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Messenger RNA-based vaccines against COVID-19 induce a robust anti-SARS-CoV-2 antibody response with potent viral neutralization activity. Antibody effector functions are determined by their constant region subclasses and by their glycosylation patterns, but their role in vaccine efficacy is unclear. Moreover, whether vaccination induces antibodies similar to those in patients with COVID-19 remains unknown. We analyze BNT162b2 vaccine-induced IgG subclass distribution and Fc glycosylation patterns and their potential to drive effector function via Fcγ receptors and complement pathways. We identify unique and dynamic pro-inflammatory Fc compositions that are distinct from those in patients with COVID-19 and convalescents. Vaccine-induced anti-Spike IgG is characterized by distinct Fab- and Fc-mediated functions between different age groups and in comparison to antibodies generated during natural viral infection. These data highlight the heterogeneity of Fc responses to SARS-CoV-2 infection and vaccination and suggest that they support long-lasting protection differently., Graphical abstract, The Fc structures of IgGs produced during infection and vaccination have important roles in shaping the immune response. Farkash et al. show that vaccine- and infection-induced anti-SARS-CoV-2 IgGs differ in their Fc regions and in the engagement of complement and Fc receptors, implying distinct effector functions and immunity.

The importance of IgG glycosylation has been known for many years not only by scientists in glycobiology but also by human pathogens that have evolved specific enzymes to modify these glycans with fundamental impact on IgG function. The rise of IgG as a major therapeutic scaffold for many cancer and immunological indications combined with the availability of unique enzymes acting specifically on IgG Fc-glycans have spurred a range of applications to study this important post-translational modification on IgG. This review article introduces why the IgG glycans are of distinguished interest, gives a background on the unique enzymatic tools available to study the IgG glycans and finally presents an overview of applications utilizing these enzymes for various modifications of the IgG glycans. The applications covered include site-specific glycan transglycosylation and conjugation, analytical workflows for monoclonal antibodies and serum diagnostics. Additionally, the review looks ahead and discusses the importance of O-glycosylation for IgG3, Fc-fusion proteins and other new formats of biopharmaceuticals.

Glycosylation is a post-translational modification essential for maintaining the structure and function of proteins. Abnormal N-glycan patterns have been found in various diseases compared to healthy controls. A decrease in terminal galactosylated N-glycans of serum IgG in rheumatoid arthritis (RA) and osteoarthritis (OA) may be involved in their immunopathogenesis. However, how glycan patterns differ between RA and OA remains unclear. Here, we identified 15 glycan forms of serum IgG from RA and OA using MALDI-TOF MS. We found that IgG galactosylation represented a suitable candidate for differentiating RA from healthy controls (AUC > 0.9). Then, we performed binary logistic regression to screen out three bisecting N-acetylglucosamine (GlcNAc) glycoforms for distinguishing between OA and RA. Combined ROC analysis of the selected glycans yielded an AUC of 0.81 between OA and RA and an AUC of 0.79 between OA and RF/ACPA negative RA. Similar results were found in the validation set. In conclusion, our analysis demonstrates that RA and OA are distinguished on the basis of their different IgG glycan patterns, which thus serve as suitable candidates as biomarkers for reliably identifying clinical conditions such as RA and OA.

Immunosuppressants and biologicals are widely used therapeutics for various chronic inflammatory diseases (CID). To gain more detailed insight into their downstream effects, we examined their impact on serum immunoglobulin G (IgG) glycosylation. We analyzed IgG subclass-specific fragment crystallizable (Fc) N-glycosylation in patients suffering from various CID using the LC-MS approach. Firstly, we compared IgG Fc N-glycosylation between 128 CID patients and 204 healthy controls. Our results replicated previously observed CID-related decrease in IgG Fc galactosylation (adjusted p-value range 1.70 × 10−2–5.95 × 10−22) and sialylation (adjusted p-value range 1.85 × 10−2–1.71 × 10−18). Secondly, to assess changes in IgG Fc N-glycosylation associated with therapy and remission status, we compared 139 CID patients receiving either azathioprine, infliximab, or vedolizumab therapy. We observed an increase in IgG Fc galactosylation (adjusted p-value range 1.98 × 10−2–1.30 × 10−15) and sialylation (adjusted p-value range 3.28 × 10−6–4.34 × 10−18) during the treatment. Furthermore, patients who reached remission displayed increased Fc galactosylation levels (p-value range 2.25 × 10−2–5.44 × 10−3) in comparison to patients with active disease. In conclusion, the alterations in IgG Fc glycosylation and the fact these changes are even more pronounced in patients who achieved remission, suggest modulation of IgG inflammatory potential associated with CID therapy.

Immunoglobulin G (IgG) glycosylation is studied in biological samples to develop clinical markers for precision medicine, for example, in autoimmune diseases and oncology. Inappropriate storage of proteins, lipids, or metabolites can lead to degradation or modification of biomolecular features, which can have a strong negative impact on accuracy and precision of clinical omics studies. Regarding the preservation of IgG glycosylation, the range of appropriate storage conditions and time frame is understudied. Therefore, we investigated the effect of storage on IgG Fc N-glycosylation in the commonly analyzed biofluids, serum and plasma. Short-term storage and accelerated storage stability were tested by incubating samples from three healthy donors under stress conditions of up to 50 °C for 2 weeks using −80 °C for 2 weeks as the reference condition. All tested IgG glycosylation features - sialylation, galactosylation, bisection, and fucosylation - remained unchanged up to room temperature as well as during multiple freeze−thaw cycles and exposure to light. Only when subjected to 37 °C or 50 °C for 2 weeks, galactosylation and sialylation subtly changed. Therefore, clinical IgG glycosylation analysis does not rely as heavily on mild serum and plasma storage conditions and timely analysis as many other omics analyses.

Es wurde bereits vielfach diskutiert, ob mütterliches Asthma bronchiale einen Risikofaktor für die Nachkommen darstellt, ebenfalls an allergischem Asthma zu erkranken. Die bisher durchgeführten Tierversuche variieren jedoch stark in der Methodik und reichen nicht aus, um die Mechanismen der maternofetalen Risikoübertragung gänzlich zu verstehen. Daher wurde in dem folgenden Maus-Modell der Einfluss einer Asthma-Exazerbation während der Schwangerschaft auf die Asthma-Suszeptibilität der Nachkommen untersucht. Ein besonderes Augenmerk lag dabei auf dem Immunglobulin G (IgG), welches in Abhängigkeit von seiner Glykosylierung pro- oder antiinflammatorisch auf den Fetus einwirken kann. Die weiblichen Mäuse wurden vor der Verpaarung entweder mit Casein (CAS) oder mit Ovalbumin (OVA) subkutan sensibilisiert. Die Kontrollgruppe erhielt eine phosphatgepufferte Salzlösung (PBS: engl. phosphate-buffered saline). Die Exazerbation während der Schwangerschaft wurde mittels einer Aerosol-Exposition des jeweiligen Allergens oder PBS ausgelöst. Bei den Nachkommen wurde nach dem gleichen Protokoll vorgegangen, jedoch verwendete man bei allen Nachkommen OVA als Allergen. Der asthmatische Phänotyp wurde mittels bronchoalveolärer Lavage (BAL), Lungenhistologie und Messung des Atemwegswiderstandes quantifiziert. Des Weiteren erfolgte eine Analyse der IgG-Glykosylierung im Serum der Muttertiere und deren allergischen Nachkommen. In den Nachkommen, die mit dem gleichen Allergen behandelt wurden wie das Muttertier (OVA-allergische Nachkommen von OVA-allergischen Muttertieren), konnte ein höheres Risiko für die Entwicklung eines hyperreagiblen Bronchialsystems im Vergleich zu den Nachkommen der Kontrollgruppe gezeigt werden. Im Gegensatz dazu zeigten die OVA-allergischen Nachkommen von CAS-allergischen Muttertieren diesen allergischen Phänotyp nicht. Während sich die CAS- und OVA-Muttertiere nicht in den Ergebnissen der BAL und der Lungenhistologie unterschieden, ließ sich ein signifikanter Unterschied in ihrer IgG-Glykosylierung aufzeigen. Die OVA-allergischen Muttertiere sowie ihre Nachkommen wiesen einen hohen Anteil an IgG auf, welches keine zusätzliche Galaktose oder Sialinsäure am Glykan des Asparagins (Asn)-297 integriert hatte. Das agalactosylierte Glykan verstärkt die proinflammatorischen Eigenschaften des IgG. Die Casein-allergischen Muttertiere zeigten ebenfalls eine Tendenz zu einer proinflammatorischen Glykosylierung des IgG, jedoch in einer deutlich geringeren Ausprägung. Das Glykosylierungsmuster spiegelte sich in ihren Nachkommen wider. Der verstärkte allergische Phänotyp der OVA-OVA-Nachkommen scheint mit der Asthma-Exazerbation des Muttertieres während der Schwangerschaft zusammenzuhängen und ist mit einem erhöhten Anteil an proinflammtorischem IgG, sowohl bei den Muttertieren als auch bei den Nachkommen, assoziiert. Da IgG transplazentar auf den Fetus übertragen werden kann, stellt dies einen möglichen Mediator der maternofetalen Risikoübertragung dar und sollte daher in zukünftigen Studien berücksichtigt werden., It has often been discussed whether maternal bronchial asthma is a risk factor for offspring to also develop allergic asthma. The animal studies conducted so far vary widely in their structure and are not sufficient to fully understand the mechanisms of maternal-fetal risk transmission. This study used a mouse model to investigate the influence of asthma exacerbation during pregnancy on asthma susceptibility of the offspring. Special attention was paid to immunoglobulin G (IgG), which can have a pro- or anti-inflammatory effect on the fetus depending on its glycosylation status. Female mice were subcutaneously sensitized with either casein (CAS) or ovalbumin (OVA) prior to mating. The control group received a phosphate-buffered saline solution (PBS). Asthma exacerbation during pregnancy was induced by aerosol exposure of the respective allergen or PBS. The same protocol was followed in the offspring, but the allergen OVA was used in all offspring. The asthmatic phenotype was investigated by bronchoalveolar lavage (BAL), histological analysis of lung inflammation and airway reactivity measurements. Similarly, an analysis of serum IgG glycosylation was performed in dams and their allergic offspring. If the mothers and their offspring were treated with the same allergen (OVA-allergic offspring of OVA-allergic mothers), the offspring showed a higher risk of developing a hyperreactive bronchial system than the offspring of the control group. However, the OVA-allergic offspring of the casein-allergic mothers were treated with a different allergen than the dam and did not show an increased allergic phenotype. While the CAS and OVA mothers did not differ in BAL and lung histology, significant differences were found in their IgG glycosylation. The OVA-allergic mothers as well as their offspring showed high levels of IgG, which did not integrate additional galactose or sialic acid in the glycan of asparagine (Asn)-297. This agalactosylated glycan enhances the proinflammatory properties of IgG. The casein-allergic mothers also showed a tendency towards proinflammatory glycosylation of IgG, but in a significantly lower degree. This was also reflected in their offspring. The increased allergic phenotype of OVA-OVA offspring seems to be related to the asthma exacerbation of the mother during pregnancy and is associated with an increased level of proinflammatory IgG in both mothers and offspring. Since IgG can be transmitted transplacentally to the fetus, this is a possible mediator of maternal-fetal risk transmission and should therefore be considered in future studies.

Human lifespan has increased significantly in the last 200 years, emphasizing our need to age healthily. Insights into molecular mechanisms of aging might allow us to slow down its rate or even revert it. Similar to aging, glycosylation is regulated by an intricate interplay of genetic and environmental factors. The dynamics of glycopattern variation during aging has been mostly explored for plasma/serum and immunoglobulin G (IgG) N-glycome, as we describe thoroughly in this chapter. In addition, we discuss the potential functional role of agalactosylated IgG glycans in aging, through modulation of inflammation level, as proposed by the concept of inflammaging. We also comment on the potential to use the plasma/serum and IgG N-glycome as a biomarker of healthy aging and on the interventions that modulate the IgG glycopattern. Finally, we discuss the current knowledge about animal models for human plasma/serum and IgG glycosylation and mention other, less explored, instances of glycopattern changes during organismal aging and cellular senescence.

Changes in immunoglobulin G (IgG) glycosylation pattern have been observed in a vast array of auto- and alloimmune, infectious, cardiometabolic, malignant, and other diseases. This chapter contains an updated catalog of over 140 studies within which IgG glycosylation analysis was performed in a disease setting. Since the composition of IgG glycans is known to modulate its effector functions, it is suggested that a changed IgG glycosylation pattern in patients might be involved in disease development and progression, representing a predisposition and/or a functional effector in disease pathology. In contrast to the glycopattern of bulk serum IgG, which likely relates to the systemic inflammatory background, the glycosylation profile of antigen-specific IgG probably plays a direct role in disease pathology in several infectious and allo- and autoimmune antibody-dependent diseases. Depending on the specifics of any given disease, IgG glycosylation read-out might therefore in the future be developed into a useful clinical biomarker or a supplementary to currently used biomarkers.

A large variation in the severity of disease symptoms is one of the key open questions in coronavirus disease 2019 (COVID-19) pandemics. The fact that only a small subset of people infected with severe acute respiratory syndrome coronavirus 2 develops severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the immunoglobulin G (IgG) glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of interindividual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing.

Background: The purpose of this paper is to evaluate the international scientific output of Igg glycosylation research by a stoichiometric analysis of the related papers published in the field of IGG glycosylation from 2009 to 2020, to explore the hot spot of IGG glycosylation and the evolution path of IGG glycosylation.Methods: Using the Web of Science core database (WoSCC) to collect the articles related to IgG glycosylation from 2009 to 2020 as the research object, using CiteSpace visualization software to analyze the co-occurrence of countries and institutions, core authors, and keywords. Cited authors and literature, Co-citation analysis of journals, obtaining cooperation maps of research countries / institutions and core authors, literature citation and clustering maps, co-occurrence maps of high-frequency subject headings, displaying related clusters, mutual influences between clusters and Keyword timeline view spectrum of the historical span of important keywords in clustering.Results: We searched 482 articles related to IgG glycosylation published in 227 journals, and observed that in the past 10 years, the number of articles published has generally increased year by year; it has been cited 13262 times, with an average of 27.5 citations per article, showing an upward trend year by year. The core team in the field of IGG glycosylation is mainly from University and research institutes in USA, Australia, Netherlands, Croatia, England, Germany, Ireland, Scotland, China and Japan. A total of 2408 authors participated in the writing of literature on Igg glycosylation, Among them, Wuhrer, Manfred、Lauc, Gordan、Irena Trbojevic Akmacic, Wei Wang and other scholars are the representatives of this research field and have important influence. There are 281 keywords under the theme of IgG glycosylation, of which there are 4 keywords with word frequency ≥100, and hot keywords that serve as bridges are pregnancy、complex、glycan、biosimilar、N-linked glycosylation、hilic-uplc、glycation and form 8 clusters. IgG glycosylation, as the "biomarker" of disease diagnosis and its mechanism has been a research hotspot in recent years.Conclusion: The use of CiteSpace software for quantitative analysis of IgG glycosylation related literature can intuitively demonstrate its development history and current research hotspots and trends, and can provide researchers with reference to the topic selection and research direction. Value and meaning.

This study aimed to investigate the association of IgG glycosylation and esophageal precancerosis for squamous cell carcinoma and determine its role in inflammation. Primary glycans selected by the least absolute shrinkage and selection operator (LASSO) algorithm were validated using univariate and multivariate logistics models plus restricted cubic spline functions. In total, 24 direct glycans and 27 derived traits were detected, among which four glycans and three derived traits were primarily selected. Then, GP5 (adjusted OR: 0.805), GP17 (adjusted OR: 1.305), G12n (adjusted OR: 1.271), Gal_1 (adjusted OR: 0.776) and Fuc (adjusted OR: 0.737) were validated and significantly associated with esophageal precancerosis. In addition, there was a consistent positive association in GP17 and G12n and a negative association in GP5, Gal_1, and Fuc by restricted cubic spline function. Compared with esophageal inflammation, GP17, G12n, and Fuc were still independently associated with precancerosis. In brief, the IgG glycosylation profile was independently associated with esophageal precancerosis beyond inflammation, which could be an early biomarker for esophageal cancer. Prevention Relevance: IgG glycosylation profile is associated with esophageal precancerosis and specific IgG glycans involves in the early stage of esophageal cancer, which is independent of inflammation.

N-glycosylation of immunoglobulin G (IgG) has been reported to change in human aging and in some age-related diseases. To further understand the molecular processes that determine these alterations, a detailed examination of individual IgG N-glycans with aging remains required. Mouse is the most commonly used model animal in studies of aging and age-related diseases, and mice have the advantage of relatively controllable genetic and environment variations compared to human. In this study, we systemically investigated the changes in serum IgG N-glycome in C57BL/6 mice during aging at 12 time points (6-80 weeks) via ultraperformance liquid chromatography with fluorescence detection. The study demonstrated several important findings. First, four chromatographic IgG N-glycan peaks were identified for the first time, including a high-mannose glycan, a monoantennary glycan, and two afucosylated glycans. Second, most of the IgG glycan levels changed significantly and presented pronounced gender-related differences from 6 to 12 weeks. Interestingly, all the IgG glycan levels tended to be similar between male and female mice at 12 weeks. Third, the level of fucosylated diantennary glycans containing one N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) decreased gradually and showed a significant negative correlation with age from 24 to 80 weeks (r = -0.716, p 0.0001), which was not sex-specific. SIGNIFICANCE: More comprehensive profile of murine IgG N-glycans by ultraperformance liquid chromatography with fluorescence detection was shown in this study with four newly identified chromatographic murine IgG N-glycan peaks. The majority of IgG N-glycans showed substantial stage-specific changes and sex-related differences during mouse aging, indicating a strict regulatory mechanism of glycan synthesis. The level of fucosylated diantennary glycans containing one Neu5Gc-linked LacNAc was significantly negatively correlated with age from 24 to 80 weeks, suggesting its great potential as an aging biomarker. The detailed characteristics of IgG N-glycosylation with aging in C57BL/6 mice demonstrated in the present study could provide essential reference data for studying the function and mechanism of IgG glycosylation in age-related researches based on C57BL/6 mouse models.

Background Neoadjuvant chemotherapy (NACT) could improve prognosis and survival quality of patients with local advanced gastric cancer (LAGC) by providing an opportunity of radical operation for them. However, no effective method could predict the efficacy of NACT before surgery to avoid the potential toxicity, time-consuming and economic burden of ineffective chemotherapy. Some research has been investigated about the correlation between serum IgG glycosylation and gastric cancer, but the question of whether IgG glycome can reflect the tumor response to NACT is still unanswered. Method Serum IgG glycome profiles were analyzed by Ultra Performance Liquid Chromatography in a cohort comprised of 49 LAGC patients of which 25 were categorized as belonging to the NACT response group and 24 patients were assigned to the non-response group. A logistic regression model was constructed to predict the response rate incorporating clinical features and differential N-glycans, while the precision of model was assessed by receiver operating characteristic (ROC) analysis. Results IgG N-glycome analysis in pretreatment serum of LAGC patients comprises 24 directly detected glycans and 17 summarized traits. Compared with IgG glycans of non-response group, agalactosylated N-glycans increased while monosialylated N-glycans and digalactosylated N-glycans decreased in the response group. We constructed a model combining patients’ age, histology, chemotherapy regimen, GP4(H3N4F1), GP6(H3N5F1), and GP18(H5N4F1S1), and ROC analysis showed this model has an accurate prediction of NACT response (AUC = 0.840) with the sensitivity of 64.00% and the specificity of 100%. Conclusion We here firstly present the profiling of IgG N-glycans in pretreatment serum of LAGC. The alterations in IgG N-glycome may be personalized biomarkers to predict the response to NACT in LAGC and help to illustrate the relationship between immunity and effect of NACT.

IgG Fc glycans were characterized in 225 healthy children and 40 children with unexplained recurrent respiratory infections, revealing age-dependent variation and suggesting a role for IgG glycosylation in immunocompetence.

Background Maternal asthma during pregnancy is considered an environmental risk factor for asthma development in children. Immunoglobulin G (IgG) antibodies that are transferred from the mother to the fetus are known to act in a pro- or anti-inflammatory manner depending on their glycosylation status. Objective Using a mouse model, we examined how maternal allergic airway inflammation during pregnancy influenced offspring experimental asthma severity, as well as maternal and offspring serum IgG antibody glycosylation patterns. Additionally, the effects of maternal and offspring exposure to the same or different allergens were investigated. Methods Female mice were either sham sensitized or sensitized to casein (CAS) or ovalbumin (OVA) before mating. Subsequently, allergic lung inflammation was induced in pregnant dams via aerosol allergen challenge (sham, CAS or OVA). After weaning, pups were subjected to an experimental asthma protocol using OVA. Asn-297 IgG glycosylation was analysed in maternal and offspring serum. Results When mothers and offspring were sensitized to the same allergen (OVA-OVA), offspring had more severe experimental asthma. This was evidenced by altered antibody concentrations, increased bronchoalveolar lavage inflammatory cell influx and decreased lung tissue and lung draining lymph node regulatory T cell percentages. When mothers and offspring were sensitized to different allergens (CAS-OVA), this phenotype was no longer observed. Additionally, maternal serum from allergic mothers had significantly higher levels of pro-inflammatory IgG1, shown by decreased galactosylation and sialylation at the Asn-297 glycosylation site. Similar glycosylation patterns were observed in the serum of adult allergic offspring from allergic mothers. Conclusions and clinical relevance We observed a strong association between maternal experimental asthma during pregnancy, increased offspring airway inflammation and pro-inflammatory IgG glycosylation patterns in mothers and offspring. IgG glycosylation is not a standard measurement in the clinical setting, and we argue that it may be an important parameter to include in future clinical studies.

Ankylosing spondylitis (AS) is a chronic inflammatory disease with poorly defined aetiologies and no curative treatments. The average delay in the diagnosis of AS is 6–8 years.1 Human leukocyte antigen B27 (HLA-B27) is a key laboratory marker for AS presenting in at least 90% of patients with AS.2 However, 63%–90% of patients with reactive arthritis3 and 19.2% of patients with psoriatic arthritis (PsA)4 are also positive for HLA-B27, indicating low specificity of HLA-B27. The risk of development of AS in an HLA-B27-positive individual is only 2%–10%,5 which suggests the limited value of HLA-B27 in supporting an AS diagnosis. Moreover, reported serum biomarkers for AS have generally exhibited low sensitivity or specificity6 (

Immunoglobulin G (IgG) is the most abundant serum antibody and is a key determinant of the humoral immune response. Its structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. IgG N-glycome patterns change with the age of individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is affected by a combination of genetic and environmental factors. However, global inter-population differences in IgG glycosylation have never been properly addressed. Here we present population-specific N-glycosylation patterns of whole IgG, analysed in 5 different populations totalling 10,482 IgG glycomes, and of IgG’s fragment crystallisable region (Fc), analysed in 2,530 samples from 27 populations sampled across the world. We observed that country of residence associates with many N-glycan features and is a strong predictor of monogalactosylation variability. IgG galactosylation also strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators. We found that subjects from developing countries had low IgG galactosylation levels, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to country-specific environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

Changes in immunoglobulin G (IgG) glycan structures are currently believed to closely related to the emergence of cancer. In this review, we summarize the current body of evidence suggesting that differences in serum IgG glycosylation patterns correspond to changes in multiple types of cancer. Modifications include IgG terminal N-link galactosylation, IgG core fucosylation, IgG terminal sialylation, and IgG terminal bisecting N-acetylglucosamine. IgG N-glycomic alterations represent promising novel biomarkers for non-invasive-cancer diagnosis, prognosis, and progression monitoring; they are characterized by high sensitivity and specificity, compensating for previously identified glycobiomarkers.

The intestine is one of the most important organs associated with the immune system. It is thought that disruption of intestinal immunity causes inflammatory bowel disease (IBD). Recent advances in immune glycobiology have provided novel insights into many human diseases. For example, studies of glycosylation remodeling in mice have underscored the importance of oligosaccharides in the pathogenesis of IBD. Furthermore, aberrant glycosylation of IgG is a good serum marker of IBD activity. In this review, we examine current understanding of the role of aberrant glycosylation in the pathogenesis of IBD in terms of our original data and recent reports.

Background: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects.Objective: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants.Methods: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns.Results: Different adjuvants induce distinct IgG(+) GC B-cell responses with specific transcriptomes and expression levels of the alpha 2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3(-) follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-inoil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-gamma(+) TFH1 cells, IL-6/IL-23-dependent IL-17A(+) T-FH17 cells, and high ratios of TFH cells to Foxp31 follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor.Conclusion: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.

Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half‐life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed‐batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high‐mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2‐F‐peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.

Global antibody glycosylation is dynamic and plays critical roles in shaping different immunological outcomes and direct antibody functionality during HIV infection. However, the relevance of global antibody or plasma glycosylation patterns to HIV persistence after antiretroviral therapy (ART) has not been characterized. First, we compared glycomes of total plasma and isolated immunoglobulin G (IgG) from HIV+ ART-suppressed, HIV+ viremic, and HIV-negative individuals. Second, in ART-suppressed individuals, we examined the associations between glycomes and (1) levels of cell-associated HIV DNA and RNA in PBMCs and isolated CD4+ T cells, (2) CD4 count and CD4%, and (3) expression of CD4+ T-cell activation markers. HIV infection is associated with persistent alterations in the IgG glycome including decreased levels of disialylated glycans, which is associated with a lower anti-inflammatory activity, and increased levels of fucosylated glycans, which is associated with lower antibody-dependent cell-mediated cytotoxicity (ADCC). We also show that levels of certain mono- and digalactosylated nonfucosylated glycomic traits (A2G1, A2G2, and A2BG2), which have been reported to be associated with higher ADCC and higher anti-inflammatory activities, exhibit significant negative correlations with levels of cell-associated total HIV DNA and HIV RNA in ART-suppressed individuals. Finally, levels of certain circulating anti-inflammatory glycans are associated with higher levels of CD4 T cells and lower levels of T-cell activation. Our findings represent the first proof-of-concept evidence that glycomic alterations, known to be associated with differential states of inflammation and ADCC activities, are also associated with levels of HIV persistence in the setting of ART suppression.

Immunoglobulin G (IgG) glycosylation is essential for function of the immune system, but the genetic and environmental factors that underlie its inter- individual variability are not well defined. The Collaborative Cross (CC) genetic resource harnesses over 90% of the common genetic variation of the mouse. By analyzing the IgG glycome composition of 95 CC strains, we made several important observations: (i) glycome variation between mouse strains was higher than between individual humans, despite all mice having the same environmental influences ; (ii) five genetic loci were found to be associated with murine IgG glycosylation ; (iii) variants outside traditional glycosylation site motifs affected glycome variation ; (iv) bisecting N- acetylglucosamine (GlcNAc) was produced by several strains although most previous studies have reported the absence of glycans containing the bisecting GlcNAc on murine IgGs ; and (v) common laboratory mouse strains are not optimal animal models for studying effects of glycosylation on IgG function.

Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.

Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.

Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality. Here we sought to determine if the glycosylation profile of serum immunoglobulin G (IgG) could distinguish patients with non-atrophic gastritis (NAG), duodenal ulcer (DU) and gastric cancer (GC). Serum IgG was released and analyzed using nano-LC–TOF mass spectrometry. Statistically significant false discovery rate (FDR)-adjusted p-values were observed for 18 glycans, eight that differed significantly between NAG and GC, three that distinguished NAG from DU, and eight that differed between DU and GC. The IgG glycosylation signature may be useful as a predictive marker for gastric cancer.

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.

N-glycosylation of immunoglobulin G (IgG) is important for its biological functions. Biantennary N-glycans attached to the heavy chains of the fragment crystallizable (Fc) moiety of the IgG molecule influence its stability and effector functions. Additionally, around 15-20% of IgG molecules carry biantennary N-glycans in the antigen-binding (Fab) fragment, potentially affecting its interaction with the antigen. Changes in the IgG N-glycosylation profile are observed in many autoimmune diseases. Mouse models are often used to study the role of IgG N-glycosylation in immunology. However, detailed studies of the mouse IgG N-glycome lag behind those of the human glycome. The aim of this study is to evaluate the biological variation of IgG N-glycans between three commonly used mouse strains. IgG was isolated from the serum of individual sex- and aged-matched mice of BALB/c, C57BL/6 and C3H strains kept in the same environment. The obtained IgG N-glycans were analysed by the capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) method, which characterizes both Fc and Fab N-glycans, and by the nano ultra-performance liquid chromatography coupled with mass spectrometer (nanoUPLC-ESI-MS) method, which provides information on subclass- specific N-glycosylation of the Fc region. We compared several derived N-glycan traits, describing the relative abundance of N-glycan structural features. We observed differences in the IgG N-glycome of the 3 strains with both methods. The profiles of the Fc-linked and the total IgG N- glycomes are different even within one strain and so are the subclass-specific profiles. These results suggest that there is a strong genetic component defining IgG N-glycosylation and that it might work in a subclass-specific manner.