Glycan Profiling Research Articles

At-line high throughput site-specific glycan profiling using targeted mass spectrometry

Protein post-translational modification (PTM) plays an important role in many biological processes; of which glycosylation is arguably one of the most complex and diverse modifications and is crucial for the safety and efficacy of biotherapeutic proteins.Mass spectrometric characterization of protein glycosylation is well established with clear advantages and disadvantages; on one hand it is precise and information-rich, as well as being relative inexpensive in terms of the reagents and consumables despite the instrumentation cost and, depending on the method, can give site specific information; on the other hand it generally suffers from low throughput, restriction to largely purified samples and is less quantitative, especially for sialylated glycan species.Here, we describe a high throughput, site-specific, targeted mass spectrometric peptide mapping approach to quickly screen/rank candidate production cell lines and culture conditions that give favourable glycosylation profiles directly from conditioned culture media for an Fc-fusion protein. The methodology is fully compatible with automation and combines the speed of ‘top-down’ mass spectrometry with the site-specific information of ‘bottom-up’ mass spectrometry. In addition, this strategy can be used for multi-attribute product quality screening/monitoring as an integral part of cell line selection and process development.

Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling [1]. However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. Comparing the N-glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, the same pool of N-glycan structures was detected in each sample type but the relative signal of 21 structures significantly differed between samples, with the high mannose class increased in enriched CM. Moreover, by comparing primary CM to hiPSC-CM collected during 20–100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation were observed and differences between primary and hiPSC-CM were revealed. Namely, >30% of the N-glycome significantly changed across these time-points of differentiation and only 23% of the N-glycan structures were shared between hiPSC-CM and primary CM. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.

C-Terminally tagged NA in replication-competent influenza A viruses reveals differences in glycan profiles between NA and HA.

Glycans attached to the viruses regulate their pathogenicity, immunogenicity, and antigenicity. We have previously shown that lectin microarray provided an easy and highly sensitive platform for analyzing glycan profiles of hemagglutinin (HA) of influenza A viruses in culture supernatants. On the other hand, the system is not applicable for neuraminidase (NA), the other viral glycoprotein of influenza A viruses, due to the limited availability of specific antibodies used to detect NA in the lectin microarray. Accordingly, we established replication-competent viruses harboring the short peptide-tag sequence at the C-terminus of NA in this study. The generated viruses underwent normal proliferation cycles and showed similar properties to the wild-type viruses. Lectin microarray analyses of the tagged NA enriched from the viral particles showed that glycan profiles of NA were mostly occupied by mannose-type glycans. Interestingly, the profiles were distinct from those of HA separated from the same particle preparation, in which core-fucosylated complex-type N-glycans terminating with non-sialylated N-acetyllactosamine were dominant. Collectively, this study provides novel platforms for the analyses of the distinction between the glycan profiles of NA and HA, and contributes to a better understanding of later stages of the viral life cycles through analyzing the glycans attached to NA.

Identification of LCA-binding Glycans as a Novel Biomarker for Esophageal Cancer Metastasis using a Lectin Array-based Strategy.

Esophageal cancer (EC) is a unique and heterogeneous disease diagnosed mostly at advanced stages. Altered glycans presented on cell surfaces are involved in the occurrence and development of malignancy. However, the effects of glycans on EC progression are largely unexplored. Here, a lectin array was utilized to detect the glycan profiling of the normal esophageal mucosal epithelial cell line and two EC cell lines. The binding of Lens culinaris lectin (LCA) to EC cells was found to be stronger than that of the normal cells. Lectin immunohistochemical staining revealed that LCA-binding glycans were markedly elevated in EC tissues compared to adjacent non-cancerous tissues. LCA staining was significantly associated with lymph node metastasis, depth of invasion, TNM stage and poor overall survival of EC patients. Added LCA to block LCA recognized glycans could inhibit the migration and invasion of EC cells. Further analysis revealed that blocking the biosynthesis of LCA-binding glycans by tunicamycin attenuated cellular migratory and invasive abilities. Additionally, a membrane glycoprotein CD147 was recognized as a binder of LCA. There was a positive correlation between LCA-binding glycans and CD147 expression in clinical samples. Interestingly, CD147 inhibition also reduced cell migration and invasion. These findings indicated that LCA-binding glycans may function as a novel indicator to predict metastasis for patients with EC.

Emerging applications of lectins in cancer detection and biomedicine

Cancer is considered highly mortal due to the frequency of late diagnosis, usually in advanced stages. Early diagnosis plays a pivotal role in reducing mortality and improving prognosis of the disease. An alteration in glycans is the hallmark of cancer. In last two decades, there has been an exponential increase in the use of lectins to mark the foremost challenging problem of detection and therapy in cancer. Plant lectins have an ability to specifically and selectively bind and target to glycosylated biological molecules with good sensitivity. This property of lectins has shown them to possess a promising and potential application within the field of cancer detection and therapy. Lectins are a special class of proteins of non-immune origin which selectively recognize and reversibly bind to carbohydrates and glycoconjugates. The aim of present paper was to discuss the current status of lectins and their nano-structures studied so far for detection and therapy of various cancers. Lectins serve as the major molecular tools for glycan profiling. Their application as nano tools is getting impedance for the diagnosis and treatment of cancer. Lectin conjugated drug delivery systems are recognized, bound, and internalized by aberrant sugar present on the cancerous cell surface. Lectins adhere to neoplastic cell membrane and cause apoptosis-induced cytotoxicity to trigger autophagy or necrosis in cancer cells without affecting the normal cells. Cytotoxicity of Nano-Lec-materials displays impressive anticancer effects in addition to early detection. Drug delivery to targeted sites using engineered nano-lectins allows enhanced efficiency and reduced toxicity to normal cells and thus improved patients’ quality of life. The present review will describe the current status of lectins and glycoconjugates in cancer detection and their applications as nano-materials in future therapeutics as drug carriers against many diseases and effective cancer targeting bullets.

Human UDP-galactose 4'-epimerase (GALE) is required for cell-surface glycome structure and function.

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE-/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

Glycan structures and their recognition roles in the human blood group ABH/Ii, Lea, b, x, y and Sialyl Lea,x active cyst glycoproteins.

Human ovarian cyst glycoproteins (HOC, cyst gps) isolated from pseudomucinous type of human ovarian cyst fluids is one of the richest and pioneer sources for studying biosynthesis, structures and functional roles of blood group ABH, Lea,b,x,y, sLea and sLex active glycoproteins. After 70+ years of exploration, four top highlights are shared. (i) an updated concept of glycotopes and their internal structures in cyst gps was composited; (ii) the unknown codes of new genes in secreted cyst gps were unlocked as Lex and Ley; (iii) recognition profiles of cyst glycans and a sialic acid-rich (18%) glycan with lectins and antibodies were shown. (iv) Co-expression of Blood Group A/ A-Leb/y and B/B-Leb/y active Glycotopes in the same glycan chains were isolated and illustrated. These are the most advanced achievements since 1980.

A streamlined workflow for twoplexing of N-linked glycan analysis using light (12C6) and heavy (13C6) isotopologues of 3-aminobenzenesulfonic acid

Comparative glycosylation analysis of biopharmaceuticals requires the development of methods that deliver the necessary throughput, support structural elucidation and relative quantitation of glycans released from therapeutics. The current study presents the development and applicability assessment of a twoplex approach using light and heavy isotopolouges of 3-aminobenzenesulfonic acid (3-ASA) under wet labeling conditions followed by UHPLC-MS analysis in data dependent acquisition mode. Excellent labelling efficiency, >90%, was achieved for both the light and heavy variants of the reagent. Glycan distributions of two human IgG lots labeled by light and heavy isotopolouges were identical, demonstrating no labeling bias introduced by either of the isotopologues. Peak area distributions of glycan profiles of two human IgG lots were compared to 2-aminobenzamide (2-AB) and RapiFluor-MS protocols. The comparison led to identical results in peak area distribution across the three dyes, but differences in chromatographic selectivity attributed to the different tags. MS1 based relative quantitation was further validated by releasing glycans from the same lot of human IgG, with glycan pools obtained labeled with light and heavy isotopologues separately, followed by mixing and clean-up of the same amount of light and heavy labeled glycan pools. MS analyses of each glycan resulted in a ratio of light and heavy XIC in the range of 0.97 ≤ x ≤ 1.05, demonstrating the method is amenable for the relative quantitation of glycans. Excellent correlation between the relative quantitation data of N-glycans from two human IgG N-glycan pools using the twoplex approach and ratios from peak area distribution calculated from the fluorescent chromatogram was observed (r = 0.986), further corroborating the reliability of the method and its potential applicability in the biopharmaceutical industry. Highly informative HCD-MS2 spectra dominated mostly by Y- and Z-type single and double glycosidic fragment ions facilitate structural interpretation of the oligosaccharides.

Identification of carbohydrate peripheral epitopes important for recognition by positive-ion MALDI multistage mass spectrometry

Carbohydrate sequences are important for various biological processes. It has recently been estimated to have 100,000–500,000 carbohydrate structures in mammalian glycome. However, the peripheral carbohydrate determinants on N- and O-glycoproteins, glycolipids, polysaccharides and secreted free sugars are limited in numbers. Among these blood-group-related antigens the ABO(H)- and Lewis-types are particularly important. Negative-ion MS/MS has been successfully used in assignment of these epitopes on free reducing sugars but cannot be applied to reduced sugars, e.g. O-glycans typically released from mucins as alditols, or in positive-ion detection of either reducing or reduced oligosaccharides. In the present study, we investigate the fragmentation features of permethylated reducing and reduced sugars under positive-ion conditions of multi-stage MALDI-MS, and propose the concept of epitope ion and epitope spectrum for determination of peripheral blood-group related epitopes on secreted human milk oligosaccharides and N-glycans as reducing sugars and O-glycans as reduced alditols in conjunction with MALDI-MS glycan profiling.

Multivariate data analysis of growth medium trends affecting antibody glycosylation.

Use of multivariate data analysis for the manufacturing of biologics has been increasing due to more widespread use of data‐generating process analytical technologies (PAT) promoted by the US FDA. To generate a large dataset on which to apply these principles, we used an in‐house model CHO DG44 cell line cultured in automated micro bioreactors alongside PAT with four commercial growth media focusing on antibody quality through N‐glycosylation profiles. Using univariate analyses, we determined that different media resulted in diverse amounts of terminal galactosylation, high mannose glycoforms, and aglycosylation. Due to the amount of in‐process data generated by PAT instrumentation, multivariate data analysis was necessary to ascertain which variables best modeled our glycan profile findings. Our principal component analysis revealed components that represent the development of glycoforms into terminally galacotosylated forms (G1F and G2F), and another that encompasses maturation out of high mannose glycoforms. The partial least squares model additionally incorporated metabolic values to link these processes to glycan outcomes, especially involving the consumption of glutamine. Overall, these approaches indicated a tradeoff between cellular productivity and product quality in terms of the glycosylation. This work illustrates the use of multivariate analytical approaches that can be applied to complex bioprocessing problems for identifying potential solutions.

In Planta Glycan Engineering and Functional Activities of IgE Antibodies.

Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities.

Type 2 Diabetes Mellitus is Associated with the Immunoglobulin G N-Glycome through Putative Proinflammatory Mechanisms in an Australian Population.

Type 2 diabetes mellitus (T2DM) is a common complex trait arising from interactions among multiple environmental, genomic, and postgenomic factors. We report here the first attempt to investigate the association between immunoglobulin G (IgG) N-glycan patterns, T2DM, and their clinical risk factors in an Australian population. N-glycosylation of proteins is one of the most frequently observed co- and post-translational modifications, reflecting, importantly, the real-time status of the interplay between the genomic and postgenomic factors. In a community-based case-control study, 849 participants (217 cases and 632 controls) were recruited from an urban community in Busselton, Western Australia. We applied the ultraperformance liquid chromatography method to analyze the composition of IgG N-glycans. We then conducted Spearman's correlation analyses to explore the association between glycan biomarker candidates and clinical risk factors. We performed area under the curve (AUC) analysis of the receiver operating characteristic curves by fivefold cross-validation for clinical risk factors, IgG glycans, and their combination. Two directly measured and four derived glycan peaks were significantly associated with T2DM, after correction for extensive clinical confounders and false discovery rate, thus suggesting that IgG N-glycan traits are highly correlated with T2DM clinical risk factors. Moreover, adding the IgG glycan profiles to fasting blood glucose in the logistic regression model increased the AUC from 0.799 to 0.859. The AUC for IgG glycans alone was 0.623 with a 95% confidence interval 0.580-0.666. In addition, our study provided new evidence of diversity in T2DM complex trait by IgG N-glycan stratification. Six IgG glycan traits were firmly associated with T2DM, which reflects an increased proinflammatory and biological aging status. In summary, our study reports novel associations between the IgG N-glycome and T2DM in an Australian population and the putative role of proinflammatory mechanisms. Furthermore, IgG N-glycomic alterations offer future prospects as inflammatory biomarker candidates for T2DM diagnosis, and monitoring of T2DM progression to cardiovascular disease or renal failure.

ALG12-CDG: novel glycophenotype insights endorse the molecular defect.

Congenital disorders of glycosylation (CDG) are genetic diseases characterized by deficient synthesis (CDG type I) and/or abnormal processing (CDG type II) of glycan moieties linked to protein and lipids. The impact of the molecular defects on protein glycosylation and in turn on the clinical phenotypes of patients with CDG is not yet understood. ALG12-CDG is due to deficiency of ALG12 α1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the endoplasmic reticulum. ALG12-CDG is a severe multisystem disease associated with low to deficient serum immunoglobulins and recurrent infections. We thoroughly investigated the glycophenotype in a patient with novel ALG12 variants and immunodeficiency. We analyzed serum native transferrin, as first line test for CDG and we profiled serum IgG and total serum N-glycans by a combination of consolidated (N-glycan analysis by MALDI MS) and innovative mass spectrometry-based protocols, such as GlycoWorks RapiFluor N-glycan analysis coupled with LC-ESI MS. Intact serum transferrin showed, as expected for a CDG type I defect, underoccupancy of N-glycosylation sites. Surprisingly, total serum proteins and IgG N-glycans showed some specific changes, consisting in accumulating amounts of definite high-mannose and hybrid structures. As a whole, ALG12-CDG behaves as a dual CDG (CDG-I and II defects) and it is associated with distinct, abnormal glycosylation of total serum and IgG N-glycans. Glycan profiling of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.

A Rapid Array-Based Approach to N-Glycan Profiling of Cultured Cells.

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, we report a rapid array-based workflow for profiling N-glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture. Approaches are demonstrated that account for background N-glycans due to serum media. Normalization procedures are shown. The method is robust and reproducible, requiring as few as 3000 cells per replicate with a 3-20% coefficient of variation to capture label-free profiles of N-glycans. Quantification by stable isotopic labeling of N-glycans in cell culture is demonstrated and adds no additional time to preparation. Utility of the method is demonstrated by measurement of N-glycan turnover rates due to induction of oxidative stress in human primary aortic endothelial cells. The developed method and ancillary tools serve as a foundational launching point for rapid profiling of N-glycans ranging from high-density arrays down to single cells in culture.

Early Stage Glycosylation Biomarkers in Alzheimer's Disease.

Alzheimer’s disease (AD) is of great cause for concern in our ageing population, which currently lacks diagnostic tools to permit accurate and timely diagnosis for affected individuals. The development of such tools could enable therapeutic interventions earlier in the disease course and thus potentially reducing the debilitating effects of AD. Glycosylation is a common, and important, post translational modification of proteins implicated in a host of disease states resulting in a complex array of glycans being incorporated into biomolecules. Recent investigations of glycan profiles, in a wide range of conditions, has been made possible due to technological advances in the field enabling accurate glycoanalyses. Amyloid beta (Aβ) peptides, tau protein, and other important proteins involved in AD pathogenesis, have altered glycosylation profiles. Crucially, these abnormalities present early in the disease state, are present in the peripheral blood, and help to distinguish AD from other dementias. This review describes the aberrant glycome in AD, focusing on proteins implicated in development and progression, and elucidates the potential of glycome aberrations as early stage biomarkers of AD.

Real-time quantification and supplementation of bioreactor amino acids to prolong culture time and maintain antibody product quality.

Real‐time monitoring of cell cultures in bioreactors can enable expedited responses necessary to correct potential batch failure perturbations which may normally go undiscovered until the completion of the batch and result in failure. Currently, analytical technologies are dedicated to real‐time monitoring of bioreactor parameters such as pH, dissolved oxygen, and temperature, nutrients such as glucose and glutamine, or metabolites such as lactate. Despite the importance of amino acids as the building blocks of therapeutic protein products, other than glutamine their concentrations are not commonly measured. Here, we present a study into amino acid monitoring, supplementation strategies, and how these techniques may impact the cell growth profiles and product quality. We used preliminary bioreactor runs to establish baselines by determining initial amino acid consumption patterns, the results of which were used to select a pool of amino acids which gets depleted in the bioreactor. These amino acids were combined into blends which were supplemented into bioreactors during a subsequent run, the concentrations of which were monitored using a mass spectrometry based at‐line method we developed to quickly assess amino acid concentrations from crude bioreactor media. We found that these blends could prolong culture life, reversing a viable cell density decrease that was leading to batch death. Additionally, we assessed how these strategies might impact protein product quality, such as the glycan profile. The amino acid consumption data were aligned with the final glycan profiles in principal component analysis to identify which amino acids are most closely associated with glycan outcomes.

Regular alteration of protein glycosylation in skeletal muscles of hibernating Daurian ground squirrels (Spermophilus dauricus).

Glycosylation is one of the most common post-translational protein modifications and is closely associated with muscle atrophy. This study aims to investigate the changes in glycan profiles in the fast-twitch extensor digitorum longus (EDL) muscles of Daurian ground squirrels (Spermophilus dauricus) during hibernation as well as the correlation between protein glycosylation and muscle atrophy prevention in hibernating animals. The results showed that there was no significant change in the muscle-to-body mass ratio, muscle fiber cross-sectional area (CSA), fiber distribution and ultrastructures in the EDL muscles of ground squirrels during hibernation. Alterations of six glycans comprising sialic acid α2-3 galactose (Sia2-3Gal) and Fucα1-2Galβ1-4Glc(NAc) in the EDL muscles were observed. In addition, the observed downregulation of sialyltransferase (ST3Gals) mRNA levels and upregulation of fucosyltransferase (FUT1 and FUT2) mRNA levels during hibernation and the subsequent restoration to normal levels during periodic interbout arousal were consistent with the changes in sialic acid and fucose modifications. Our results indicate that changes in ST3Gals and FUTs in the EDL muscles of Daurian ground squirrels during hibernation can alter sialylation and fucosylation of muscle glycoproteins, which may protect the skeletal muscles of hibernating Daurian ground squirrels from disuse atrophy.

Glycosylation of biosimilars: Recent advances in analytical characterization and clinical implications

Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms.In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products.

1-(2-Aminoethyl)-3-methyl-1H-imidazol-3-ium tetrafluoroborate: synthesis and application in carbohydrate analysis

Abstract Reductive alkylation of the carbonyl group of carbohydrates with fluorescence or ionizing labels is a prerequisite for the sensitive analysis of carbohydrates by chromatographic and mass spectrometric techniques. Herein, 1-(2-aminoethyl)-3-methyl-1H-imidazol-3-ium tetrafluoroborate ([MIEA][BF4]) was successfully synthesized using tert-butyl N-(2-bromoethyl)carbamate and N-methylimidazole as starting materials. MIEA+ was then investigated as a multifunctional oligosaccharide label for glycan profiling and identification using LC-ESI-ToF and by MALDI-ToF mass spectrometry. The reductive amination of this diazole with carbohydrates was exemplified by labeling N-glycans from the model glycoproteins horseradish peroxidase, RNase B, and bovine lactoferrin. The produced MIEA+ glycan profiles were comparable to the corresponding 2AB labeled glycan derivatives and showed improved ESI-MS ionization efficiency over the respective 2AB derivatives, with detection sensitivity in the low picomol to the high femtomol range.

A Comparison of the Oligosaccharide Structures of Antithrombin Derived from Plasma and Recombinant Using POTELLIGENT® Technology

Human antithrombin (AT) has two isoforms of which the predominant α-form is glycosylated on all four possible glycosylation sites and the lower abundant β-isoform lacks the oligosaccharide on Asn135. The main oligosaccharide structure of human AT consists of biantennary complex-type oligosaccharides lacking a core fucose. Generally, Chinese hamster ovary (CHO) cells produce recombinant human AT (rhAT) with core-fucosylated oligosaccharides. However, rhAT lacking core-fucose oligosaccharides can be produced by POTELLIGENT® technology, which uses FUT8 knockout CHO cells in production. The rhAT has more variable glycan structures, such as tetra-antennary complex type, high-mannose type, and mannose 6-phosphate species as minor components compared to plasma-derived human AT (phAT). In addition, the site-specific glycan profile was different between two ATs. We evaluated the effect of these properties on efficacy and safety based on a comparison of rhAT made by that technology with phAT in terms of their respective oligosaccharide structures, site-specific oligosaccharide profiles, and the ratio of α- and β-forms. Although some structural differences were found between the rhAT and phAT, we concluded that these differences have no significant effect on the efficacy and safety of rhAT.