Capillary electrophoresis analysis of industrial galactooligosaccharides.

Galactooligosaccharides are added to infant formula to simulate some of the benefits associated with human milk oligosaccharides, in particular to modulate the gut microbiota. During our study the galactooligosaccharide content of an industrial GOS ingredient was determined by differential enzymatic digestion using amyloglucosidase and ß-galactosidase. The resulting digests were fluorophore labeled and analyzed by capillary gel electrophoresis with laser induced fluorescence detection. Quantification of the results were based on a lactose calibration curve. Utilizing this approach, the galactooligosaccharide concentration of the sample was determined as 37.23 g/100 g, very similar to earlier HPLC results, but requiring only 20 min separation time. The CGE-LIF method in conjunction with the differential enzymatic digestion protocol demonstrated in this paper offers a rapid and easy to use method to measure galactooligosaccharides and should be applicable to the determination of GOS in infant formulas and other products.

Immobilized exoglycosidase matrix mediated solid phase glycan sequencing.

Full characterization of the attached carbohydrate moieties of glycoproteins is of high importance for both the rapidly growing biopharmaceutical industry and the biomedical field. In this paper we report the design and production of three important 6HIS-tagged exoglycosidases (neuraminidase, ß-galactosidase and hexosaminidase) to support rapid solid phase N-glycan sequencing with high robustness using immobilized enzymes. The exoglycosidases were generated in bacterial expression systems with high yield. Oriented immobilization via the 6HIS-tag portion of the molecules supported easy accessibility to the active sites and consequently high digestion performance. The three exoglycosidases were premixed in an appropriate matrix format and processed in a low-salt buffer to support long term storage. The digestion efficiencies of the immobilized enzymes were demonstrated by using solid phase sequencing in conjunction with capillary electrophoresis analysis of the products on a commercial glycoprotein therapeutic (palivizumab) and human serum derived fluorophore labeled glycans.

Enhanced Recombinant Protein Production of Soluble, Highly Active and Immobilizable PNGase F.

High resolution analysis of N-glycans can be performed after their endoglycosidase mediated removal from proteins. N-glycosidase F peptide (PNGase F) is one the most frequently used enzyme for this purpose. Because of the significant demand for PNGase F both in basic and applied research, rapid and inexpensive methods are of great demand for its large-scale production, preferably in immobilizable form to solid supports or surfaces. In this paper, we report on the high-yield production of N-terminal 6His-PNGase F enzyme in a bacterial Escherichia coli SHuffle expression system. The activity profile of the generated enzyme was compared to commercially available PNGase F enzymes, featuring higher activity for the former. The method described here is thus suitable for the cost-effective production of PNGase F in an active, immobilizable form.

Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product.

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.

N-glycosylation structure - function characterization of omalizumab, an anti-asthma biotherapeutic product.

Omalizumab, a glycoprotein based biotherapeutics, is one of the most frequently used targeted antibody biopharmaceutical to reduce asthma exacerbations, improve lung function and reduce oral corticosteroid use. The effector function and clearance time of such glycoprotein drugs is affected by their N-glycosylation, that defines the required administration frequency to improve the quality of life in appropriately selected patients. Therefore, the glycosylation of biologics is an important critical quality attribute (CQA). The profile of asparagine linked carbohydrates is greatly dependent on the manufacturing process. Even a small deviation may have a major effect on the structure and therefore the function of the biotherapeutic product. For this reason, comprehensive N-glycosylation analysis is of high importance during production and release. Capillary electrophoresis (CE) is one of the frequently used tools to characterize protein therapeutics and utilized by the biopharmaceutical industry for protein and glycan level analysis, which are key parts both for drug development and quality control. To reveal important structure - function relationships, characterization of omalizumab is presented using capillary SDS gel electrophoresis with UV detection at the protein level and capillary gel electrophoresis with laser induced fluorescent detection at the N-linked carbohydrate level. This latter technique was also used for oligosaccharide sequencing for glycan structure validation. The results suggested no ADCC function - structure relationship due to the mostly core fucosylated biantennary glycans found. However, the presence of the high mannose structures probably affects the clearance rate of the drug.

N-Glycosylation Profiling of Human Blood in Type 2 Diabetes by Capillary Electrophoresis: A Preliminary Study.

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis bioanalytical technique, for a high-resolution determination of the N-glycan profile in human blood samples of patients with type 2 diabetes (T2D). To achieve the profile information of these complex oligosaccharides, linked by asparagine to hIgG in the blood, the glycoproteins of the samples needed to be cleaved, labelled, and purified with sufficient yield and selectivity. The resulting samples were analyzed by capillary electrophoresis, with laser-induced fluorescence detection. After separation parameter optimization, the capillary electrophoresis technique was implemented for efficient N-glycan profiling of whole blood samples from the diabetic patients. Our results revealed that there were subtle differences between the N-glycan profiles of the diabetic and control samples; in particular, two N-glycan structures were identified as potential glycobiomarkers that could reveal significant changes between the untreated/treated type 2 diabetic and control samples. By analyzing the resulting oligosaccharide profiles, clinically relevant information was obtained, revealing the differences between the untreated and HMG-CoA reductase-inhibitor-treated diabetic patients on changes in the N-glycan profile in the blood. In addition, the information from specific IgG N-glycosylation profiles in T2D could shed light on underlying inflammatory pathophysiological processes and lead to drug targets.

Multilevel capillary gel electrophoresis characterization of new antibody modalities.

Capillary gel electrophoresis-based methods were applied to comprehensively characterize two development phase new modality monoclonal antibodies including a glycoengineered and a bispecific test compound. The samples were subjected to multilevel characterization at the intact (both by SDS-SGE and cIEF) as well as the reduced protein and the released N-glycan levels. SDS capillary gel electrophoresis analysis showed excellent separation of the light and heavy chains of both samples. The bispecific antibody required a special temperature gradient denaturation process and a longer capillary to resolve its two light chain fragments. Separation of PNGase F digested antibodies revealed migration time shifts, suggesting the presence of N-linked glycosylation on the corresponding subunits. For efficient glycan removal, the highly glycosylated glycoengineered monoclonal antibody was trypsin digested prior to the endoglycosidase treatment. The released glycans were profiled by capillary gel electrophoresis after APTS labeling and their oligosaccharide structures were identified by exoglycosidase based carbohydrate sequencing. Finally, capillary isoelectric focusing shed light on the charge heterogeneity of the test compounds, providing important complementary information. A flowchart was established for workflow optimization.

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control.

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2'-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2'- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.

Ultrafast high-resolution analysis of human milk oligosaccharides by multicapillary gel electrophoresis.

There is recently growing interest towards synthesized human milk oligosaccharides (HMOs) as baby formula additives, and interestingly also as dietary supplements for adults. Currently quite a few manufacturers synthesize HMOs, however, their analysis is challenging, both in resolution and speed. In this paper an ultrafast high-resolution method is introduced for the separation of HMOs by multicapillary gel electrophoresis. Two gel compositions were evaluated with complementary resolving power. One was a conventionally used industrial standard carbohydrate separation matrix, resolving oligosaccharides according to their charge to hydrodynamic volume ratios. The other one was a borate-buffered dextran gel, which utilized the secondary equilibrium of the borate-vicinal diol complexation to enhance resolution. Considering the rapid analysis time and multiplexing (12-channel system), a 96 well sample plate can be analyzed in less than 80 min with the conventional type carbohydrate separation matrix and in less than one hour with the borate-buffered dextran gel. Exploiting the one fluorophore per molecule labeling stoichiometry, the limit of detection (S/N > 3) and limit of quantitation (S/N > 10) were determined as 0.025 and 0.100 mg/mL, respectively, with good linearity. Based on the calibration plot, the quantities of several low concentration HMOs were determined from a human milk sample.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Modeling of the Desialylated Human Serum N-glycome for Molecular Diagnostic Applications in Inflammatory and Malignant Lung Diseases.

BACKGROUND: Immunoglobulin G and A, transferrin, haptoglobin and alpha-1- antitrypsin represent approximately 85% of the human serum glycoproteome and their N-glycosylation analysis may lead to the discovery of important molecular disease markers. However, due to the labile nature of the sialic acid residues, the desialylated subset of the serum N-glycoproteome has been traditionally utilized for diagnostic applications. OBJECTIVE: Creating a five-protein model to deconstruct the overall N-glycosylation fingerprints in inflammatory and malignant lung diseases. METHODS: The N-glycan pool of human serum and the five high abundant serum glycoproteins were analyzed. Simultaneous endoglycosidase/sialidase digestion was followed by fluorophore labeling and separation by CE-LIF to establish the model. Pooled serum samples from patients with COPD, lung cancer (LC) and their comorbidity were all analyzed. RESULTS: Nine significant (>1%) asialo-N-glycan structures were identified both in human serum and the standard protein mixture. The core-fucosylated-agalacto-biantennary glycan differentiated COPD and LC and both from the control and the comorbidity groups. Decrease in the core-fucosylated-agalacto-biantennary-bisecting, monogalacto and bigalacto structures differentiated all disease groups from the control. The significant increase of the fucosylated-galactosylated-triantennary structure was highly specific for LC, to a medium extent for COPD and a lesser extent for comorbidity. Also, some increase in the afucosylated-galactosylated-biantennary structure in all three disease types and afucosylated-galactosylated-triantennary structures in COPD and LC were observed in comparison to the control group. CONCLUSION: Our results suggested that changes in the desialylated human serum Nglycome hold glycoprotein specific molecular diagnostic potential for malignant and inflammatory lung diseases, which can be modeled with the five-protein mixture.

Multilevel Characterization of Antibody-Ligand Conjugates by CESI-MS.

AIM: To demonstrate the capabilities of our new capillary electrophoresis - mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. BACKGROUND: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. OBJECTIVE: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. MATERIALS AND METHODS: Capillary electrophoresis coupled with electrospray ionization mass spectrometry was used to analyze the antibody-ligand conjugates. RESULTS: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. CONCLUSION: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

Expanding the capillary electrophoresis-based glucose unit database of the GUcal app.

GUcal is a standalone application for automatically calculating the glucose unit (GU) values for separated N-glycan components of interest in an electropherogram and suggests their tentative structures by utilizing an internal database. We have expanded the original database of GUcal by integrating all publicly available capillary electrophoresis (CE) data in the GlycoStore collection (https://www.glycostore.org) and with in-house measured GU values. The GUcal app is freely available online (https://www.gucal.hu) and readily facilitates CE-based high throughput GU value determination for first line structural elucidation.

Comparative analysis of the human serum N-glycome in lung cancer, COPD and their comorbidity using capillary electrophoresis.

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to their comorbidity. In this pilot study the N-glycosylation profile of pooled human serum samples (90 patients each) from lung cancer, COPD and comorbidity (LC with COPD) patients were investigated in comparison to healthy individuals (control) by capillary gel electrophoresis with high sensitivity laser-induced fluorescence detection. Sample preparation was optimized for human serum samples introducing a new temperature adjusted denaturation protocol to prevent precipitation and increased endoglycosidase digestion time to assure complete removal of the N-linked carbohydrates. The reproducibility of the optimized method was <3.5%. Sixty-one N-glycan structures were identified in the pooled control human serum sample and the profile was compared to pooled lung cancer, COPD and comorbidity of COPD with lung cancer patient samples. One important finding was that no other sugar structures were detected in any of the patient groups, only quantitative differences were observed. Based on this comparative exercise, a panel of 13 N-glycan structures were identified as potential glycobiomarkers to reveal significant changes (>33% in relative peak areas) between the pathological and control samples. In addition to N-glycan profile changes, alterations in the individual N-glycan subclasses, such as total fucosylation, degree of sialylation and branching may also hold important glycobiomarker values.

Sample Preparation Scale-Up for Deep N-glycomic Analysis of Human Serum by Capillary Electrophoresis and CE-ESI-MS.

We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.

Imaging Laser-Induced Fluorescence Detection at the Taylor Cone of Electrospray Ionization Mass Spectrometry.

Laser-induced fluorescence detection (LIF) is a powerful tool for the quantitative analysis of fluorescent molecules, widely used in glycan analysis with fluorophore labeled carbohydrates where each species has a common response factor. Electrospray ionization mass spectrometry (ESI-MS), on the other hand, while revealing important structural information about individual analytes, generally can have different response factors for different species. For simpler and improved quantitation with ESI-MS, laser-induced fluorescent images were collected at the Taylor cone of the electrospray interface, enabling simultaneous and robust optical (quantitative) and MS (qualitative) detection of fluorophore labeled sugars. The performance of this universally applicable, interface design independent imaging laser-induced fluorescent (iLIF) system was demonstrated using capillary electrophoresis (CE)-ESI-MS in the analysis of aminopyrene-trisulfonate labeled linear maltooligosaccharides and branched glycans from human immunoglobulin. The limit of detection (LOD) of the iLIF system was in this case 40 attomole. The intra- and interday quantitative (peak area) reproducibilities of the system (RSD) were 4.15% and 6.79%, respectively.

Evaporative fluorophore labeling of carbohydrates via reductive amination.

As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF). Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation.

Glycosimilarity assessment of biotherapeutics 1: Quantitative comparison of the N-glycosylation of the innovator and a biosimilar version of etanercept.

The carbohydrate moieties on the polypeptide chains in most glycoprotein based biotherapeutics and their biosimilars play essential roles in such major mechanisms of actions as antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, anti-inflammatory functions and serum clearance. In addition, alteration in glycosylation may influence the safety and efficacy of the product. Glycosylation, therefore, is considered as one of the important critical quality attributes of glycoprotein biotherapeutics, and consequently for their biosimilar counterparts. Thus, the carbohydrate moieties of such biopharmaceuticals (both innovator and biosimilar products) should be closely scrutinized during all stages of the manufacturing process. In this paper we introduce a rapid, capillary gel electrophoresis based process to quantitatively assess the glycosylation aspect of biosimilarity (referred to as glycosimilarity) between the innovator and a biosimilar version of etanercept (Enbrel® and Benepali®, respectively), based on their N-linked carbohydrate profiles. Differences in sialylated, core fucosylated, galactosylated and high mannose glycans were all quantified. Since the mechanism of action of etanercept is TNFα binding, only mannosylation was deemed as critical quality attribute for glycosimilarity assessment due to its influence on serum half-life.

Quantitative assessment of mAb Fc glycosylation of CQA importance by capillary electrophoresis.

The attached carbohydrates at the highly conserved asparagine-linked glycosylation site in the CH 2 domain of the fragment crystallizable (Fc) region of monoclonal antibody therapeutics can play an essential role in their mechanism of action, including ADCC, CDC, anti-inflammatory functions, and serum half-life. Thus, this particular glycosylation represents one of the important critical quality attributes (CQA) of therapeutic monoclonal antibodies, which should be closely monitored and controlled during all stages of biopharmaceutical manufacturing. To study Fc glycosylation related quantitative critical quality attributes, the N-glycan pool of adalimumab (Humira® ) was spiked with increasing amounts of mannose-5 oligosaccharide, a glycan with high CQA importance. The method enabled precise quantitative CQA assessment with high detection sensitivity.

Effect of the flow profile on separation efficiency in pressure-assisted reversed-polarity capillary zone electrophoresis of anions: Simulation and experimental evaluation.

Capillary electrophoresis connected to electrospray ionization mass spectrometry is a promising combination to analyze complex biological samples. The use of sheathless electrospray ionization interfaces, such as a porous nanoelectrospray capillary emitter, requires the application of forward flow (either by pressure or electroosmosis) to maintain the electrospray process. The analysis of solute molecules with strong negative charges (e.g., aminopyrenetrisulfonate labeled glycans) necessitates a reversed-polarity capillary electrophoresis separation mode, in which case the electroosmotic flow is counter current, thus pressure assistance is necessary. In this study, we compared the effect of forced convection with and without counter electroosmotic flow on the resulting separation efficiency in capillary electrophoresis based on flow profile simulations by computational fluid dynamics technique and by actual experiments. The efficiencies of the detected peaks were calculated from the resulting electropherograms and found approximately 950 000 plates/m for electrophoresis with counter electroosmotic flow, 20 000 plates/m with pressure only (such as would be in open tubular liquid chromatography), and 480 000 plates/m for electrophoresis with simultaneous counter electroosmotic flow and forward pressure assistance, which validates the simulation data.