Application of compositional models for glycan HILIC data

Glycoconjugates constitute a major class of biomolecules which include glycoproteins, glycosphingolipids and proteoglycans. The enzymatic process in which glycans (sugar chains) are linked to proteins or lipids is called glycosylation. Glycosylation is involved in many biological processes, both physiological and pathological, inlcuding host-pathogen interactions, tumour invasion, cell trafficking and signalling. Changes in glycan structure are thought be be at least partly responsible for the development of inflammation, infection, arteriosclerosis, immune defects and autoimmunity. Such changes have been observed in human diseases such as diabetes mellitus, rheumatoid arthritis and Alzheimer’s Disease. Aberrant patterns of glycosylation are also a universal feature of cancer cells. The field of glycobiology thus shows great potential for the discovery of glycan biomarkers for disease diagnosis and prognosis. Here we focus specifically on N-glycans, that is, glycans attached to protein molecules via a nitrogen atom. This class of glycans is the best characterized. High-throughput HILIC analysis is a well-established technique for the separation and quantification of N-linked glycans released from glycoproteins. HILIC analysis quantifies theN-glycan structures in serum via a chromatogram, which is subsequently standardized and integrated. The generated data for each sample is a set of relative HILIC peak areas and as a result, the data is compositional. To-date, most statistical analyses of these glycan data fail to account for their compositional nature. We compare and contrast three compositional data models for the glycan HILIC data: the Dirichlet, Nested Dirichlet and Logistic Normal models, with the intention of providing tools for the statistical analysis of compositional data analysis in the glycobiology field. We use these three models for classification of disease/control cases in ovarian and lung cancer diagnosis applications. We discuss and compare these models in terms of their classification performance and goodness-of-fit

The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody

Microcarriers are widely used for the large-scale culture of attachment-dependent cells with increased cell densities and, ultimately, higher product yield. In these processes, the specific culture conditions can affect the quality of the product, which is closely related to its glycosylation pattern. Furthermore, the lack of studies in the area reinforces the need to better understand the effects of microcarrier culture in product glycosylation. Consequently, in this work, the glycosylation profile of a monoclonal antibody (mAb) produced by adherent CHO-K1 cells grown in Cytodex 3 was evaluated under different conditions, and compared to that obtained of typical adherent cultures. It was found that microcarrier cultures result in a glycosylation profile with different characteristics from T-flask cultures, with a general increase in galactosylation and decrease in fucosylation levels, both with a potentially positive impact on mAb activity. Sialylation also varied but without a general tendency. This study then showed that the specific culture conditions used in microcarrier culture influence the mAb glycan profile, and each functional element (galactose, core fucose, sialic acid) is independently affected by these conditions. In particular, great reductions of fucosylation (from 79 to 55%) were obtained when using half volume at inoculation, and notable decreases in sialylation (from 23 to 2%) and glycoform heterogeneity (from 20 to 11 glycoforms) were observed for shake flask culture, potentially associated with the improved cell densities achieved in these culture vessels.Fundação para a Ciência e a Tecnologia (FCT

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

The IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)–UPLC, reversed phase (RP)–UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC–UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques

Region-Specific Characterization of N-Glycans in the Striatum and Substantia Nigra of an Adult Rodent Brain

N-glycan alterations in the nervous system can result in different neuropathological symptoms such as mental retardation, seizures, and epilepsy. Studies have reported the characterization of N-glycans in rodent brains, but there is a lack of spatial resolution as either the tissue samples were homogenized or specific proteins were selected for analysis of glycosylation. We hypothesize that region-specific resolution of N-glycans isolated from the striatum and substantia nigra (SN) can give an insight into the establishment and pathophysiological degeneration of neural circuitry in Parkinson’s disease. Specific objectives of the study include isolation of N-glycans from the rat striatum and SN; reproducibility, resolution, and relative quantitation of N-glycome using ultra-performance liquid chromatography (UPLC), weak anion exchange-UPLC, and lectin histochemistry. The total N-glycomes from the striatum and SN were characterized using database mining (GlycoStore), exoglycosidase digestions, and liquid chromatography-mass spectrometry. It revealed significant differences in complex and oligomannose type N-glycans, sialylation (mono-, di-, and tetra-), fucosylation (tri-, core, and outer arm), and galactosylation (di-, tri-, and tetra-) between striatum and SN N-glycans with the detection of phosphorylated N-glycans in SN which were not detected in the striatum. This study presents the most comprehensive comparative analysis of relative abundances of N-glycans in the striatum and SN of rodent brains, serving as a foundation for identifying “brain-type” glycans as biomarkers or therapeutic targets and their modulation in neurodegenerative disorders

A comparative study of free oligosaccharides in the milk of domestic animals

This study was conducted to provide a comprehensive analysis of the oligosaccharides in the milk of a variety of important domestic animals including cow, goat, sheep, pig, horse, and dromedary camel. Using an analytical workflow which included ultra-performance hydrophilic interaction liquid chromatography with fluorescence detection (UPLC-HILIC-FLD) and coupling to a quadrupole time of flight (qTOF) mass spectrometer (MS), detailed oligosaccharide libraries were established. The partial or full characterization of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencing with linkage- and sugar- specific exoglycosidases. Relative peak quantification of the 2-AB labelled oligosaccharides provided additional information. Milks from domestic animals contained a much larger variety of complex oligosaccharides than was previously assumed and thirteen of these structures were previously identified in human milk. The direct comparison of the oligosaccharide mixtures could contribute to a better understanding of possible differences in their biological effects and highlight the potential value of animal milks for commercial oligosaccharide extraction.Fil: Albrecht, Simone. National Institute for Bioprocessing, Research and Training. NIBRT GlycoScience Group; IrlandaFil: Lane, Jonathan A.. Teagasc Food Research Centre; IrlandaFil: Mariño, Karina Valeria. National Institute for Bioprocessing, Research and Training. NIBRT GlycoScience Group; Irlanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Al Busadah, Khalid A.. King Faisal University. Camel Research Center; Arabia SauditaFil: Carrington, Stephen D.. University College Dublin. Veterinary Sciences Centre; IrlandaFil: Hickey, Rita M.. Teagasc Food Research Centre; IrlandaFil: Rudd, Pauline M.. National Institute for Bioprocessing, Research and Training. NIBRT GlycoScience Group; Irland

UniCarb-DB: a database resource for glycomic discovery

Summary: Glycosylation is one of the most important post-translational modifications of proteins, known to be involved in pathogen recognition, innate immune response and protection of epithelial membranes. However, when compared to the tools and databases available for the processing of high-throughput proteomic data, the glycomic domain is severely lacking. While tools to assist the analysis of mass spectrometry (MS) and HPLC are continuously improving, there are few resources available to support liquid chromatography (LC)-MS/MS techniques for glycan structure profiling. Here, we present a platform for presenting oligosaccharide structures and fragment data characterized by LC-MS/MS strategies. The database is annotated with high-quality datasets and is designed to extend and reinforce those standards and ontologies developed by existing glycomics databases. Availability: http://www.unicarb-db.org Contact: [email protected]

Genome-wide association study identifies _FUT8_ and _ESR2_ as co-regulators of a bi-antennary N-linked glycan A2 (GlcNAc~2~Man~3~GlcNAc~2~) in human plasma proteins

HPLC analysis of N-glycans quantified levels of the biantennary glycan (A2) in plasma proteins of 924 individuals. Subsequent genome-wide association study (GWAS) using 317,503 single nucleotide polymorphysms (SNP) identified two genetic loci influencing variation in A2: FUT 8 and ESR2. We demonstrate that human glycans are amenable to GWAS and their genetic regulation shows sex-specific effects with _FUT 8_ variants explaining 17.3% of the variance in pre-menopausal women, while _ESR2_ variants explained 6.0% of the variance in post-menopausal women

The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins

Introduction: Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins. Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context. Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no ‘gold-standard’ approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein

GlycoDigest: a tool for the targeted use of exoglycosidase digestions in glycan structure determination

Summary: Sequencing oligosaccharides by exoglycosidases, either sequentially or in an array format, is a powerful tool to unambiguously determine the structure of complex N- and O-link glycans. Here, we introduce GlycoDigest, a tool that simulates exoglycosidase digestion, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. The tool allows the targeted design of glycosidase enzyme mixtures by allowing researchers to model the action of exoglycosidases, thereby validating and improving the efficiency and accuracy of glycan analysis. Availability and implementation: http://www.glycodigest.org. Contact: [email protected] or [email protected]