Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow.

Analytical methods fr direct quantitative N-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled N-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-N-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent N-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS m/z peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.

Real-time monitoring of the sialic acid biosynthesis pathway by NMR.

Sialic acids are part of the outermost component of the glycocalyx of all vertebrates; as such, they are fundamental markers in physiological and pathological processes. In this study, we introduce a real-time assay to monitor individual enzymatic steps of sialic acid biosynthesis, either with recombinant enzymes, in particular using UDP-N-acetylglucosamine 2-epimerase (GNE) or N-acetylmannosamine kinase (MNK), or in cytosolic rat liver extract. Using state-of-the-art NMR techniques, we are able to follow the characteristic signal of the N-acetyl methyl group, which displays different chemical shifts for the biosynthesis intermediates UDP-N-acetylglucosamine, N-acetylmannosamine (and its 6-phosphate) and N-acetylneuraminic acid (and its 9-phosphate). Pseudo 2- and 3-D NMR demonstrated that in rat liver cytosolic extract, the phosphorylation reaction of MNK is exclusive for N-acetylmannosamine generated by GNE. Thus, we speculate that phosphorylation of this sugar from other sources (e.g. external application to cells) or N-acetylmannosamine derivatives often applied in metabolic glycoengineering is not conducted by MNK but by a yet unknown sugar kinase. Competition experiments with the most prevalent neutral carbohydrates demonstrated that of these, only N-acetylglucosamine slowed N-acetylmannosamine phosphorylation kinetics, suggesting an N-acetylglucosamine-preferring kinase as the acting enzyme.

Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept.

Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel® and its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126 kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity.

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment.

Eptacog alfa (NovoSeven®) is a vitamin K-dependent recombinant Factor VIIa produced by genetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues. After activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino acids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten glutamic acids of the N-terminal moiety are γ-carboxylated, Asn145 and Asn322 are N-glycosylated, and Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted for the originator and the first biosimilar AryoSeven™ to evaluate comparable bioengineering. Physicochemical properties were analyzed based on mass spectrometry, including intact mass, PTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their N-glycan profiles. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed N-glycosylation sites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and Ser52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry (TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of both biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating comparable bioactivity. Consequently, careful process optimization led to a stable production process of the biopharmaceuticals.

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans.

Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of ß1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor.

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

Werner Reutter: A Visionary Pioneer in Molecular Glycobiology.

A creative pioneer: Werner Reutter (1937-2016) was a scientist who both made fundamental discoveries in glycobiology and reached out to disciplines beyond his core field. Many of his former colleagues and students will remember his desire to exchange research ideas, which ultimately contributed to the birth of new research fields.

Comparability study of Rituximab originator and follow-on biopharmaceutical.

Immunglobolin G (IgG)-based biopharmaceuticals are emerging on the pharmaceuticals market due to their high target selectivity in different diseases. In parallel, a growing interest by other companies to produce similar or highly similar follow-on biologics exits, once the patent of blockbuster biotherapeutics is about to expire. In correlation to their complex structure, an analytical challenge is facing the approval of these biosimilars. Health authorities (e.g. FDA and EMA) have issued several guidelines to define critical quality attributes during manufacturing process changes. In the current study, physicochemical characterization using state-of-the-art analytics was applied to analyse intact mass, post-translational modifications (PTMs) and higher order structure of Rituximab and one of its biosimilars. Intact mass analysis, middle-up approach as well as subunit analysis revealed similar glycoforms but additional lysine variants in the biosimilar. The N-glycosylation site was confirmed for both, the originator and the biosimilar. PTMs and higher order structure were confirmed to be similar. A special focus was given to N-glycosylation due to its potential to monitor the batch-to-batch consistency and alteration during the production bioprocess. Comparison of the N-glycosylation profiles obtained from three batches of the biosimilar and the reference product showed quantitative variations, although the N-glycans were qualitatively similar. Furthermore, a head-to-head comparability of functional properties was performed to investigate the impact of glycosylation alteration and PTMs on potency within the biosimilar batches and between originator and follow-on biodrug. The data affirm that the difference is still in the acceptable range for biosimilarity.

Small Molecules Targeting Human N-Acetylmannosamine Kinase.

N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.