Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose ß-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose ß-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product.

Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Cross-platform comparison of glycan microarray formats.

Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell-cell interactions. To date, the majority of data gathered on glycan expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.

Divergent mechanisms of interaction of Helicobacter pylori and Campylobacter jejuni with mucus and mucins.

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.

Construction of a natural mucin microarray and interrogation for biologically relevant glyco-epitopes.

Mucins are the principal components of mucus, and mucin glycosylation has important roles in defense, microbial adhesion, immunomodulation, inflammation, and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogeneous glycans can substitute one mucin molecule, and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken, and deer) and from two human-derived cell lines, were purified. Following optimization of mucin printing and construction of a novel mucin microarray, the glycoprofiles of the whole mucins on the microarray were compared using a panel of lectins and one antibody. Accessible glyco-motifs of GI mucins varied according to species and localization of mucin origin, with terminal fucose, the sialyl T-antigen, and N-linked oligosaccharides identified as potentially important. The occurrence of T- and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal N-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial-host interactions and fertility.

Neoglycoprotein and Glycoprotein Printing on a Hydrogel Functionalized Microarray Surface and Incubation with Labeled Lectins.

Glycan microarrays are widely used to elucidate carbohydrate binding specificity and affinity of various analytes including proteins, microorganisms, cells, and tissues. Glycan microarrays comprise a wide variety of platforms, differing in surface chemistry, presentation of carbohydrates, carbohydrate valency, and detection strategies, all of which impact on analyte performance. This chapter describes detailed methods for printing neoglycoprotein and glycoprotein microarrays on hydrogel-coated slides and incubation of these glycan microarrays with fluorescently labeled lectins.

Preparation and Fluorescent Labeling of Cell-Derived Micelles and Profiling on Glycan Microarrays.

Mammalian cell surface lectins mediate many important biological interactions which regulate physiological processes and therefore profiling mammalian cells on glycan microarray is of interest. However, many whole mammalian cells are not compatible with glycomics microarray formats and instead cell-derived micelles are prepared and profiled instead of whole cells as they can accurately represent the parental cell glycome. In this chapter, we describe the preparation of cell-derived micelles from mammalian cells, their labeling using a membrane-incorporating dye, and their profiling on a glycan microarray platform.

Bacterial Staining and Profiling for Glycan Interactions on Glycan Microarrays for t-Test Calculation.

The use of glycan microarrays to study carbohydrate interactions of bacterial cells is of great interest owing to the key roles these interactions play in bacterial colonization and infection of a host. In this chapter, the methods to fluorescently stain Gram-positive or Gram-negative bacteria and profiling them for glycan interactions using glycan microarrays are described in detail. The application of the Student's t-test to glycan microarray data using an example data set comparing glycan microarray binding of an Acinetobacter baumannii wild type and mutant strain is also described in step-by-step detail.

Sugar Coating: Utilisation of Host Serum Sialoglycoproteins by Schistosoma mansoni as a Potential Immune Evasion Mechanism.

Parasitic helminths resort to various mechanisms to evade and modulate their host's immune response, several of which have been described for Schistosoma mansoni. We recently reported the presence of sialic acid residues on the surface of adult S. mansoni extracellular vesicles (EVs). We now report that these sialylated molecules are mammalian serum proteins. In addition, our data suggest that most sialylated EV-associated proteins do not elicit a humoral response upon injection into mice, or in sera obtained from infected animals. Sialic acids frequently terminate glycans on the surface of vertebrate cells, where they serve important functions in physiological processes such as cell adhesion and signalling. Interestingly, several pathogens have evolved ways to mimic or utilise host sialic acid beneficially by coating their own proteins, thereby facilitating cell invasion and providing protection from host immune effectors. Together, our results indicate that S. mansoni EVs are coated with host glycoproteins, which may contribute to immune evasion by masking antigenic sites, protecting EVs from removal from serum and aiding in cell adhesion and entry to exert their functions.

Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism.

Half maximal inhibitory concentration (IC50) is a measurement often used to compare the efficiency of various carbohydrates and their derivatives for inhibition of lectin binding to particular ligands. IC50 values can be calculated using experimental data from various platforms including enzyme-linked immunosorbent assay- (ELISA-)type microtiter plate assays, isothermal titration calorimetry (ITC), or glycan microarrays. In this chapter, we describe methods to fluorescently label a lectin, to carry out a lectin binding inhibition experiment on glycan microarrays, and to calculate the IC50 value of a binding inhibitory molecule using GraphPad Prism software. In the example used to illustrate the method in this chapter, IC50 calculation is demonstrated for inhibition of Maackia amurensis agglutinin (MAA) binding to 3'sialyl-N-acetyllactosamine (3SLN) using free lactose.

Mucin Purification and Printing Natural Mucin Microarrays.

Mucin glycosylation is the key facilitator of microbial attachment and nutrition and it varies according to biological location, health and disease status, microbiome composition, infection, and multiple other factors. Mucin glycans have also been reported to attenuate pathogen virulence and mediate biofilm dispersal. With the labor intensive and time-consuming purification required for natural mucins and their low quantitative yield from biological sources, natural mucin microarrays provide a convenient and multiplexed platform to study mucin glycosylation and interactions. In this chapter we describe the purification of natural mucins, using sputum as an example biological source, and the printing of natural mucin microarrays.

Comparison of Single and Repeated Dosing of Anti-Inflammatory Human Umbilical Cord Mesenchymal Stromal Cells in a Mouse Model of Polymicrobial Sepsis.

Mesenchymal stromal cells (MSCs) ameliorate pre-clinical sepsis and sepsis-associated acute kidney injury (SA-AKI) but clinical trials of single-dose MSCs have not indicated robust efficacy. This study investigated immunomodulatory effects of a novel MSC product (CD362-selected human umbilical cord-derived MSCs [hUC-MSCs]) in mouse endotoxemia and polymicrobial sepsis models. Initially, mice received intra-peritoneal (i.p.) lipopolysaccharide (LPS) followed by single i.p. doses of hUC-MSCs or vehicle. Next, mice underwent cecal ligation and puncture (CLP) followed by intravenous (i.v.) doses of hUC-MSCs at 4 h or 4 and 28 h. Analyses included serum/plasma assays of biochemical indices, inflammatory mediators and the AKI biomarker NGAL; multi-color flow cytometry of peritoneal macrophages (LPS) and intra-renal immune cell subpopulations (CLP) and histology/immunohistochemistry of kidney (CLP). At 72 h post-LPS injections, hUC-MSCs reduced serum inflammatory mediators and peritoneal macrophage M1/M2 ratio. Repeated, but not single, hUC-MSC doses administered at 48 h post-CLP resulted in lower serum concentrations of inflammatory mediators, lower plasma NGAL and reversal of sepsis-associated depletion of intra-renal T cell and myeloid cell subpopulations. Hierarchical clustering analysis of all 48-h serum/plasma analytes demonstrated partial co-clustering of repeated-dose hUC-MSC CLP animals with a Sham group but did not reveal a distinct signature of response to therapy. It was concluded that repeated doses of CD362-selected hUC-MSCs are required to modulate systemic and local immune/inflammatory events in polymicrobial sepsis and SA-AKI. Inter-individual variability and lack of effect of single dose MSC administration in the CLP model are consistent with observations to date from early-phase clinical trials.

Periodic acid-Schiff's reagent assay for carbohydrates in a microtiter plate format.

Microtiter plate colorimetric assays are widely used for analysis of carbohydrates and glycoconjugates. However, mucins are often not easily detected, as they have low neutral sugar content. We have adapted and optimised the periodic acid-Schiff's reagent (PAS) staining for microtiter plate assay by examining five factors: concentration and volume of periodic acid, oxidation time, volume of Schiff's reagent, and color development time. This assay requires just 25 µl of sample, utilises standardised Schiff's reagent, and has decreased assay time (140 min to completion). Seventeen monosaccharides (acidic, neutral, basic, phosphorylated, and deoxy) and four disaccharides were assessed. PAS-positive carbohydrates (amino, N-acetylamino, deoxy, and certain neutral monosaccharides, and sialic acids) responded linearly within a 10-100 nmol range approximately, which varied for each carbohydrate. The assay response for fetuin and porcine gastric mucin (PGM) was linear up to 150 µg (highest concentration tested), with no response from nonglycosylated protein. A lower response for asialofetuin was observed, but desialylated PGM preparations were similar or higher in response than their sialylated counterparts. The simplicity and low sample consumption of this method make it an excellent choice for screening or quantitation of chromatographic fractions containing carbohydrates and glycoconjugates, especially in the case of mucins.

Analysis of Schistosoma mansoni Extracellular Vesicles Surface Glycans Reveals Potential Immune Evasion Mechanism and New Insights on Their Origins of Biogenesis.

Parasitic helminths are master manipulators of host immunity. Their strategy is complex and involves the release of excreted/secreted products, including extracellular vesicles (EVs). The protein and miRNA contents of EVs have been characterised for many parasitic helminths but, despite reports suggesting the importance of EV surface carbohydrate structures (glycans) in the interactions with target cells and thus subsequent effector functions, little is known about parasite EV glycomics. Using lectin microarrays, we identified several lectins that exhibit strong adhesion to Schistosoma mansoni EVs, suggesting the presence of multiple glycan structures on these vesicles. Interestingly, SNA-I, a lectin that recognises structures with terminal sialic acid, displayed strong affinity for S. mansoni EVs, which was completely abolished by neuraminidase treatment, suggesting sialylation in the EV sample. This finding is of interest, as sialic acids play important roles in the context of infection by aiding immune evasion, affecting target recognition, cell entry, etc., but are not thought to be synthesised by helminths. These data were validated by quantitative analysis of free sialic acid released from EVs following treatment with neuraminidase. Lectin histochemistry and fluorescence in situ hybridisation analyses on whole adult worms suggest the involvement of sub-tegumental cell bodies, as well as the digestive and excretory systems, in the release of EVs. These results support previous reports of EV biogenesis diversity in trematodes and potentially highlight new means of immune modulation and evasion employed by schistosomes.

Correction: Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators.

Correction for 'Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators' by Marie Le Berre et al., Mol. Omics, 2021, 17, 338-346, DOI: 10.1039/D0MO00193G.

Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators.

Oestrus is the period in the sexual cycle of female mammals where they become most receptive to mating and are most fertile. Efficient detection of oestrus is a key component in successful reproductive livestock management programmes. Oestrus detection in cattle is most often performed by visual observation, such as mounting behaviour and standing heat, to facilitate more successful prediction of optimal time points for artificial insemination. This time-consuming method requires a skilled, diligent observer. Biological measurements using easily accessible biomolecules in the cervico-vaginal mucus could provide an alternative strategy to physical methods of oestrus detection, providing an inexpensive means of rapidly and accurately assessing the onset of oestrus. In this study, glycosylation changes in cervico-vaginal mucus from three heifers following oestrus induction were investigated as a proof of concept to assess whether potential glycosylation-based trends could be useful for oestrus stage indication. Mucus collected at different time points following oestrus induction was immobilised in a microarray format and its glycosylation interrogated with a panel of fluorescently labelled lectins, carbohydrate-binding proteins with different specificities. Individual animal-specific glycosylation patterns were observed, however each pattern followed a similar trend around oestrus. This unique oestrus-associated glycosylation was identified by a combination of relative binding of the lectins SNA-I and WFA for each animal. This alteration in cervico-vaginal mucus glycosylation could potentially be exploited in future to more accurately identify optimal fertilisation intervention points compared to visual signs. More effective oestrus biomarkers will lead to more successful livestock reproductive programmes, decreasing costs and animal stress.

Glycobiomimics and glycobiosensors.

Following steady advances in analytical technologies, our knowledge in glycomics is now increasing rapidly. Over the last decade, specific glycans have been described that are associated with a range of diseases, such as cancer and inflammation, with host-pathogen interactions and with various stages during stem cell development and differentiation. Simultaneously, deeper structural insight has been gained on glycosylated biopharmaceutical protein therapeutics manufactured in CHO (Chinese-hamster ovary) and other cell systems. This glycomic information is highly relevant for clinicians and biomanufacturing industries as a new class of glycobiomarkers emerges. However, current methods of glycoanalysis are primarily research tools and are not suitable for point-of-care on-site detection and analysis, or sensor devices. Lectin-based glycan detection provides the most promising approach to fill these gaps. However, the limited availability of lectins with high specificity and sensitivity for specific glycan motifs presents one of the main challenges in building reliable glycobiosensors. Recent reports have demonstrated the use of recombinant protein engineering, phage display and aptamer technologies in the production of lectin mimics, as well as the construction of biosensors that are capable of rapidly detecting glycan motifs at low levels in both a labelled and label-free manner. These are primarily proof-of-principle reports at this stage, but some of the approaches, either alone or in combination, will lead to functional glycobiosensors in the coming years which will be valuable tools for the clinical, biopharmaceutical and life science research communities.

Glyco-biosensors: recent advances and applications for the detection of free and bound carbohydrates.

The field of biosensor development now encompasses several areas specifically geared toward the rapid and sensitive detection, identification, and quantification of target analytes. In contrast to the more mature research and development of nucleic acid and protein biosensors, the development of 'glyco-biosensors' for detecting carbohydrates and conjugates of carbohydrates (glycoconjugates) is at a relatively nascent stage. The application of glyco-biosensors aims to open novel analytical and diagnostic avenues, encompassing industrial bioprocesses, biomedical and clinical applications. This area of research has been greatly aided by advancement brought by interdisciplinary mergers of engineering, biology, chemistry and physical sciences and enabling the miniaturization of detection platforms. In this review, we briefly introduce the need for glyco-biosensors, discuss current analytical technologies, and examine advances in glyco-biosensor approaches aimed at the detection and/or quantification of glycoconjugates or carbohydrates derived from glycoconjugates since 2005.

Lectin microarray profiling and monosaccharide analysis of bovine milk immunoglobulin G oligosaccharides during the first 10 days of lactation.

Immunoglobulin G (IgG) in bovine milk is credited with ensuring efficient passive immunity for newborn calves. Bovine milk IgG glycosylation may also have positive impacts on the health of nonbovine consumers of cow's milk. Milk IgG's glycosylation contributes to effector function and may also protect it from protease digestion, allowing IgG to reach the intestine for absorption. However, relatively little is known about changes in milk IgG oligosaccharide presentation and composition over early lactation. In this work, IgG was isolated from milk pooled from three cows at four time points over the first 10 days of lactation postparturition. Purified IgG was labeled with a fluorescent dye and interrogated with a microarray consisting of 48 carbohydrate-binding proteins (lectins) from plant, fungal, and bacterial sources. Lectin microarray profiles suggested that only subtle changes in the glycosylation of IgG occurred during days 2 and 3 of lactation, but by day 10, the lectin profile diverged from the other three time points. Monosaccharide analysis carried out after hydrolysis confirmed that the ratios of oligosaccharide components remained relatively stable through day 3 and also that sialylation was substantially reduced by day 10. The differences that were observed for glycosylation suggest that different functionalities associated with IgG glycosylation may be required in the first days of life.

Advanced glycation end products as biomarkers in systemic diseases: premises and perspectives of salivary advanced glycation end products.

Advanced glycation end products (AGEs) are glycated proteins associated with high dry temperature food processing, coloring and flavor modification of food products. Previous studies on diet-related disease support the role of the glycation products as biomarkers in local and general proinflammatory response. Exogenous and endogenous AGEs are involved in chronic low-level inflammation, which underlies the onset of metabolic syndrome influenced by food intake, there by demonstrating their implication in diet-related pathologies. Although studies have revealed a strong association between the accumulation of AGEs and the occurrence/worsening of metabolic diseases, their routine use for the diagnosis or monitoring of local and general disease has not yet been reported.