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High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).

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ProNGF (nerve growth factor) is a precursor of NGF and a signaling peptide exerting opposite effects on neuronal cells, i.e., apoptotic or neuritogenic. The conflicting biological activity of proNGF depends on the relative levels of two membrane receptors, TrkA and p75NTR. The effect of proNGF depends on the expression levels of these receptor proteins and their affinity to proNGF. Since the affinity of proteins has been studied with various recombinant proteins, it is worth comparing the affinity of these proteins within one experiment with the same method. This study examined the affinity between a recombinant proNGF and p75NTR expressed in common systems: bacterial, insect, and mammalian cells. The extracellular domain of p75NTR expressed in the insect or mammalian systems bound to native mature NGF, with a higher affinity for the insect receptor. The uncleavable proNGF was expressed in the three systems and they showed neuritogenic activity in PC12 cells. These recombinant proteins were used to compare their binding affinity to p75NTR. The insect p75NTR showed a higher binding affinity to proNGF than the mammalian p75NTR. The insect p75NTR bound proNGF from the insect system with the highest affinity, then from the mammalian system, and the lowest from the bacterial system. Conversely, the mammalian p75NTR showed no such preference for proNGF. Because the recombinant proNGF and p75NTR from different expression systems are supposed to have the same amino acid sequences, these differences in the affinity depend likely on their post-translational modifications, most probably on their glycans. Each recombinant proNGF and p75NTR in various expression systems exhibited different mobilities on SDS-PAGE and reactivities with glycosidases and lectins.

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A mucin-type glycoprotein extracted from various species of jellyfish (JF) is named qniumucin (Q-mucin). Compared with general mucins, most of which are from mammals including humans, Q-mucin can be collected on a relatively large scale with high yield. Owing to its simple structure with low heterogeneity, Q-mucin has a potential to be developed into material mucins which opens various applications valuable to humans. On the basis of our present knowledge, here, we describe our protocol for the extraction of Q-mucin, which can be extracted from any JF species worldwide. Experimental protocols to identify the structure of Q-mucin are also introduced.

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INTRODUCTION: Prostate-specific membrane antigen (PSMA) is a US Food and Drug Administration-approved theranostic target for prostate cancer (PCa). Although PSMA is known to be glycosylated, the composition and functional roles of its N-linked glycoforms have not been fully characterized. METHODS: PSMA was isolated from pooled seminal plasma from low-risk grade Groups 1 and 2 PCa patients. Intact glycopeptides were analyzed by mass spectrometry to identify site-specific glycoforms. RESULTS: We observed a rich distribution of PSMA glycoforms in seminal plasma from low and low-intermediate-risk PCa patients. Some interesting generalities can be drawn based on the predicted topology of PSMA on the plasma membrane. The glycoforms at ASN-459, ASN-476, and ASN-638 residues that are located at the basal domain facing the plasma membrane in cells, are predominantly high mannose glycans. ASN-76 which is located in the interdomain region adjacent to the apical domain of the protein shows a mixture of high mannose glycans and complex glycans, whereas ASN-121, ASN-195 and ASN-336 that are located and are exposed at the apical domain of the protein predominantly possess complex sialylated and fucosylated N-linked glycans. These highly accessible glycosites display the greatest diversity in isoforms across the patient samples. CONCLUSIONS: Our study provides novel qualitative insights into PSMA glycoforms that are present in the seminal fluid of PCa patients. The presence of a rich diversity of glycoforms in seminal plasma provides untapped potential for glycoprotein biomarker discovery and as a clinical sample for noninvasive diagnostics of male urological disorders and diseases including PCa. Specifically, our glycomics approach will be critical in uncovering PSMA glycoforms with utility in staging and risk stratification of PCa.

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Macroheterogeneity in follicle-stimulating hormone (FSH) ß-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of females under 35 years of age, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate whether the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro mouse follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion and oocyte quality compared with FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions and cell-to-cell communication within 24 h in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro by increasing cell-to-cell communication early in folliculogenesis, and that the shift in in vivo abundance from FSH21 to FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

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Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

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Introduction: Chikungunya virus (CHIKV) is a re-emerging mosquito transmitted alphavirus of global concern. Neutralizing antibodies and antibody Fc-effector functions have been shown to reduce CHIKV disease and infection in animals. However, the ability to improve the therapeutic activity of CHIKV-specific polyclonal IgG by enhancing Fc-effector functions through modulation of IgG subclass and glycoforms remains unknown. Here, we evaluated the protective efficacy of CHIKV-immune IgG enriched for binding to Fc-gamma receptor IIIa (FcγRIIIa) to select for IgG with enhanced Fc effector functions. Methods: Total IgG was isolated from CHIKV-immune convalescent donors with and without additional purification by FcγRIIIa affinity chromatography. The enriched IgG was characterized in biophysical and biological assays and assessed for therapeutic efficacy during CHIKV infection in mice. Results: FcγRIIIa-column purification enriched for afucosylated IgG glycoforms. In vitro characterization showed the enriched CHIKV-immune IgG had enhanced human FcγRIIIa and mouse FcγRIV affinity and FcγR-mediated effector function without reducing virus neutralization in cellular assays. When administered as post-exposure therapy in mice, CHIKV-immune IgG enriched in afucosylated glycoforms promoted reduction in viral load. Discussion: Our study provides evidence that, in mice, increasing Fc engagement of FcγRs on effector cells, by leveraging FcγRIIIa-affinity chromatography, enhanced the antiviral activity of CHIKV-immune IgG and reveals a path to produce more effective therapeutics against these and potentially other emerging viruses.

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Follicle-stimulating hormone (FSH), together with luteinizing hormone (LH) and human chorionic gonadotropin (hCG), plays a fundamental role in human reproduction. The discovery of FSH and other gonadotropins was a defining moment in our understanding of reproduction and led to the development of many treatments for infertility. In this regard, exogenous FSH has been used to treat infertility in women for decades. Today, several recombinant and highly purified urinary forms of FSH are used in medically assisted reproduction (MAR). However, differences in the macro- and micro-heterogeneity of FSH result in a variety of FSH glycoforms, with glycoform composition determining the bioactivity (or potency), pharmacokinetic/pharmacodynamic (PK/PD) profiles, and clinical efficacy of the different forms of FSH. This review illustrates how the structural heterogeneity of FSH glycoforms affects the biological activity of human FSH products, and why potency does not predict effects in humans in terms of PK, PD, and clinical response.

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Many biotherapeutics such as monoclonal antibodies (mAbs) consist of various glycoforms, which can have different PK properties upon administration to animals and human. As a result, it is necessary to monitor the abundance of glycoforms and limit lot-to-lot variability during the manufacturing process. However, limited information is known about the clearance of mAb glycoforms from ocular space upon intravitreal injection. We present here an assessment of glycoform clearance of a biotherapeutic mAb (IgG1) from rabbit vitreous humor, aqueous humor and retina tissue using LC/MS. The results show that G0, G0F and G1F have similar T1/2, while mannose-5 has a longer T1/2 and is cleared slower in rabbit ocular space, which contradicted with what has been reported in the literature in which Mann5 was cleared faster systematically.

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Aberrant protein glycosylation is an active pathological alteration related to the progression of cancers. The speed of progression varies among individuals, increasing the difficulties of prognosis assessment. Hence, evaluating variation in glycosylation using patients themselves as their own controls is a potential way to reduce the impact of individual differences on progression monitoring. Here, following a longitudinal follow-up study involving 125 lung cancer (LC) patients with progressive disease, we isolated disease-specific IgG from serum using polyacrylamide gel electrophoresis, obtained IgG glycoform ratios using mass spectrometry, and then set a fold-change cutoff of 1.5 to utilize the intra-individual variation in IgG glycosylation to monitor PD. We found that the serial monitoring of 15 types of glycoform ratios provided an effective way for monitoring LC progression. Over 1.5-fold changes in glycoform ratios relative to the first observed value were detected in 117 of 125 LC patients (93.6%). Our established method predicted LC progression 55.8 (IQR 31.1-90.1) weeks earlier than imaging examination did. In summary, intra-individual variation in IgG glycoform ratios is useful to monitor LC progression, expanding our knowledge about the relationship between IgG glycosylation and cancer prognosis. The raw data files are available via the ProteomeXchange Consortium with the identifier PXD037541.

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OBJECTIVE: The purpose of this study was to examine the levels of cerebrospinal fluid (CSF) apolipoprotein E (apoE) species in Alzheimer's disease (AD) patients. METHODS: We analyzed two CSF cohorts of AD and control individuals expressing different APOE genotypes. Moreover, CSF samples from the TgF344-AD rat model were included. Samples were run in native- and SDS-PAGE under reducing or non-reducing conditions (with or without ß-mercaptoethanol). Immunoprecipitation combined with mass spectrometry or western blotting analyses served to assess the identity of apoE complexes. RESULTS: In TgF344-AD rats expressing a unique apoE variant resembling human apoE4, a ~35-kDa apoE monomer was identified, increasing at 16.5 months compared with wild-types. In humans, apoE isoforms form disulfide-linked dimers in CSF, except apoE4, which lacks a cysteine residue. Thus, controls showed a decrease in the apoE dimer/monomer quotient in the APOE ε3/ε4 group compared with ε3/ε3 by native electrophoresis. A major contribution of dimers was found in APOE ε3/ε4 AD cases, and, unexpectedly, dimers were also found in ε4/ε4 AD cases. Under reducing conditions, two apoE monomeric glycoforms at 36 kDa and at 34 kDa were found in all human samples. In AD patients, the amount of the 34-kDa species increased, while the 36-kDa/34-kDa quotient was lower compared with controls. Interestingly, under reducing conditions, a ~100-kDa apoE complex, the identity of which was confirmed by mass spectrometry, also appeared in human AD individuals across all APOE genotypes, suggesting the occurrence of aberrantly resistant apoE aggregates. A second independent cohort of CSF samples validated these results. CONCLUSION: These results indicate that despite the increase in total apoE content the apoE protein is altered in AD CSF, suggesting that function may be compromised.

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Follicle-stimulating hormone (FSH), an α/ß heterodimeric glycoprotein hormone, consists of functionally significant variants resulting from the presence or absence of either one of two FSHß subunit N-glycans. The two most abundant variants are fully-glycosylated FSH24 (based on 24 kDa FSHß band in Western blots) and hypo-glycosylated FSH21 (21 kDa band, lacks ßAsn24 glycans). Due to its ability to bind more rapidly to the FSH receptor and occupy more FSH binding sites than FSH24, hypo-glycosylated FSH21 exhibits greater biological activity. Endoglycosidase F1-deglycosylated FSH bound to the complete extracellular domain of the FSH receptor crystallized as a trimeric complex. It was noted that a single biantennary glycan attached to FSHα Asn52 might preemptively fill the central pocket in this complex and prevent the other two FSH ligands from binding the remaining ligand-binding sites. As the most active FSH21 preparations possessed more rapidly migrating α-subunit bands in Western blots, we hypothesized that Asn52 glycans in these preparations were small enough to enable greater FSH21 receptor occupancy in the putative FSHR trimer model. Highly purified hFSH oligosaccharides derived from each FSH subunit, were characterized by electrospray ionization-ion mobility-collision-induced dissociation (ESI-IM-CID) mass spectrometry. FSHß glycans typically possessed core-linked fucose and were roughly one third bi-antennary, one third tri-antennary and one third tetra-antennary. FSHα oligosaccharides largely lacked core fucose and were bi- or tri-antennary. Those αAsn52 glycans exhibiting tetra-antennary glycan m/z values were found to be tri-antennary, with lactosamine repeats accounting for the additional mass. Selective αAsn52 deglycosylation of representative pituitary hFSH glycoform Superdex 75 gel filtration fractions followed by ESI-IM-CID mass spectrometry revealed tri-antennary glycans predominated even in the lowest molecular weight FSH glycoforms. Accordingly, the differences in binding capacity of the same receptor preparation to different FSH glycoforms are likely the organization of the FSH receptor in cell membranes, rather than the αAsn52 oligosaccharide.

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Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is a powerful tool for the characterization and identification of the macro- and microheterogeneity of a glycoprotein in a bottom-up approach. This chapter describes in detail the sample preparation procedures using a purified biological sample, prostate-specific antigen, as a model protein, including proteolytic digestion (trypsin). In addition, insights are provided into the strengths of using capillary electrophoresis for obtaining isomer separation of differently linked sialic acids. Lastly, approaches and potential pitfalls for the integration and quantitation of glycopeptide signals from the obtained CZE-MS data are discussed.

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Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

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Regulatory authorities place stringent guidelines on the removal of contaminants during the manufacture of biopharmaceutical products. Monoclonal antibodies, Fc-fusion proteins, and other mammalian cell-derived biotherapeutics are heterogeneous molecules that are validated based on the production process and not on molecular homogeneity. Validation of clearance of potential contamination by viruses is a major challenge during the downstream purification of these therapeutics. Virus filtration is a single-use, size-based separation process in which the contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filtration is routinely used as part of the overall virus clearance strategy. Compromised performance of virus filters due to membrane fouling, low throughput and reduced viral clearance, is of considerable industrial significance and is frequently a major challenge. This review shows how components generated during cell culture, contaminants, and product variants can affect virus filtration of mammalian cell-derived biologics. Cell culture-derived foulants include host cell proteins, proteases, and endotoxins. We also provide mitigation measures for each potential foulant.

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Proteomics studies allow for the determination of the identity, amount, and interactions of proteins under specific conditions that allow the biological state of an organism to ultimately change. These conditions can be either beneficial or detrimental. Diseases are due to detrimental changes caused by either protein overexpression or underexpression caused by as a result of a mutation or posttranslational modifications (PTM), among other factors. Identification of disease biomarkers through proteomics can be potentially used as clinical information for diagnostics. Common biomarkers to look for include PTM. For example, aberrant glycosylation of proteins is a common marker and will be a focus of interest in this review. A common way to analyze glycoproteins is by glycoproteomics involving mass spectrometry. Due to factors such as micro- and macroheterogeneity which result in a lower abundance of each version of a glycoprotein, it is difficult to obtain meaningful results unless rigorous sample preparation procedures are in place. Microheterogeneity represents the diversity of glycans at a single site, whereas macroheterogeneity depicts glycosylation levels at each site of a protein. Enrichment and derivatization of glycopeptides help to overcome these limitations. Over the time range of 2016 to 2020, several methods have been proposed in the literature and have contributed to drastically improve the outcome of glycosylation analysis, as presented in the sampling surveyed in this review. As a current topic in 2020, glycoproteins carried by pathogens can also cause disease and this is seen with SARS CoV2, causing the COVID-19 pandemic. This review will discuss glycoproteomic studies of the spike glycoprotein and interacting proteins such as the ACE2 receptor.

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Dried blood spot (DBS) is the standard specimen for the newborn screening of inborn errors of metabolism (IEM) by tandem mass spectrometry. Availability of DBS for the mass spectrometric analysis of the diagnostic marker proteins, transferrin (Tf) and apolipoprotein CIII (apoCIII), of congenital disorders of glycosylation (CDG) was examined. Recovery of Tf from DBS was only slightly reduced compared with fresh serum. Although oxidation of the core polypeptides was observed, glycans of Tf and apoCIII were unaffected by storage of DBS in the ambient environment for at least 1 month. The combination of DBS and the triple quadrupole mass spectrometer used for IEM screening was sufficient to characterize the aberrant glycoprofiles of Tf and apoCIII in CDG. DBS or dried serum spot on filter paper can reduce the cost of sample transportation and potentially promote mass spectrometric screening of CDG.

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INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

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Monoclonal antibodies (mAbs) are one of the cornerstones of modern medicine, across an increasing range of therapeutic areas. All therapeutic mAbs are glycoproteins, i.e., their polypeptide chain is decorated with glycans, oligosaccharides of extraordinary structural diversity. The presence, absence, and composition of these glycans can have a profound effect on the pharmacodynamic and pharmacokinetic profile of individual mAbs. Approaches for the glycoengineering of therapeutic mAbs-the manipulation and optimisation of mAb glycan structures-are therefore of great interest from a technological, therapeutic, and regulatory perspective. In this review, we provide a brief introduction to the effects of glycosylation on the biological and pharmacological functions of the five classes of immunoglobulins (IgG, IgE, IgA, IgM and IgD) that form the backbone of all current clinical and experimental mAbs, including an overview of common mAb expression systems. We review selected examples for the use of small molecule inhibitors of glycan biosynthesis for mAb glycoengineering, we discuss the potential advantages and challenges of this approach, and we outline potential future applications. The main aim of the review is to showcase the expanding chemical toolbox that is becoming available for mAb glycoengineering to the biology and biotechnology community.

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The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a profound impact on the safety and efficacy of therapeutic IgG monoclonal antibodies (mAbs). Fc glycosylation of a mAb is an established critical quality attribute (CQA), and its oligosaccharide profile is required to be thoroughly characterized by state-of-the-art analytical methods. The Fc oligosaccharides are highly heterogeneous, and the differentially glycosylated species (glycoforms) of IgG express unique biological activities. Glycoengineering is a promising approach for the production of selected mAb glycoforms with improved effector functions, and non- and low-fucosylated mAbs exhibiting enhanced antibody-dependent cellular cytotoxicity activity have been approved or are under clinical evaluation for treatment of cancers, autoimmune/chronic inflammatory diseases, and infection. Recently, the chemoenzymatic glycoengineering method that allows for the transfer of structurally defined oligosaccharides to Asn-linked GlcNAc residues with glycosynthase has been developed for remodeling of IgG-Fc oligosaccharides with high efficiency and flexibility. Additionally, various glycoengineering methods have been developed that utilize the Fc oligosaccharides of IgG as reaction handles to conjugate cytotoxic agents by "click chemistry", providing new routes to the design of antibody-drug conjugates (ADCs) with tightly controlled drug-antibody ratios (DARs) and homogeneity. This review focuses on current understanding of the biological relevance of individual IgG glycoforms and advances in the development of next-generation antibody therapeutics with improved efficacy and safety through glycoengineering.

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