2-Dimensional ultra-high performance liquid chromatography and DMT-MM derivatization paired with tandem mass spectrometry for comprehensive serum N-glycome characterization.

Glycosylation is a prominent co- and post-translational modification which contributes to a variety of important biological functions. Protein glycosylation characteristics, particularly N-glycosylation, are influenced by changes in one's pathological state, such as through the presence of disease, and as such, there is great interest in N-glycans as potential disease biomarkers. Human serum is an attractive source for N-glycan based biomarker studies as circulatory proteins are representative of one's physiology, with many serum proteins containing N-glycosylation. The difficulty in comprehensively characterizing the serum N-glycome arises from the absence of a biosynthetic template resulting in great structural heterogeneity and complexity. To help overcome these challenges we developed a 2-dimensional liquid chromatography platform which utilizes offline weak anion exchange (WAX) chromatography in the first dimension and hydrophilic interaction liquid chromatography (HILIC) in the second dimension to separate N-glycans by charge, corresponding to degree of sialylation, and size, respectively. Performing these separations offline enables subsequent derivatization with 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) for sialic acid linkage determination and the identification of sialic acid linkage isomers. Subsequent tandem mass spectrometry analysis revealed the identification of 212 complete and partial N-glycan structures including low abundant N-glycans containing acetyl and sulphate modifications. The identifications obtained through this platform were then applied to N-glycans released from a set of stage 3 gastric cancer serum samples obtained from patients before (pre-op) and after (post-op) tumour resection to investigate how the serum N-glycome can facilitate differentiation between the two pathological states.

ADP-dependent glucokinase regulates energy metabolism via ER-localized glucose sensing.

Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division. Endoplasmic reticulum (ER)-localized ADP-dependent glucokinase (ADPGK) has been shown to play a critical role in T cell receptor activation-induced remodeling of energy metabolism, however the underlying mechanisms remain unclear. Therefore, we established and characterized in vitro and in vivo models for ADPGK-deficiency using Jurkat T cells and zebrafish. Upon activation, ADPGK knockout Jurkat T cells displayed increased cell death and ER stress. The increase in cell death resulted from a metabolic catastrophe and knockout cells displayed severely disturbed energy metabolism hindering induction of Warburg phenotype. ADPGK knockdown in zebrafish embryos led to short, dorsalized body axis induced by elevated apoptosis. ADPGK hypomorphic zebrafish further displayed dysfunctional glucose metabolism. In both model systems loss of ADPGK function led to defective N- and O-glycosylation. Overall, our data illustrate that ADPGK is part of a glucose sensing system in the ER modulating metabolism via regulation of N- and O-glycosylation.

Salivary N-glycosylation as a biomarker of oral cancer: A pilot study.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann-Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC.

Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry.

Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using cation exchange chromatography is routinely used as a fingerprint of the distribution of posttranslational modifications present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identification of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high-resolution native mass spectrometry.

Glycosylation Analysis of Therapeutic Glycoproteins Produced in CHO Cells.

In the last decades, the number of approved therapeutic proteins drugs is increasing exponentially and a large number of new therapeutic entities are progressing through clinical trials, solidifying biologics as the most promising class of pharmaceuticals on the market. Several cell lines are available for biopharmaceutical processes but mammalian cells are preferred since they give fewer problems for immunogenicity as they produce human-like post-translational modifications (PTMs). Glycosylation is the most common and complex (for both bioprocess engineering and quality control) of these modifications. Obtaining the desired glycosylation pattern is crucial for therapeutic proteins as it can impact significantly stability, half-life and safety as well as driving molecular processes, modifying the way drug interacts with patients' cells. As a consequence, glycosylation (like other PTMs) needs to be regulated and accurately analyzed during biopharmaceutical production. Herein we describe and discuss the analytical approaches for glycosylation analysis of therapeutic glycoproteins produced in CHO (Chinese Hamster Ovary) cells. This chapter will describe glycoprotein purification after separation from producing cell lines, N-glycan release and their variants fine structural characterization through mass spectrometry techniques.

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.

Quantitative glycomics using liquid phase separations coupled to mass spectrometry.

Post-translational modification of proteins by the attachment of glycans is governed by a variety of highly specific enzymes and is associated with fundamental impacts on the parent protein's physical, chemical and biological properties. The inherent connection between cellular physiology and specific glycosylation patterns has been shown to offer potential for diagnostic and prognostic monitoring of altered glycosylation in the disease state. Conversely, glycoprotein based biopharmaceuticals have emerged as dominant therapeutic strategies in the treatment of intricate diseases. Glycosylation present on these biopharmaceuticals represents a major critical quality attribute with impacts on both pharmacokinetics and pharmacodynamics. The structural variety of glycans, based upon their non-template driven assembly, poses a significant analytical challenge for both qualitative and quantitative analysis. Labile monosaccharide constituents, isomeric species and often low sample availability from biological sources necessitates meticulous sample handling, ultra-high-resolution analytical separation and sensitive detection techniques, respectively. In this article a critical review of analytical quantitation approaches using liquid phase separations coupled to mass spectrometry for released glycans of biopharmaceutical and biomedical significance is presented. Considerations associated with sample derivatisation strategies, ionisation, relative quantitation through isotopic as well as isobaric labelling, metabolic/enzymatic incorporation and targeted analysis are all thoroughly discussed.

Polyclonal Immunoglobulin G N-Glycosylation in the Pathogenesis of Plasma Cell Disorders.

The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients [MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5)] were characterized to map glycan structures in distinct disease phases of multiple myeloma. N-Glycan profiles from MGUS resembled normal control. The abundance of neutral glycans containing terminal galactose was highest in SMM, while agalactosylated glycans and fucosylated glycans were lowest in MM. Three afucosyl-biantennary-digalactosylated-sialylated species (A2G2S1, A2BG2S1, and A2BG2S2) decreased 2.38-, 2.4-, and 4.25-fold, respectively, from benign to active myeloma. Increased light chain sialylation was observed in a longitudinal case of transformation from MGUS to MM. Bisecting N-acetylglucosamine was lowest in the CR group, while highest in relapsed disease. Gene expression levels of FUT 8, ST6GAL1, B4GALT1, RECK, and BACH2 identified from publicly available GEP data supported the glycomic changes seen in MM compared to control. The observed differential glycosylation underlined the heterogeneity of the myeloma spectrum. This study demonstrates the feasibility of mapping glycan modifications on the IgG molecule and provides proof of principle that differential IgG glycosylation patterns can be successfully identified in plasma cell disorders.

Twoplex 12/13 C6 aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MSE workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral 12 C6 and 13 C6 aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex 12 C6 /13 C6 aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.

Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13C6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12C6 'light' and 13C6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

Quantitative host cell protein analysis using two dimensional data independent LC-MS(E).

Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed. Mass spectrometry methods provide rationale for targeted removal strategies through the provision of both qualitative and quantitative HCP information. A high pH, low pH, reversed-phase data independent 2D-LC-MS(E) proteomic platform was applied to determine HCP repertoires in the Protein A purified monoclonal antibody (mAb) samples as a function of culture harvest time, elution buffer used for DSP and also following inclusion of additional DSP steps. Critical quality attributes (CQAs) were examined for mAbs purified with different Protein A elution buffers to ensure that the selected buffers not only minimized the HCP profile but also exhibited no adverse effect on product quality. Results indicated that an arginine based Protein A elution buffer minimized the levels of HCPs identified and quantified in a purified mAb sample and also demonstrated no impact on product CQAs. It was also observed that mAbs harvested at later stages of cell culture contained higher concentrations of HCPs but that these were successfully removed by the addition of DSP steps complementary to Protein A purification. Taken together, our results showed how mass spectrometry based methods for HCP determination in conjunction with careful consideration of processing parameters such as harvest time, Protein A elution buffers, and subsequent DSP steps can reduce the HCP repertoire of therapeutic mAbs.

Unraveling the glyco-puzzle: glycan structure identification by capillary electrophoresis.

State-of-the-art high-resolution separation techniques play an important role in the full structural elucidation of glycans. Capillary electrophoresis (CE) offers a rapid yet simple method for exhaustive carbohydrate profiling. CE is a versatile analytical platform, which can be operated in several separation modes, simply by altering separation conditions during operation. For in-depth glycan structural analysis, CE has also gained significantly from the additional resolution introduced by complementary and orthogonal separation techniques such as ion exchange or hydrophilic interaction chromatography. Commercially available mass spectrometry (MS) interfaces have not only brought this information-rich detection technique within reach, but CE also represents an expedient highly efficient separation inlet for MS, capable of separating isobaric oligosaccharide isomers prior to MS detection and MS/MS fragmentation based identification. This Perspective gives a sophisticated impression of the versatility of capillary electrophoresis for deep structural elucidation of carbohydrates derived from glycoproteins of biomedical interest. Different separation modes for the analysis of both charged and neutral glycans, such as influencing electroosmotic flow, using complexation/interaction based secondary equilibria, and the use of charged and neutral labels are compared. The merits of introducing orthogonal and complementary techniques, such as exoglycosidase digestion arrays, analytical/preparative chromatography and mass spectrometric detection, extending the dynamic range and resolution of CE are all thoroughly discussed.

Analysis of haptoglobin N-glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis.

A CE-based method was introduced to compare the N-glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease, and lung cancer patients by means of two haptoglobin-specific monoclonal antibodies. Haptoglobin N-glycans were then enzymatically released, fluorescently labeled, and profiled by CE. Disease-associated changes of core and antennary fucosylation were identified by targeted exoglycosidase digestions and their levels were compared in the different patient groups. Terms such as core- and arm-fucosylation degree, as well as branching degree, were introduced for easier characterization of the changes and statistical analysis was used to examine which structures were responsible for the observed differences. Increased level of α1-6 fucosylated tri-antennary glycans was found in all disease groups compared to the control. Elevated amounts of core- and arm-fucosylation on tetra-antennary glycans were detected in the lung cancer group compared to the chronic obstructive pulmonary disease group. A larger scale study is necessary to confirm and validate these preliminary findings in the glycosylation changes of haptoglobin, so could then be used as biomarkers in the diagnosis of malignant and inflammatory lung diseases.

Influence of molecular configuration and conformation on the electromigration of oligosaccharides in narrow bore capillaries.

Capillary electrophoresis enables fast, high efficiency separations of oligosaccharides, wherein positional and/or linkage isomers, bearing the same charge-to-mass ratio, can readily be separated based on hydrodynamic radius differences. Fundamental electrophoretic mobility theory was used to investigate the correlation between changes in hydrodynamic volume equivalent radius and corresponding electrophoretic characteristics of oligosaccharides with different molecular properties. Fluorescently derivatized isomeric malto-, cello-, and isomaltooligosaccharide ladders, differing only in their linkage type of α1→4, ß1→4, and α1→6, respectively, as well as a sterically larger N-acetylchitooligosaccharide ladder were used as model compounds. Mere differences in glycosidic linkage type or anomericity of isomeric oligo-glucoses had a decisive impact on their electromigration behavior, thus reflecting discrepancies in hydrodynamic radii and associated molecular conformations. The impact of hydrogen bridges, and associated availability of hydroxyl groups, on the molecular conformations, was investigated by hydrophilic interaction liquid chromatography. The experimentally observed electrophoretic and chromatographic differences between isomeric oligo-glucoses strongly suggested that special attention must be given when homooligosaccharide ladders are employed for normalization and comparability purposes, for example, in glucose unit calculation based structural elucidation.

Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation.

N-glycans attached to the C(H)2 domains of the Fc or the antigen binding regions of IgG play an important role in stabilizing and modulating antibody activity. Exhaustive elucidation of 32 IgG N-glycans using a combination of weak anion exchange enrichment and exoglycosidase array digestion with subsequent profiling exceeded 48 h. Pursuing increased throughput and associated structural annotation confidence, we compared the 1.7 µm hydrophilic interaction phase for UPLC with CE-LIF for the rapid and comprehensive characterization of N-glycans released from healthy human serum polyclonal IgG. Combination of the data individually generated using each technique demonstrated that complete structural annotation was possible within a total analysis time of 20 min due to the advantageous orthogonality of the separation mechanisms. The parallel use of both analytical techniques provides a powerful platform for rapid and comprehensive analysis of IgG N-glycosylation present on therapeutic antibodies or on antibodies of biomedical or pathological significance.

Identification of N-glycans displaying mannose-6-phosphate and their site of attachment on therapeutic enzymes for lysosomal storage disorder treatment.

Characterization of mono- and bis-mannose-6-phosphate (M6P) bearing oligosaccharides present on acid hydrolase enzymes poses a considerable analytical challenge. In the current paper, we investigated the use of UPLC profiling on a 1.7 µm HILIC phase and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) combined with exoglycosidase digestion and weak anion exchange fractionation for the characterization of M6P bearing glycans on recombinant ß-glucuronidase expressed in Chinese Hamster Ovary (CHO) cells. Using this multidimensional approach a number of peaks were observed to resist digestion, suggesting the presence and blocking activity of the M6P tag. To investigate further, mixed oxide affinity purification on a combined TiO(2)/ZrO(2) resin facilitated the selective enrichment of oligosaccharides bearing mono- or diphospho-esters that corresponded to those peaks previously identified to resist exoglycosidase digestion. Alkaline phosphatase digestion identified Man(6)GlcNAc(2) and Man(7)GlcNAc(2) glycans as the primary carriers of the M6P tag. Site-specific glycoproteomic analysis revealed that Man(7)GlcNAc(2)-M6P oligosaccharides were present at asparagine 272 and 420, while asparagine 631 displayed Man(6)GlcNAc(2)-M6P. The analytical strategy applied herein represents a novel yet simple approach for the qualitative and semiquantitative structural characterization of M6P containing oligosaccharides on therapeutic enzymes.

Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 µm hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, haptoglobin, transferrin, and α1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and α1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease progression. The speed and selectivity offered by the sub-2 µm HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome.

Two variable semi-empirical and artificial neural-network-based modeling of peptide mobilities in CZE: the effect of temperature and organic modifier concentration.

This work was focused on investigating the effects of two separation influencing parameters in CZE, namely temperature and organic additive concentration upon the electrophoretic migration properties of model tripeptides. Two variable semi-empirical (TVSE) models and back-propagation artificial neural networks (ANN) were applied to predict the electrophoretic mobilities of the tripeptides with non-polar, polar, positively charged, negatively charged and aromatic R group characteristics. Previously published work on the subject did not account for the effect of temperature and buffer organic modifier concentration on peptide mobility, in spite of the fact that both were considered to be influential factors in peptide analysis. In this work, a substantial data set was generated consisting of actual electrophoretic mobilities of the model tripeptides in 30 mM phosphate buffer at pH 7.5, at 20, 25, 30, 35 and 40 degrees C and at four different organic additive containing running buffers (0, 5, 10 and 15% MeOH) applying two electric field strengths (12 and 16 kV) to assess our mobility predicting models. Based on the Arrhenius plots of natural logarithm of mobility versus reciprocal absolute temperature of the various experimental setups, the corresponding activation energy values were derived and evaluated. Calculated mobilities by TVSE and back-propagation ANN models were compared with each other and to the experimental data, respectively. Neural network approaches were able to model the complex impact of both temperature and organic additive concentrations and resulted in considerably higher predictive power over the TVSE models, justifying that the effect of these two factors should not be neglected.