptides was used [102, 103] (Figure 4a). In this study, glycopeptide enrichment by
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Although the IgA1 HR O-glycoforms vary with respect to the number of monosaccharides
attached, such as GalNAc, Gal, and sialic acid, the population of glycoforms can be
determined by using trypsin-digested glycopeptide analysis with MALDI MS or LC-MS
[95, 105–107]. Quantitative analysis of O-glycan microheterogeneity and attachment sites
should be obtained for comprehensive understanding of the pathogenic forms of IgA in
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IgAN. IgA1 HR, rich in Ser, Thr, and Pro, contains clustered O-glycans and shows high
resistance to proteases, including trypsin. Tryptic digests of IgA1 HR generate a 38-mer
peptide with 9 potential sites for O-glycosylation. Thus, comprehensive analysis of IgA1 HR
O-glycoforms is challenging due to large molecular mass and multiple glycosylation sites.
To address the problem, HRMS was applied to characterize O-glycoform
microheterogeneity [108, 109]. Determination of sites with attached aberrant O-glycans was
achieved by activated ion electron capture dissociation (AI-ECD) FTICR MS/MS [108]. The
AI-ECD FTICR MS/MS protocol was subsequently demonstrated using three distinct IgA-
specific proteases (Clostridium ramosum strain AK183, Streptococcus pneumoniae strain
TIGR4, Haemophilus influenzae strain HK50) to reduce the molecular masses of precursor
ions to obtain sufficient fragmentation of c or z ions to assign all O-glycosylation sites [110]
(Figure 3b, 4). An extracted ion chromatogram (XIC) of some IgA1 O-glycoforms showed
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multi-modal peaks, indicating structural isomers based on glycan attachment sites [111].
Combined quantification of IgA1 HR O-glycoforms and localization of O-glycan attachment
sites, including structural isomers, quantifies the sites of O-glycan heterogeneity (Figure 4b)
[111, 112]. To increase the throughput toward analysis of clinical samples from IgAN
patients, an automated program for quantitative analysis of the HR O-glycopeptide profiles
was developed. Furthermore, sites with Gd O-glycans were unambiguously identified by
electron transfer/higher-energy collision dissociation EThcD MS/MS following a removal of
galactosylated O-glycans [113]. This novel protocol enabled quantitative assignment of Gd
sites. The most frequent Gd site was T236, followed by S230, T233, T228, and S232. Future
improvement of these analytical methods will enable the analysis of clinical samples and the
understanding of pathogenic IgA1 HR O-glycoforms in IgAN.
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epithelial cells is generally glycosylated with larger branched core 2 O-glycans often
extended by polylactosamine and, to a lesser extent, capped core 1 O-glycans, whereas the
truncated core 1 structures and terminal or sialylated GalNAc are absent. In contrast, breast-
cancer cell lines produce MUC-1 with altered glycosylation, generally characterized by
high-density glycosylation and reduction in the O-glycan chain lengths [114]. MUC-1 is
overexpressed and aberrantly glycosylated in many types of adenocarcinomas [115–117].
The aberrant MUC-1 glycoforms can apparently induce immune responses, as many patients
with adenocarcinoma have antibodies specific for these cancer-associated MUC-1
glycoforms (for review see [118, 119]). Some observations indicated that such antibodies
may be protective [120, 121], making MUC-1 a possible therapeutic target for
immunotherapy [119, 122–126]. Specifically, MUC-1 (glyco)peptides or MUC-1 variants
with multiple tandem repeats have been tested in clinical trials for the induction of immune
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3.4. Virus glycoconjugates for vaccine development targeting HIV-1 and influenza
infections
Many enveloped viruses, including HIV-1 and influenza virus, encode envelope
glycoproteins. Some of these glycoproteins have important functions in virus biology.
Moreover, some of the glycosylated motifs are recognized by the host’s immune system and,
thus, can trigger a response against the glycosylated compound(s) of the virus(es). These
glycoproteins are of great interest to vaccinologists who have been working on the
development of vaccines against HIV-1 and influenza.
Recent progress in HIV-1 vaccine development has been driven by better understanding of
the HIV-1 envelope (Env) glycoprotein and the impact its glycans play in immune responses
and, conversely, virus immune escape and protection from immune recognition. Viral
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proteins are dependent upon the host cellular machinery for protein synthesis and any
subsequent post-translational modifications, including glycosylation. The HIV-1 Env
glycoprotein, a trimer consisting of gp120 and gp41 non-covalently associated subunits, has
multiple sites of N-glycosylation. The densely glycosylated surface of the HIV-1 Env
glycoprotein, often referred to as a “glycan shield”, is host-dependent and yet successful in
evading immune responses. Longitudinal sampling of HIV-1-infected individuals revealed
changes in the locations of clustered N-glycans due to the high mutation rate of HIV-1,
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creating an ever-shifting surface topology of this glycan shield. In spite of this evasion
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mechanism, some individuals with chronic HIV-1 infection develop broadly neutralizing
antibodies that recognize multiple HIV strains. Several of the epitopes recognized by the
broadly neutralizing antibodies have been mapped to the HIV-1 Env glycoprotein segments
with highly conserved sites of N-glycosylation. These glycoprotein epitopes are the focus of
several vaccine development projects for HIV-1 (for review see [134–139]).
The influenza virus also uses protein glycosylation as a key component of its host-cell entry
and immune evasion. Specifically, the surface proteins of the viral particles, hemagglutinin
and neuraminidase, play vital roles in infection. The number of N-glycans in the
hemagglutinin globular domain changes as it circulates in the human population. Although
the N-glycans on hemagglutinin are not as densely clustered as those found on HIV-1 Env,
they play a role in immune evasion by masking antigenic sites from antibodies (for review
see [140]). However, soluble C-type lectins, such as the mannose-binding lectin and
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surfactant protein, are known to recognize influenza hemagglutinin glycans and promote
innate immune responses by macrophages and other immune mechanisms [141, 142]. Viral
hemagglutinin protein binds sialic acid groups of cellular surface proteins to achieve viral
attachment and entry. Human influenza viruses bind preferentially to α2,6-linked sialic acid,
whereas avian influenza viruses use α2,3-linked sialic acid. Amino acids at key positions in
the hemagglutinin receptor that affect binding specificity have been identified in the seasonal
human and avian viruses [143]. The influenza neuraminidase is vital for viral entry into the
respiratory tract to bypass the sialic acid-rich mucus proteins. Neuraminidase is also
required for release of newly formed viral particles from the cell surface of host cells [144].
Thus, drugs that are structural mimics of sialic acid can inhibit the activity of influenza
neuraminidase and represent a viable treatment option (for review see [145]).
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can elicit broadly protective humoral responses against HIV-1 as well as influenza and other
enveloped viruses [165–172].
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and porcine pancreas [173]. Currently, most therapeutic glycoproteins are produced as
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IgG.
4.1. N-Glycosylation patterns of monoclonal antibodies and the approach for glycan
analysis
Among protein therapeutics approved by FDA, there are many mAbs (Table 1). It is
estimated that 30% of newly licensed drugs in the next decade will be based on antibodies
and their derivatives [174]. Most therapeutic mAbs are of the IgG class and contain a
glycosylation site in the Fc region of each heavy chain, such as the human IgG [175, 176].
There are also glycosylation sites in the Fab region in rare cases such as cetuximab at Asn88
of the VH region [175–179].
The glycosylation patterns of mAbs mostly depend on producing cell lines, although other
parameters of the fermentation process (e.g., glucose content, dissolved oxygen, bioreactor
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pH, sodium butyrate, ammonia content, CO2 concentration, temperature, and production
scale) can also impact the final glycan patterns to some extent [180]. From the viewpoint of
immunogenicity, the type of cell line used to produce a mAb is important. Most approved
mAbs are produced using Chinese hamster ovary (CHO) cells because of their ability to
produce human-like glycosylation. Some are produced in murine myeloma cell lines (e.g.,
NS0 or SP2/0). Products produced using murine cell-lines contain non-human glycan
moieties, such as N-glycolylneuraminic acid and α-Gal, which are immunogenic in humans.
Indeed, cetuximab, which is produced in the SP2/0 cell-line, has been reported to induce
anaphylaxis in some of the recipients [181].
terminal Gal, can bind to asialoglycoprotein receptor, whereas Fc high-mannose glycans can
bind to mannose-binding lectin. These interactions may impact half-life of these proteins in
the circulation [175, 176, 182, 183]. Glycosylation of the antibody in the Fc region can also
alter the effector functions of the antibody. For example, IgG glycoforms with glycans
decorated with terminal GlcNAc may activate the complement system though the lectin
pathway, IgG with Gal and sialic acid may bind C1q, whereas other types of glycosylation
may impact complement-dependent cellular cytotoxicity (CDC) [184]. Furthermore,
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(ADCC) via increased binding of the IgG Fc portion to FcγRIIIa on the natural killer cells
[185]. As IgG glycosylation of Fc and Fab is crucial for pharmacokinetics and antibody
effector functions, close monitoring of these characteristics during manufacturing is
required.
To fulfill this requirement, many different methods for glycosylation analysis have been
developed. Methods for glycosylation analysis of mAbs are classified into three main
strategies, as described in section 2 (Figure 2), 1) intact molecule, 2) glycopeptide, and 3)
released glycan. For a top-down or middle-down approach, an intact IgG molecule after
reduction of disulphide bonds (or after digestion with endoproteinase LysC, Pepsin, or IdeS
for middle-down) is directly analyzed by LC-HR MS [186, 187].
trypsin, LysC or pronase. In-depth analysis of IgG glycans in a site-specific manner can be
achieved [186, 188, 189]. To overcome ion suppression of glycopeptide signals by peptides,
enrichment of glycopeptides by HPLC or HILIC is required before MS assay. Reusch et al.
tested a high-throughput workflow for monitoring Fc-IgG glycosylation using LC-ESI-MS
analysis after IgG purification by Protein A in immobilized 96-well plates and extraction of
glycopeptides by HILIC beads [190].
IgG mediates pro- and anti-inflammatory activities through the engagement of its Fc
fragment with distinct FcγRs [194]. Human and mouse FcγRs consist of several activating
receptors and one inhibitory receptor, FcγRIIB. After IVIG therapy, FcγRIIB is upregulated
on myeloid cells and the number of FcγRIIB-expressing myeloid cells is increased [195].
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N-glycosylation of the IgG Fc region was found to affect IVIG anti-inflammatory function
via upregulating expression of FcγRIIB in the effector cells. About a decade ago, both
removal of the Asn297-linked sugar moiety and removal of only the terminal sialic acid
residues from IgG in IVIG preparations showed a loss of the anti-inflammatory activity of
IVIG [196]. Recently, dendritic cell-specific intercellular adhesion molecule-3-grabbing
non-integrin (DC-SIGN)-related protein 1 (SIGNR1) and its human counterpart DC-SIGN
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were identified as glycosylation-specific receptors for the IgG Fc region with terminal sialic
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Fokkink et al. investigated the variation in IgG Fc N-glycosylation between different batches
and brands of IVIG products using the LC-MS technique [198]. The rates of sialylation of
Fc N-glycans (range, min-max) were 13.5–18.4% in IgG1 and 16.7–21.5% in IgG2/3.
Insufficient sialylation of Fc N-glycans requires extremely high doses of IVIG to be
administered to patients (2g / kg body weight) for sufficient clinical effect. For reduction of
cost and adverse events caused by excessive IVIG administration, the development of an
IVIG biomimetic was expected.
More recently, hexametric human Fc-fusion protein (hexa-Fc), a biomimetic substitute for
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IVIG, was developed. Mekhaiel et al. succeeded in manufacturing polymerized human IgG1
based Fc-fusions. The 18-amino-acid tailpiece from human IgM together with a five-amino-
acid linker was added to the C-terminus of IgG Fc-fusions mutated with His310 to Leu to
achieve a polymeric form [199]. After forming multimers, the affinity to FcRs of hexa-Fc
was enhanced compared to that of IgG1-Fc monomers [199].
With regards to the interaction between hexa-Fc, the higher affinity of hexa-Fc to soluble
recombinant human DC-SIGN compared to that of IVIG suggested that alteration of N-
glycosylation affected the high affinity of hexa-Fc and DC-SIGN [200]. The difference in N-
glycosylation patterns between hexa-Fc and IVIG was investigated by released N-glycans
analysis using MALDI-TOF-MS. Hexa-Fc was found to be rich in high mannose glycans in
comparison with IVIG [200]. Although the affinity of hexa-Fc to DC-SIGN (KD of 1.26 μM)
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was lower compared to HIV gp120 (KD of 4.39 μM), a well-studied DC-SIGN ligand known
to carry high amounts of N-linked high mannose oligosaccharides, they demonstrated that
measured off-rate of hexa-Fc from DC-SIGN were relatively slow (Koff of 8.39 × 10−4s−1)
[200]. This suggests that once hexa-Fc is bound to DC-SIGN, the binding of the complex is
stable.
Etanercept (ETN) is a fusion protein consisting of two copies of the tumor necrosis factor-α
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receptor (TNFR) and the Fc portion of human IgG1. ETN is produced in CHO cells and
consequently bears high level of glycosylation. There are four N- and 26 O-glycosylation
sites in the dimeric TNFR domain, as well as two N-glycosylation sites in the IgG Fc portion
(Figure 5) [202]. Recently, Wohlschlager et al. reported a new approach for unraveling the
glycoform heterogeneity of ETN using high-resolution native MS combined with enzymatic
digestion [202]. Intact ETN was observed in a charge-state distribution from m/z 4800 to
6500 corresponding to charges of 25+ to 20+. By deconvoluting the raw mass spectra of
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Ohyama et al. Page 15
intact ETN in the most abundant charge state, they observed at least 70 distinguishable
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protein signals within mass range from 125 to 131 kDa. This deconvoluted spectra obtained
by native MS of intact proteins has the potential to be a fingerprinting tool for assessment of
batch-to-batch variability. By virtue of the large number of existing glycan combinations on
ETN, simplification of its structure by applying glycosidases and/or proteases was required.
To determine N-glycosylation of TNFR and Fc domains separately, ETN was digested by
IdeS at the hinge region (Figure 5 b-1 and b-2). The TNFR domain underwent further
digestion by sialidase and O-glycosidase to acquire simplified spectra. To pinpoint the exact
N-glycan structures on Asn149 and Asn171, which are the N-glycosylation sites of TNFR
domain, bottom-up analysis was performed using digestion with AspN followed by HPLC-
MS/MS analysis (Figure 5 b-3). By combining top-down and bottom-up analyses, MS data
showed that the TNFR domain most likely has the core N-glycan at Asn149 decorated with
Gal2(GlcNAc)2Man3(GlcNAc)2 (A2G2) and with Gal2(GlcNAc)2Man3(Fuc)(GlcNAc)2
(A2G2F) at the Asn171 glycan. With regard to determination of N-glycosylation of the Fc
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domain, the native MS result showed concordant results with bottom-up analysis of tryptic
digested glycopeptides and (GlcNAc)2Man3(Fuc)(GlcNAc)2 (A2G0F) / A2G0F and
A2G0F / (Gal)(GlcNAc)2Man3(Fuc)(GlcNAc)2 (A2G1F) were assigned as the most
prominent decorations of the glycan attached at Asn317 (Figure 5 b-3). Assessment of O-
glycoforms was performed via the native MS approach after PNGase-F digestion with or
without sialidase digestion. With both PNGase-F and sialidase digestion (Figure 5 a-2), 14–
23 core 1 glycan unites were assigned in the deconvoluted spectrum as well as 17–21 O-core
variants with different numbers of sialic acid residues that were detected in the deconvoluted
spectrum with only PNGase-F digestion (Figure 5 a-1). The typical ratio of N-
acetylneuraminic acid (NeuAc) content per O-glycan core was 1.2–1.3. Twelve attachment
sites were identified by Houel et al. using ETD (Figure 5 a-3)[203]. As seen from these
reports, native MS analysis has a rapid fingerprinting tool to provide valuable information on
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true glycoform heterogeneity at the intact protein level. However, specific deglycosylation
and digestion by IdeS are required to increase accuracy and to find out site specific
glycoform in such a highly complicated glycosylation analysis. Furthermore, integration of
middle-down and bottom-up proteomics data is necessary for in-depth characterization that
would include modified sites.
Asn83, and a single O-glycosylation site at Ser126 [205]. The main N-glycan structures of
rhEPOs are complex-type tetra-sialylated tetra-antennary glycans [206, 207]. The
glycosylation of EPO, especially sialic acid-containing carbohydrate content, influences its
biological activity and pharmacokinetics. Darbepoetin-α, which is a glycosylation analog of
rhEPO, best describes this phenomenon. With five amino-acid substitutions (Ala30, His32,
Pro87, Trp88, Pro90 to Asn30, Thr32, Val87, Asn88, Thr90), two additional N-glycosylation
motifs were added into the EPO protein [208]. By this modification, Darbepoetin-α attains
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Ohyama et al. Page 16
two additional N-glycans (five in total) with higher sialic acid content compared to rhEPO.
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Higher sialic acid content extends serum half-life as it reduces serum clearance, but also
imparts lower affinity for EPO receptors on the surface of red-blood cell precursors. Despite
the negative impact of higher sialic acid content on the biological activity, the extension of
half-life overcomes the reduced receptor-binding activity and enhances in vivo efficacy [208,
209].
Jiang et al. introduced the procedure of site-specific qualitative and quantitative analysis of
N-and O-glycoforms in rhEPO. As there is no trypsin cleavage site between Asn24 and
Asn38 and the resulting peptide 21EAENITTGCAEHCSLNENITVPDTK45 is too large for
MS analysis, endoproteinase Glu-C digestion was combined with trypsin digestion. As a
result, rhEPO was cleaved at Glu (E) / Lys (K) / Arg (R). After enrichment of the intact
glycopeptides by ZIC-HILIC material, enriched glycopeptides were analyzed by LC-ESI-
MS. Site assignments for glycans were mainly achieved by accurate mass detection of Y1
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5. Conclusion
Protein structure and biological function are altered by the addition or a change of
glycosylation, both in pathogenic and therapeutic proteins. Although new analytical methods
and techniques are regularly introduced in the field of glycoproteomics, comprehensive
analysis of glycoprofiling still remains a scientific challenge, as the methods for elucidating
the structure of composite glycans and modification sites are just beginning to be
standardized across the analytical community rather than being the analytical specialty of a
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relative few. This standardization is being driven by the biotechnology and pharmaceutical
companies with interest in having their therapeutic candidates approved by regulatory
agencies. Along with this standardization and push in methodology, the opportunity to
analyze native populations of glycoproteins isolated from disease states is increasing as well.
While general characterization of glycoproteins by releasing the glycans and analyzing
proteolytic digests is quickly becoming standard, the ability to assess glycoprotein
heterogeneity with intact or minimally cleaved glycoproteins has the potential to become the
method of choice in the future. Quantitative methods for assessing glycoprotein
heterogeneity is the area that still needs the greatest development. As these methods are
being improved and more widely disseminated, there will be a greater need for the
standardization of how glycoprotein analysis is to be reported and compared across
preparations and laboratories. Glycoprotein/glycopeptide standards will need to be
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developed as references. We expect that innovative analytical strategies will not only
discover glycoforms of improved protein therapeutics or the glycan pattern related to disease
pathogenesis, but will also lead to better understanding of the biological mechanisms and
functional significance of protein glycosylation. High-throughput analysis will be required
for the translation of glycoproteomic analyses to clinical research and application in
precision medicine.
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Ohyama et al. Page 17
6. Expert Opinion
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The expression patterns of some glycosyltransferases and glycosidases, the enzymes that
compose the glycosylation pathways, are often altered in pathological conditions, such as
cancer and autoimmune diseases [45–48]. Identification of the specific glycoforms in a
disease and clarifying their relationship with clinical prognosis could potentially lead to the
development of new biomarkers with diagnostic and prognostic significance. Furthermore,
detecting the antigenic glycoforms that cause disease informs development of novel
therapeutic drugs and vaccines, e.g., MUC1-peptide vaccines for cancer [127]. The
glycosylation patterns of therapeutic proteins are important for their pharmacokinetic
properties, biological functions, and safety. The glycoengineering of therapeutic proteins is
becoming important for improving their therapeutic effect. Furthermore, in the near future,
many biosimilars will be developed, and their structure (including primary structures, high-
order structures, and PTMs), function, and safety have to be monitored through the
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development process. The detailed analysis of glycosylation in the reference product and
comparison with glycosylation in newly developed biosimilar should be reported.
Furthermore, lot-to-lot variability assessment of products may also be required.
To reach these goals, high-throughput analyses with high resolution are required. For highly
accurate analysis, including quantitation of glycoform heterogeneity and localization of
glycan-modified sites, digestion protocols using various proteases and separation and
enrichment techniques should be considered to achieve sufficient intensity of glycopeptide
ions for quantification and/or fragmentation [49, 205]. Furthermore, a combination of
various glycosidases can be applied to simplify the glycan structures and make it easier to
analyze the glycoform and glycan attachment sites [110, 113]. Sample preparation is an
important factor for high throughput analysis. Better separation of target glycoproteins
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and/or efficient enrichment of glycopeptides improves throughput [49, 190]. The top-down
or middle-down approach is one of the most promising techniques for high-throughput
analysis, but further improvement is needed for sensitivity and detailed analysis of attached
glycans. The analytical software should be optimized for glycoproteins, which contain
multiple N- and/or O-glycosylation sites. Recent advances of MS-based proteomic
techniques provide the establishment of a workflow for unbiased quantitative analysis of
proteins in crude samples [210]. Furthermore, this analytical platform enables the discovery
and validation of biomarkers, simultaneously using shotgun proteomics in great depth
among different cohorts [211]. We also note that the quality of sample biobanking should
always be considered, as glycans on proteins may be affected by sampling and storage
conditions. Thus, a robust and high-throughput analytical platform should be applied to
clinical samples to define pathological forms of glycoproteins and develop and characterize
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new biomarkers to fully extend the impact of the emerging field of “glycomedicine” [1–3].
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Ohyama et al. Page 18
with genomic information has been applied to cancer research and has revealed new
molecular subtypes of cancers that were not previously identified by genome sequencing
[212, 213]. Thus, a multi-layer approach (genomics, proteomics, and metabolomics) using
clinical samples has become a powerful combination of tools to connect genomic analyses
with clinical phenotypes. Furthermore, a glycoproteogenomic approach indicates that
protein glycosylation patterns associated with genetic variants can classify individuals [214].
A recent study showed that serum levels of abnormally glycosylated IgA1 were associated
with regulation of expression of a specific glycosyltransferase [215, 216].
Glycoproteogenomic analyses using MS techniques offer a tremendous potential to elucidate
the pathogenesis of diseases related to aberrant protein glycosylation.
Acknowledgments
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Funding
The works described in this manuscript that were completed in our laboratories was supported by the JSPS
KAKENHI (grant numbers 16K09632, 19K08715, and 19K08691); by a Grant-in-Aid for Practical Research
Project for Renal Diseases, from the Japan Agency for Medical Research and Development; by Takeda Science
Foundation; by Aichi Jinzou Foundation; and in part by NIH grants DK078244, GM098539, DK105124,
AI149431, and DK082753.
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Article highlights
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Figure 1.
Biosynthesis of common N- and O-glycans.
a) N-glycan synthesis pathway. The mature glycan precursor attached to dolichol phosphate
(Dol-P) is transferred to the Asn residue of the Asn-X-Ser/Thr peptide sequence in the
endoplasmic reticulum (ER) by oligosaccharyltransferase (OST). After cleavage of three
glucose (Glc) residues and one mannose (Man) residue, properly folded glycoprotein with
oligomannose N-glycan (gray shadow with one asterisk*) is transferred to the Golgi
apparatus. In the Golgi apparatus, the structure of GlcNAcMan5GlcNAc2 is generated by
further mannose removal and addition of GlcNAc. By additional trimming by α-
mannosidase Ⅱ (α-Man II) and addition of second GlcNAc by GlcNAc-transferase-II (GnT-
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II), the precursor of all complex N-glycans is generated. Further branching and capping of
antennae is performed by some transferases to synthesize complex N-glycans (gray shadow
with three asterisks***): GlcNAc-transferase III-V (GnT-III-V), α1–6-fucosyltransferase
(FUT8), β1,4-galactosyltransferases (Gal-T), α2,3-sialyltransferase (α2,3, Sialyl-T) and
α2,6-sialyltransferase (α2,6 Sialyl-T). Hybrid N-glycans are formed if the
GlcNAcMan5GlcNAc2 glycan is not trimmed by α-mannosidase II and then extended by
Gal-T and Sialyl-T (gray shadow with two asterisks**).
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GlcNAc-transferase I, GnT- I.
b) O-glycan synthesis pathway. O-GalNAc are added to the protein in α-linkage to Ser/Thr
by a polypeptide GalNAc-transferase GALNT) in the Golgi apparatus. Four core structures
of O-GalNAc glycans are generated by extension of glycans by different enzymes. Less
extended O-GalNAc glycans, which are linked to Ser/Thr, are called Tn antigen, T antigen,
and Sialyl-Tn antigen and are associated with several types of cancer.
N-acetylgalactosamine, GalNAc; galactose, Gal; N-acetylglucosamine, GlcNAc; N-
acetylneuraminic acid, NeuAc; core 1 β1–3-galactosyltransferase 1, C1GALT1; molecular
chaperone of C1GALT1, COSMC; core 2 β1–6-N-acetylglucosaminyltransferases 1,
C2GnT-1, GCNT1; β−1,4-galactosyltransferase, B4GALT; α2–6-sialyltransferase,
ST6GALNAC1; α2–3-sialyltransferases, ST3GAL1; β1–3-N-acetylglucosaminyltransferase
3, B3GNT3; core 3 β1–3 N-acetylglucosaminyltransferase 6, B3GNT6; M-type N-
Acetylglucosaminyl transferase 3, GCNT3; β1,3-galactosyltransferase 5, B3GALT5.
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Figure 2.
The overall strategy for the analysis of protein glycosylation. (Left) For intact glycoprotein
analysis, a glycoprotein is directly subjected to high-resolution hybrid MS analysis after
reduction of disulfide bonds. (Right) For glycopeptide analysis, the glycoprotein is digested
with a protease(s) and the resultant glycopeptides are enriched prior to MS analysis. Glycans
are released by enzymatic or chemical methods, either from glycopeptides or from
glycoproteins. As shown in the lower panel, various useful technologies already exist and
are rapidly advancing.
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Figure 3.
Schematic representation of IgG1 and IgA1/2 with the cleavage sites of different proteases.
a) Scheme of IgG1 structure is shown in the top left. The example of enzymatic digestion for
separation of IgG Fab (antigen-binding fragment) and IgG Fc (crystallizable fragment)
moieties are shown in the top right. Digestion of IgG by pepsin and papain occurs below and
above the disulfide bond, respectively. Conversely, IdeS and IdeZ proteases digest IgG at a
specific site between Gly-Gly of the IgG hinge region, below the disulfide bond. Novel
workflow of site-specific N-glycan analysis of IgG, which was reported by Bondt et al. [49],
is shown in the bottom of the figure. IgG was captured and digested by IdeS protease on
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beads. The F(ab’)2 fragment was collected in the flow-through (FT), and the Fc portion was
collected after elution by 100 mM HCl.
b) Schemes of IgA1 and IgA2 structure are shown in the top. Backbone peptide of IgA hinge
region (HR) produced by trypsin is shown in the middle. O-glycosylation mainly occurs at
the serine (S) and threonine (T) residues indicated in red. Digestion sites in the IgA1 HR by
IgA-specific proteases (Clostridium ramosum AK183, Streptococcus pneumoniae TIGR4,
Haemophilus influenzae HK50) are also shown. N-glycopeptides of IgA1/2 produced by
trypsin digestion are shown at the bottom. N-glycan attachment sites are shown in red.
Closed circles represent N-glycans. Open circles represent O-glycans. constant region 1,
CH1; constant region 2, CH2; constant region 3, CH3; heavy-chain variable region, VH;
light-chain variable region, VL
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Figure 4.
Comparison of two analytical work flow for IgA-glycopeptides.
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a) High-throughput strategy for analysis of IgA N- and O- glycopeptides. This is the method
reported by Bondt et al [102]. Purified IgA was digested by trypsin after reduction and
alkylation. *To improve separation accuracy, two-step Hydrophilic interaction
chromatography (HILIC) enrichment was performed, which enabled site-specific N-
glycopeptide detection. The merit obtained by this method is high-throughput analysis. By
using ultrahigh resolution matrix assisted laser dissociation (MALDI)-Fourier transform ion
cyclotron resonance (FTICR)-MS, detection accuracy was improved and sialic acid
fragmentation was reduced. The representative mass spectra after two-step HILIC
enrichment were shown in the middle. This MS spectrum was obtained from ref 102. under
Creative Commons Attribution 4.0 International License (https://creativecommons.org/
licenses/by/4.0/).
O-glycopeptide, Q1; non-truncated, T1; truncated Asn340 containing glycopeptide, U1;
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the attachment sites was demonstrated by Takahashi et al. using LC-electron transfer
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Figure 5.
The workflow for Etanercept N- and O-glycosylation analysis.
a) The analysis workflow for O-glycosylation of Etanercept. a-1) By native MS analysis
after peptide N-glycosidase F (PNGase-F) digestion, the number of O-glycan core variants
with different numbers of sialic acids was detected, and the ratio of NeuAc residues to O-
glycan core was elucidated. a-2) After PNGase-F and sialidase digestion, the number of O-
glycans was detected by native MS. a-3) attachment sites of O-glycans were analyzed by
electron transfer dissociation (ETD) fragmentation after digestion by trypsin and sialidase.
N-acetylneuraminic acid, NeuAc
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Table 1.
Glycosylation of biological products in the list of the US Food and Drug Administration’s Center for Drug Evaluation and Research
Therapeutic proteins Proprietary name Definition Production system Class Total glycosylation sites Main target disease
Ohyama et al.
6 N-linked
Abatacept Orencia Anti-CTLA4 CHO IgG1 Rheumatoid arthritis
4 O-linked
Adalimumab Humira Anti-TNFα CHO IgG1 2 N-linked Rheumatoid arthritis
Neovascular age-related macular
Aflibercept Eylea/ zaltrap Anti-VEGF CHO IgG1 10 N-linked
degeneration, colon cancer
Agalsidase β Fabrazyme Agalsidase β CHO 3 N-linked Fabry disease
Alemtuzumab Campath, Lemtrada Anti-CD52 CHO IgG1 2 N-linked Chronic lymphocytic leukemia
Alglucosidase α Myozyme Alglucosidase α CHO 6 N-linked Pompe disease
Alirocumab Praluent Anti-PCSK9 CHO IgG1 2 N-linked Hyperlipidemia
Alteplase Activase Tissue plasminogen activator CHO 3 N-glycan Acute ischemic stroke
Perinatal/infantile- and juvenile-onset
Asfotase α Strensiq Asfotase α CHO IgG1 12 N-glycan
hypophosphatasia
Avelumab Bavencio Anti-PD-L1 CHO IgG1 2 N-linked Non-small cell lung cancer
Basiliximab Simulect Anti-IL-2R α Sp2/0 IgG1 2 N-linked Kidney transplantation
Becaplermin Regranex PDGF-BB yeast 2 N-linked Wound care of diabetic foot
6 N-linked
Belatacept Nulojix Anti-CTLA-4 CHO IgG1 Kidney transplantation
4 O-linked
Belimumab Benlysta Anti-B-cell activating factor NS0 IgG1 2 N-linked Systemic lupus erythematosus
Benralizumab Fasenra Anti-IL-5R CHO IgG1 2 N-linked (defucosylated) Asthma
Bevacizumab Avastin Anti-VEGF-A CHO IgG1 2 N-linked Colon cancer, lung cancer, etc
Bezlotoxumab Zinplava Anti-C. difficile toxins A and B CHO IgG1 2 N-linked Clostridium difficile infections
Brodalumab Siliq Anti-IL-17R CHO IgG2 2 N-linked Psoriasis
Expert Rev Proteomics. Author manuscript; available in PMC 2021 May 28.
Burosumab-twza Crysvita Anti-FGF23 CHO IgG1 2 N-linked X-linked hypophosphatemia
Cemiplimab Libtayo Anti-PD-1 CHO IgG4 2 N-linked Cutaneous squamous cell carcinoma
Cerliponase α Brineura Human tripeptidyl peptidase-1 CHO 5 N-linked TPP1 deficiency
Cetuximab Erbitux Anti-EGFR Sp2/0 IgG1 4 N-linked Metastatic colon cancer
Daclizumab Zinbryta Anti-IL-2R NS0 IgG 2 N-linked Multiple sclerosis
Daratumumab Darzalex Anti-CD38 CHO IgG1 2 N-linked Multiple myeloma
5 N-linked
Darbepoetin α Aranesp Erythropoietin CHO Renal anemia
1 O-linked
Denosumab Prolia Anti-RANKL CHO IgG2 2 N-linked Osteoporosis
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Therapeutic proteins Proprietary name Definition Production system Class Total glycosylation sites Main target disease
Dinutuximab Unituxin Anti-disialoganglioside CHO IgG1 2 N-linked Neuroblastoma
Dulaglutide Trulicity GLP-1 analog CHO IgG4 2 N-linked Diabetes
Dupilumab Dupixent Anti-IL-4R α CHO IgG4 2 N-linked Atopic dermatitis, asthma
Ohyama et al.
Expert Rev Proteomics. Author manuscript; available in PMC 2021 May 28.
Ibalizumab Trogarzo Anti-CD4 receptors NS0 IgG4 2 N-linked HIV infection
Ibritumomab tiuxetan Zevalin Anti-CD20 CHO IgG1 2 N-linked Non-Hodgkin lymphomas
Idursulfase Elaprase Human iduronate-2-sulfatase Human cells 8 N-linked Mucopolysaccharidosis II
Infliximab Infliximab Anti-TNF-α CHO IgG1 2 N-linked Rheumatoid arthritis
Inotuzumab ozogamicin Besponsa Anti-CD22 CHO IgG4 2 N-linked Acute lymphoblastic leukemia
Interferon α2b Intron A Interferon α2b CHO 1 O-linked Hepatitis C virus infection
Interferon β1a Rebif Interferon β1a CHO 1 N-linked Multiple sclerosis
Ixekizumab Taltz Anti-IL-17A receptor Human cells IgG4 2 N-linked Psoriasis
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Therapeutic proteins Proprietary name Definition Production system Class Total glycosylation sites Main target disease
Lanadelumab Takhzyro Anti-plasma kallikrein CHO IgG1 2 N-linked Hereditary angioedema
Laronidase Aldurazyme Alpha-L-iduronidase CHO 6 N-linked Mucopolysaccharidosis I
Mepolizumab Nucala Anti-IL-5 CHO IgG1 2 N-linked Eosinophilic asthma
Ohyama et al.
Mogamulizumab Poteligeo Anti-CCR4 CHO IgG1 2 N-linked (defucosylated) Cutaneous T-cell lymphoma
Natalizumab Tysabri Anti-integrin α4-subunit NS0 IgG4 2 N-linked Multiple sclerosis
Necitumumab Portrazza Anti-EGFR NS0 IgG1 2 N-linked Non-small cell lung cancer
Nivolumab Opdivo Anti-PD-1 CHO IgG4 2 N-linked Non-small cell lung cancer
Obinutuzumab Gazyva Anti-CD20 CHO IgG1 2 N-linked (defucosylated) Chronic lymphocytic leukemia
Ocrelizumab Ocrevus Anti-CD20 CHO IgG1 2 N-linked Multiple sclerosis
Ofatumumab Arzerra anti-CD20 NS0 IgG1 2 N-linked Chronic lymphocytic leukemia
Olaratumab Lartruvo Anti-PDGFR-α NS0 IgG1 4 N-linked Soft tissue sarcoma
Omalizumab Xolair Anti-IgE CHO IgG1 2 N-linked Asthma
Palivizumab Synagis Anti-F protein of RSV NS0 IgG1 2 N-linked RSV infection
Panitumumab Vectibix Anti-EGFR CHO IgG2 2 N-linked Colorectal cancer
Pembrolizumab Keytruda Anti-PD-1 CHO IgG4 2 N-linked Melanoma, lung cancer, etc
Pertuzumab Perjeta HER2 CHO IgG1 2 N-linked Breast cancer
Polatuzumab vedotin Polivy Anti-CD79b CHO IgG1 2 N-linked Diffuse large B-cell lymphoma
Ramucirumab Cyramza Anti-VEGFR2/KDR NS0 IgG1 2 N-linked Gastric adenocarcinoma
Ravulizumab Ultomiris Anti-complement component 5 CHO IgG2 2 N-linked Paroxysmal nocturnal hemoglobinuria
Anti-protective antigen component of
Raxibacumab raxibacumab NS0 IgG1 2 N-linked Anthrax
B. anthracis toxin
Reslizumab Cinqair Anti-IL-5 NS0 IgG4 2 N-linked Asthma
Rilonacept Arcalyst IL-1R and the IL-1R accessory protein CHO IgG1 2 N-linked Cryopyrin-associated periodic syndromes
Risankizumab Skyrizi Anti-IL-23 p19 CHO IgG1 2 N-linked Plaque psoriasis
Expert Rev Proteomics. Author manuscript; available in PMC 2021 May 28.
Rituximab Rituxan Anti-CD20 CHO IgG1 2 N-linked Non-Hodgkin Lymphomas
Romosozumab Evenity Anti-sclerostin CHO IgG2 2 N-linked Postmenopausal women osteoporosis
2 N-linked
Sargramostim Leukine Recombinant GM-CSF Yeast cells Neutropenia
2–4 O-linked
Sarilumab Kevzara Anti-IL6-Rα CHO IgG1 2 N-linked Rheumatoid arthritis
Sebelipase α Kanuma Human lysosomal acid lipase Egg white 6 N-linked Lysosomal acid lipase deficiency
Secukinumab Cosentyx Anti-IL-17A CHO IgG1 2 N-linked Psoriasis
Siltuximab Sylvant Anti-IL6 CHO IgG1 2 N-linked Castleman’s disease
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Therapeutic proteins Proprietary name Definition Production system Class Total glycosylation sites Main target disease
2 N-linked
Tenecteplase TNK-tPA CHO Acute myocardial infarction
1 O-linked fucose
Tildrakizumab Ilumetri Anti-IL-23 CHO IgG1 2 N-linked Psoriasis
Tocilizumab Actemra Anti-IL6R CHO IgG1 2 N-linked Rheumatoid arthritis
Ohyama et al.
The list of biological products was generated from Purple book (https://www.fda.gov/drugs/therapeutic-biologics-applications-bla/purple-book-lists-licensed-biological-products-reference-product-
exclusivity-and-biosimilarity-or) on December 2019. Glycosylation of each products was referred from Drugs. com (https://www.drugs.com/), DRUGBANK (https://www.drugbank.ca/), and EUROPEAN
MEDICINES AGENCY (https://www.ema.europa.eu/en).
cytotoxic T-lymphocyte associated protein 4, CTLA4; chinese hamster ovary, CHO; tumor necrosis factor, TNF; vascular endothelial growth factor, VEGF; proprotein convertase subtilicin/kexin type 9,
PCSK9; programmed death-ligand 1, PD-L1; interleukin-2 receptor, IL-2R; platelet-derived growth factor-BB, PDGF-BB; murine myeloma cells, Sp2/0; murine myeloma cells, NS0; interleukin-5 receptor,
IL-5R; vascular endothelial growth factor A, VEGF-A; interleukin-17 receptor, IL-17R; fibroblast growth factor 23, FGF23; programmed cell death-1, PD-1; epidermal growth factor receptor, EGFR;
receptor activator of NF-κB ligand, RANKL; glucagon-like peptide-1, GLP-1; interleukin-4 receptor, IL-4R; signaling lymphocyte activation marker Family member 7, SLAMF7; calcitonin gene-related
peptide, CGRP; interleukin-23, IL-23; interleukin-17A, IL-17A; interleukin-5, IL-5; CC chemokine receptor 4, CCR4; platelet-derived growth factor receptor-α, PDGFR-α; fusion protein of respiratory
syncytial virus, F protein of RSV; human epidermal growth factor receptor 2 protein, HER2; vascular endothelial growth factor receptor 2 /kinase insert domain-containing receptor, VEGFR2/KDR;
interleukin-5, IL-5; interleukin-1 receptor, IL-1R; interleukin-23p 19; IL-23p 19; granulocyte macrophage colony-stimulating factor, GM-CSF; interleukin-6 receptor α, IL-6Rα; interleukin-17A, IL-17A;
interleukin-6, IL-6; Tenecteplase-tissue plasminogen activator, TNK-tPA; interleukin-12, IL-12.
Expert Rev Proteomics. Author manuscript; available in PMC 2021 May 28.
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