Erythropoietin -glycosylation of Therapeutic Formulations Quantified and Characterized: An Interlab Comparability Study of High-Throughput Methods.

Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO -glycosylation influences important pharmacological parameters, prominently serum half-life.

Elevated concentrations of Neu5Ac and Neu5,9Ac in human plasma: potential biomarkers of cardiovascular disease.

Cardiovascular disease (CVD) is a group of health conditions affecting the heart and vascular system with very high prevalence and mortality rates. The presence of CVD is characterised by high levels of inflammation which have previously been associated with increased plasma concentrations of N-acetyl neuraminic acid (Neu5Ac). While Neu5Ac has been studied in the context of CVD, Neu5,9Ac has not, despite being the second most abundant sialic acid in human plasma.

Serum N-Glycomic Biomarkers Predict Treatment Escalation in Inflammatory Bowel Disease.

Biomarkers to guide clinical decision making at diagnosis of inflammatory bowel disease [IBD] are urgently needed. We investigated a composite serum N-glycomic biomarker to predict future disease course in a discovery cohort of 244 newly diagnosed IBD patients. In all, 47 individual glycan peaks were analysed using ultra-high performance liquid chromatography, identifying 105 glycoforms from which 24 derived glycan traits were calculated.

Generation and Characterization of Native and Sialic Acid-Deficient IgE.

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC).

Sialic Acid Derivatization of Fluorescently Labeled -Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography-Fluorescence Detection-Mass Spectrometry.

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS.

Nanoparticle Biomolecular Corona-Based Enrichment of Plasma Glycoproteins for N-Glycan Profiling and Application in Biomarker Discovery.

Biomolecular corona formation has emerged as a recurring and important phenomenon in nanomedicine that has been investigated for potential applications in disease diagnosis. In this study, we have combined the "personalized protein corona" with the N-glycosylation profiling that has recently gained considerable interest in human plasma biomarker discovery as a powerful early warning diagnostic and patient stratification tool. We envisioned that the protein corona formation could be exploited as an enrichment step that is critically important in both proteomic and proteoglycomic workflows.

Development of Polyamine Lassos as Polyamine Transport Inhibitors.

Nine- and twelve-membered triaza-macrocycles were appended to one end of homospermidine to make polyamine lassos. These compounds were shown to be potent polyamine transport inhibitors (PTIs) using pancreatic ductal adenocarcinoma L3.6pl cells, which have high polyamine transport activity.

Probing the glycans accessibility in the nanoparticle biomolecular corona.

Hypothesis: Following blood administration, the pristine surface of nanoparticles (NPs) associates with biomolecules from the surrounding environment forming the so-called "biomolecular corona". It is well accepted that the biomolecular corona dramatically affects the NP fate in the biological medium while the pristine surface is no longer available for binding. Recent studies have shown that the glycans associated with the proteins forming the corona have a role in the NP interaction with macrophages, but the glycan identities remain unknown.

Development of an exoglycosidase plate-based assay for detecting α1-3,4 fucosylation biomarker in individuals with HNF1A-MODY.

Maturity-onset diabetes of the young due to hepatocyte nuclear factor-1 alpha variants (HNF1A-MODY) causes monogenic diabetes. Individuals carrying damaging variants in HNF1A show decreased levels of α1-3,4 fucosylation, as demonstrated on antennary fucosylation of blood plasma N-glycans. The excellent diagnostic performance of this glycan biomarker in blood plasma N-glycans of individuals with HNF1A-MODY has been demonstrated using liquid chromatography methods.

Quantitative Standards of 4-O-Acetyl- and 9-O-Acetyl-N-Acetylneuraminic Acid for the Analysis of Plasma and Serum.

N-Acetylneuraminic acid (sialic acid, Neu5Ac) is one of a large, diverse family of nine-carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac , as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards.

Glycoengineering of Therapeutic Antibodies with Small Molecule Inhibitors.

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.

Automation of Immunoglobulin Glycosylation Analysis.

The development of reliable, affordable, high-resolution glycomics technologies that can be used for many samples in a high-throughput manner are essential for both the optimization of glycosylation in the biopharmaceutical industry as well as for the advancement of clinical diagnostics based on glycosylation biomarkers. We will use this chapter to review the sample preparation processes that have been used on liquid-handling robots to obtain high-quality glycomics data for both biopharmaceutical and clinical antibody samples. This will focus on glycoprotein purification, followed by glycan or glycopeptide generation, derivatization and enrichment.

N-glycan Characterization by Liquid Chromatography Coupled with Fluorimetry and Mass Spectrometry.

Human blood plasma and serum have been a source of biomarkers for the indication and progression of many diseases for a few decades now. Human blood plasma is also an excellent source material to enable patients to monitor their health, with a multitude of biomarkers detectable for the assessment of health status. Blood sampling kits are increasingly available for use in the home with no specialist clinical skills required to obtain good quality samples for pathology lab analysis.

Equal mixing time enables scale-down and optimization of a CHO cell culture process using a shaken microbioreactor system.

The advancement of microbioreactor technology in recent years has transformed early- and mid-stage process development. The monitoring and control capabilities of microbioreactors not only promote the quick accumulation of process knowledge but has also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtitre plates. This study seeks to establish a framework for the micro-Matrix microbioreactor (Applikon-Biotechnology BV) as process development tool.

A semi-automated, high throughput approach for O-glycosylation profiling of in vitro established cancer cell lines by MALDI-FT-ICR MS.

The study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is known that alterations in O-glycosylation are involved in the development and progression of cancer. Their easy accessibility makes in vitro established cell lines suitable and useful models for studying biological mechanisms in disease.

Sialic acid as a potential biomarker for cardiovascular disease, diabetes and cancer.

Cardiovascular disease (CVD), diabetes and cancer pose increasing global healthcare burdens. New biomarkers could enable earlier diagnosis of these diseases, leading to more effective treatment and lower associated healthcare burden. Elevated sialic acid concentration in plasma and serum has been positively correlated with the presence of CVDs, diabetes and the development of malignant tumors.

Interlaboratory evaluation of plasma N-glycan antennary fucosylation as a clinical biomarker for HNF1A-MODY using liquid chromatography methods.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker.

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin-mediated clearance.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance.

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins.

L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLe) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear.

A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories.

A Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Assay for the Relative Quantitation of Antennary Fucosylated -Glycans in Human Plasma.

Changes in the abundance of antennary fucosylated glycans in human total plasma -glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation.

Improved and semi-automated reductive β-elimination workflow for higher throughput protein O-glycosylation analysis.

Protein O-glycosylation has shown to be critical for a wide range of biological processes, resulting in an increased interest in studying the alterations in O-glycosylation patterns of biological samples as disease biomarkers as well as for patient stratification and personalized medicine. Given the complexity of O-glycans, often a large number of samples have to be analysed in order to obtain conclusive results. However, most of the O-glycan analysis work done so far has been performed using glycoanalytical technologies that would not be suitable for the analysis of large sample sets, mainly due to limitations in sample throughput and affordability of the methods.

High-throughput Serum -Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum -glycosylation in a high-throughput manner.