Glycan Composition Research Articles

Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Evaluation of Prebiotic Glycan Composition in Human Milk and Infant Formula: Profile of Galacto-Oligosaccharides and Absolute Quantification of Major Milk Oligosaccharides by UPLC-Cyclic IM-MS and UPLC-MS/MS.

Prebiotic oligosaccharides have attracted immense interest in the infant formula (IF) industry due to their unique health benefits for infants. There is a need for the reasonable supplementation of prebiotics in premium IF products. Herein, we characterized the profile of galacto-oligosaccharides (GOS) in human milk (HM) and IF using ultrahigh-performance liquid chromatography-cyclic ion mobility-mass spectrometry (UPLC-cIM-MS) technique. Additionally, we further performed a targeted quantitative analysis of five essential HM oligosaccharides (HMOs) in HM (n = 196), IF (n = 50), and raw milk of IF (n = 10) by the high-sensitivity UPLC-MS/MS method. HM exhibited a more abundant and variable HMO composition (1183.19 to 2892.91 mg/L) than IF (32.91 to 56.31 mg/L), whereas IF contained extra GOS species and non-negligible endogenous 3'-sialyllactose. This also facilitated the discovery of secretor features within the Chinese population. Our study illustrated the real disparity in the prebiotic glycome between HM and IF and provided crucial reference for formula improvement.

Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains.

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

Glycoprofiling of proteins as prostate cancer biomarkers: A multinational population study.

The glycoprofiling of two proteins, the free form of the prostate-specific antigen (fPSA) and zinc-α-2-glycoprotein (ZA2G), was assessed to determine their suitability as prostate cancer (PCa) biomarkers. The glycoprofiling of proteins was performed by analysing changes in the glycan composition on fPSA and ZA2G using lectins (proteins that recognise glycans, i.e. complex carbohydrates). The specific glycoprofiling of the proteins was performed using magnetic beads (MBs) modified with horseradish peroxidase (HRP) and antibodies that selectively enriched fPSA or ZA2G from human serum samples. Subsequently, the antibody-captured glycoproteins were incubated on lectin-coated ELISA plates. In addition, a novel glycoprotein standard (GPS) was used to normalise the assay. The glycoprofiling of fPSA and ZA2G was performed in human serum samples obtained from men undergoing a prostate biopsy after an elevated serum PSA, and prostate cancer patients with or without prior therapy. The results are presented in the form of an ROC (Receiver Operating Curve). A DCA (Decision Curve Analysis) to evaluate the clinical performance and net benefit of fPSA glycan-based biomarkers was also performed. While the glycoprofiling of ZA2G showed little promise as a potential PCa biomarker, the glycoprofiling of fPSA would appear to have significant clinical potential. Hence, the GIA (Glycobiopsy ImmunoAssay) test integrates the glycoprofiling of fPSA (i.e. two glycan forms of fPSA). The GIA test could be used for early diagnoses of PCa (AUC = 0.83; n = 559 samples) with a potential for use in therapy-monitoring (AUC = 0.90; n = 176 samples). Moreover, the analysis of a subset of serum samples (n = 215) revealed that the GIA test (AUC = 0.81) outperformed the PHI (Prostate Health Index) test (AUC = 0.69) in discriminating between men with prostate cancer and those with benign serum PSA elevation.

Aluminum exposure alters the pedal mucous secretions of the chocolate-band snail, Eobania vermiculata (Gastropoda: Helicidae).

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 μM of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. RESEARCH HIGHLIGHTS: Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

Identification and characterization of intact glycopeptides in human urine

Glycoproteins in urine have the potential to provide a rich class of informative molecules for studying human health and disease. Despite this promise, the urine glycoproteome has been largely uncharacterized. Here, we present the analysis of glycoproteins in human urine using LC–MS/MS-based intact glycopeptide analysis, providing both the identification of protein glycosites and characterization of the glycan composition at specific glycosites. Gene enrichment analysis reveals differences in biological processes, cellular components, and molecular functions in the urine glycoproteome versus the urine proteome, as well as differences based on the major glycan class observed on proteins. Meta-heterogeneity of glycosylation is examined on proteins to determine the variation in glycosylation across multiple sites of a given protein with specific examples of individual sites differing from the glycosylation trends in the overall protein. Taken together, this dataset represents a potentially valuable resource as a baseline characterization of glycoproteins in human urine for future urine glycoproteomics studies.

Coupling extracellular glycan composition with metagenomic data in papermill and brewery anaerobic granular sludges

Glycans are crucial for the structure and function of anaerobic granular sludge in wastewater treatment. Yet, there is limited knowledge regarding the microorganisms and biosynthesis pathways responsible for glycan production. In this study, we analysed samples from anaerobic granular sludges treating papermill and brewery wastewater, examining glycans composition and using metagenome-assembled genomes (MAGs) to explore potential biochemical pathways associated with their production. Uronic acids were the predominant constituents of the glycans in extracellular polymeric substances (EPS) produced by the anaerobic granular sludges, comprising up to 60 % of the total polysaccharide content. MAGs affiliated with Anaerolineacae, Methanobacteriaceae and Methanosaetaceae represented the majority of the microbial community (30–50 % of total reads per MAG). Based on the analysis of MAGs, it appears that Anaerolinea sp. and members of the Methanobacteria class are involved in the production of exopolysaccharides within the analysed granular sludges. These findings shed light on the functional roles of microorganisms in glycan production in industrial anaerobic wastewater treatment systems.

Characterization of milk oligosaccharide and sialic acid content and their influence on brain sialic acid in a lean mouse model for gestational diabetes

Oligosaccharides and sialic acids (Sia) are bioactive components in milk that contribute to newborn development and health. Hyperglycemia in pregnancy (HIP) can have adverse effects on both mother and infant. HIP is associated with low-grade systemic inflammation. Inflammation influenced glycan composition, particularly of Sia-containing structures. We hypothesize that HIP and high-fat diet influence milk oligosaccharide composition, particularly sialylated oligosaccharides. Furthermore, we propose that milk Sia content influences pup brain Sia content. To test these hypotheses we (i) characterize mouse milk oligosaccharides and Sia concentrations in mouse milk of a GDM mouse model with dietary fat intake intervention; and (ii) determine Sia levels in offspring brains.The concentrations of oligosaccharides and Sia in mouse milk and offspring's brains were quantified using UPLC-FLD analysis. Analyses were performed on surplus samples from a previous study, where HIP was induced by combining high-fat diet (HF) feeding and low-dose streptozotocin injections in C57Bl/6NTac female mice. The previous study described the metabolic effects of HIP on dams and offspring.We detected 21 mouse milk oligosaccharides, including 9 neutral and 12 acidic structures using UPLC-MS. A total of 8 structures could be quantified using UPLC-FLD. Maternal HIP and HF diet during lactation influenced sialylated oligosaccharide concentrations in mouse milk and total and free sialic acid concentrations. Sia content in offspring brain was associated with total and free Neu5Gc in mouse milk of dams, but no correlations with HIP or maternal diet were observed.

Functional redundancy revealed by the deletion of the mimivirus GMC-oxidoreductase genes.

The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion. In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase, the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then we deleted the GMC-oxidoreductase, the most abundant in the layer of fibrils, to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared with the wild-type viruses under laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Because the glycosylation machinery and glycan composition of members of different clades are different, we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family Mimiviridae, suggesting this may be the major evolutionary force behind their giant genomes.

Semisynthesis of homogeneous spike RBD glycoforms from SARS-CoV-2 for profiling the correlations between glycan composition and function

ABSTRACT Vaccines have been the primary remedy in the global fight against coronavirus disease 2019 (COVID-19). The receptor-binding domain (RBD) of the spike protein, a critical viral immunogen, is affected by the heterogeneity of its glycan structures and relatively low immunogenicity. Here, we describe a scalable synthetic platform that enables the precise synthesis of homogeneously glycosylated RBD, facilitating the elucidation of carbohydrate structure–function relationships. Five homogeneously glycosylated RBDs bearing biantennary glycans were prepared, three of which were conjugated to T-helper epitope (Tpep) from tetanus toxoid to improve their weak immune response. Relative to natural HEK293-derived RBD, synthetic RBDs with biantennary N-glycan elicited a higher level of neutralising antibodies against SARS-CoV-2 in mice. Furthermore, RBDs containing Tpep elicited significant immune responses in transgenic mice expressing human angiotensin-converting enzyme 2. Our collective data suggest that trimming the N-glycans and Tpep conjugation of RBD could potentially serve as an effective strategy for developing subunit vaccines providing efficient protection.

Taxonomic and functional stability overrules seasonality in polar benthic microbiomes.

Coastal shelf sediments are hot spots of organic matter mineralization. They receive up to 50% of primary production, which, in higher latitudes, is strongly seasonal. Polar and temperate benthic bacterial communities, however, show a stable composition based on comparative 16S rRNA gene sequencing despite different microbial activity levels. Here, we aimed to resolve this contradiction by identifying seasonal changes at the functional level, in particular with respect to algal polysaccharide degradation genes, by combining metagenomics, metatranscriptomics, and glycan analysis in sandy surface sediments from Isfjorden, Svalbard. Gene expressions of diverse carbohydrate-active enzymes changed between winter and spring. For example, β-1,3-glucosidases (e.g. GH30, GH17, GH16) degrading laminarin, an energy storage molecule of algae, were elevated in spring, while enzymes related to α-glucan degradation were expressed in both seasons with maxima in winter (e.g. GH63, GH13_18, and GH15). Also, the expression of GH23 involved in peptidoglycan degradation was prevalent, which is in line with recycling of bacterial biomass. Sugar extractions from bulk sediments were low in concentrations during winter but higher in spring samples, with glucose constituting the largest fraction of measured monosaccharides (84% ± 14%). In porewater, glycan concentrations were ~18-fold higher than in overlying seawater (1107 ± 484 vs. 62 ± 101μg C l-1) and were depleted in glucose. Our data indicate that microbial communities in sandy sediments digest and transform labile parts of photosynthesis-derived particulate organic matter and likely release more stable, glucose-depleted residual glycans of unknown structures, quantities, and residence times into the ocean, thus modulating the glycan composition of marine coastal waters.

The glycosylation landscape of prostate cancer tissues and biofluids.

An overview of the role of glycosylation in prostate cancer (PCa) development and progression is presented, focusing on recent advancements in defining the N-glycome through glycomic profiling and glycoproteomic methodologies. Glycosylation is a common post-translational modification typified by oligosaccharides attached N-linked to asparagine or O-linked to serine or threonine on carrier proteins. These attached sugars have crucial roles in protein folding and cellular recognition processes, such that altered glycosylation is a hallmark of cancer pathogenesis and progression. In the past decade, advancements in N-glycan profiling workflows using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) technology have been applied to define the spatial distribution of glycans in PCa tissues. Multiple studies applying N-glycan MALDI-MSI to pathology-defined PCa tissues have identified significant alterations in N-glycan profiles associated with PCa progression. N-glycan compositions progressively increase in number, and structural complexity due to increased fucosylation and sialylation. Additionally, significant progress has been made in defining the glycan and glycopeptide compositions of prostatic-derived glycoproteins like prostate-specific antigen in tissues and biofluids. The glycosyltransferases involved in these changes are potential drug targets for PCa, and new approaches in this area are summarized. These advancements will be discussed in the context of the further development of clinical diagnostics and therapeutics targeting glycans and glycoproteins associated with PCa progression. Integration of large scale spatial glycomic data for PCa with other spatial-omic methodologies is now feasible at the tissue and single-cell levels.

Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions.

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.

Specificity determinants revealed by the structure of glycosyltransferase Campylobacter concisus PglA.

In selected Campylobacter species, the biosynthesis of N-linked glycoconjugates via the pgl pathway is essential for pathogenicity and survival. However, most of the membrane-associated GT-B fold glycosyltransferases responsible for diversifying glycans in this pathway have not been structurally characterized which hinders the understanding of the structural factors that govern substrate specificity and prediction of resulting glycan composition. Herein, we report the 1.8 Å resolution structure of Campylobacter concisus PglA, the glycosyltransferase responsible for the transfer of N-acetylgalatosamine (GalNAc) from uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) to undecaprenyl-diphospho-N,N'-diacetylbacillosamine (UndPP-diNAcBac) in complex with the sugar donor GalNAc. This study identifies distinguishing characteristics that set PglA apart within the GT4 enzyme family. Computational docking of the structure in the membrane in comparison to homologs points to differences in interactions with the membrane-embedded acceptor and the structural analysis of the complex together with bioinformatics and site-directed mutagenesis identifies donor sugar binding motifs. Notably, E113, conserved solely among PglA enzymes, forms a hydrogen bond with the GalNAc C6″-OH. Mutagenesis of E113 reveals activity consistent with this role in substrate binding, rather than stabilization of the oxocarbenium ion transition state, a function sometimes ascribed to the corresponding residue in GT4 homologs. The bioinformatic analyses reveal a substrate-specificity motif, showing that Pro281 in a substrate binding loop of PglA directs configurational preference for GalNAc over GlcNAc. This proline is replaced by a conformationally flexible glycine, even in distant homologs, which favor substrates with the same stereochemistry at C4, such as glucose. The signature loop is conserved across all Campylobacter PglA enzymes, emphasizing its importance in substrate specificity.

Novel Selective Quantification of Zinpentraxin Alfa Biotherapeutic in the Presence of Endogenous Isomer in Plasma Samples of Idiopathic Pulmonary Fibrosis Patients Using Immunoaffinity LC–MS

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal interstitial lung disease that affects three million patients worldwide and currently without an effective cure. Zinpentraxin alfa, a recombinant human pentraxin-2 (rhPTX-2) protein, has been evaluated as a potential drug candidate for the treatment of IPF. Clinical pharmacokinetic analysis of zinpentraxin alfa has been challenging historically due to interference from serum amyloid P component (SAP), an endogenous human pentraxin-2 protein. These molecules share an identical primary amino acid sequence and glycan composition; however, zinpentraxin alfa possesses α2,3-linked terminal sialic acid residues while SAP is an α2,6-linked isomer. By taking advantage of this only structural difference, we developed a novel assay strategy where α2,3-sialidase was used to selectively hydrolyze α2,3-linked sialic acid residues, resulting in desialylated zinpentraxin alfa versus unchanged sialylated SAP, following an immunoaffinity capture step. Subsequent tryptic digestion produced a unique surrogate asialo-glycopeptide from zinpentraxin alfa and allowed specific quantification of the biotherapeutic in human plasma. In addition, a common peptide shared by both molecules was selected as a surrogate to determine total hPTX-2 concentrations, i.e., sum of zinpentraxin alfa and SAP. The quantification methods for both zinpentraxin alfa and total hPTX-2 were validated and used in pharmacokinetic assessment in IPF patients. The preliminary results suggest that endogenous SAP levels remained largely constant in IPF patients throughout the treatment with zinpentraxin alfa. Our novel approach provides a general bioanalytical strategy to selectively quantify α2,3-sialylated glycoproteins in the presence of their corresponding α2,6-linked isomers.Graphical

Monitoring the Secretion and Activity of Alpha-1 Antitrypsin in Various Mammalian Cell Types.

Overexpression of recombinant protein in mammalian cells is widely used for producing biologics, as protein maturation and post-translational modifications are similar to human cells. Some therapeutics, such as mRNA vaccines, target nonnative cells that may contain inefficient secretory machinery. For example, gene replacement therapies for alpha-1 antitrypsin (AAT), a glycoprotein normally produced in hepatocytes, are often targeted to muscle cells due to ease of delivery. In this chapter, we define methods for expressing AAT in representative cell types such as Huh-7; hepatocytes; Chinese hamster ovarian cells (CHO), a common host to produce biologics; and C2C12, a muscle progenitor cell line. Methods for metabolically labeling AAT to monitor secretion in these cell lines are described along with the use of proteostasis activators to increase the amount of AAT secreted in both C2C12 myoblasts and differentiated myotubes. Assays to assess the activity and glycan composition of overexpressed AAT are also presented. The usage of the proteostasis activator SAHA provided a 40% improvement in expression of active AAT in muscle-like cells and may be an advantageous adjuvant for recombinant production of proteins delivered by mRNA vaccines.

Site- and Structure-Specific Glycosylation Signatures of Bovine, Caprine, Porcine, and Human Milk-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-bound vesicles released by living cells. As vesicles for macromolecule transmission and intercellular communication, EVs are broadly applied in clinical diagnosis and biomimetic drug delivery. Milk-derived EVs (MEVs) are an ideal choice for scale-up applications because they exhibit biocompatibility and are easily obtained. Herein, intact glycopeptides in MEVs from bovines, caprines, porcines, and humans were comprehensively analyzed by high-resolution mass spectrometry using the sceHCD, followed by the EThcD fragment method, revealing that protein glycosylation is abundant and heterogeneous in MEVs. The dominant glycans in all MEVs were sialic acid-modified N-linked glycans (over 50%). A couple of species-specific glycans were also characterized, which are potentially markers of different original EVs. Interestingly, the Neu5Gc-modified glycans were enriched in caprine milk-derived EVs (58 ± 2%). Heterogeneity of MEV protein glycosylation was observed for glycosites and glycan compositions, and the structural heterogeneity of protein glycosylation was also identified and validated. The glycosignatures of EV biogenesis- and endocytosis-related proteins (CD63 and MFGE8) were significantly different in these four species. Overall, we comprehensively characterized the glycosylation signature of MEVs from four different species and provided insight into protein glycosylation related to drug target delivery.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation.

N-linked glycosylation is one of the most important post-translational modifications of monoclonal antibodies (mAbs) and is considered to be a critical quality attribute (CQA), as the glycan composition often has immunomodulatory effects. Since terminal galactose residues of mAbs can affect antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC) activation, serum half-life, and antiviral activity it has to be monitored, controlled and modulated to ensure therapeutic effects. The ability of small noncoding microRNAs (miRNAs) to modulate glycosylation in Chinese hamster ovary (CHO) production cells was recently reported establishing miRNAs as engineering tools for modulation of protein glycosylation. In this study, we report the characterization and validation of miRNAs as engineering tools for increased (mmu-miR-452-5p, mmu-miR-193b-3p) or decreased (mmu-miR-7646-5p, mmu-miR-7243-3p, mmu-miR-1668, mmu-let-7c-1-3p, mmu-miR-7665-3p, mmu-miR-6403) degree of galactosylation. Furthermore, the biological mode of action regulating gene expression of the galactosylation pathway was characterized as well as their influence on bioprocess-related parameters. Most important, stable plasmid-based overexpression of these miRNAs represents a versatile tool for engineering N-linked galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production.

Comparative Analysis of Human Milk Glycosphingolipids from Different Secretor Mothers Using HILIC-MS/MS.

Glycosphingolipids participate in brain development, intestinal tract maturation, and defense against gut pathogens. Here, we performed a qualitative and quantitative comparison of milk glycosphingolipids from secretors and nonsecretors. Hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry was employed, along with an internal standard, to resolve the complications presented by the fact that glycosphingolipids are structurally diverse, varying in glycan composition and ceramide. In total, 101 glycosphingolipids were detected, of which 76 were reported for the first time, including fucose-modified neutral glycosphingolipids. Seventy-eight glycosphingolipids differed significantly between secretor and nonsecretor milk (p < 0.05), resulting in higher levels of certain neutral species (p < 0.001) but lower levels of fucose-modified monosialylated and disialylated species in secretor mothers (p < 0.01). In both milk types, the most abundant glycosphingolipids were of the monosialylated type, followed by disialylated, neutral, and trisialylated ones. Notably, fucose-modified monosialylated glycosphingolipids accounted for the highest proportion.