In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 µg/mL propidium iodide in the separation medium (LOD 2 µg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 µg/mL, but using the local maximum at 100 µg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Ionic liquids in capillary electrophoresis analysis of proteins and carbohydrates.

Ionic liquids (ILs), as non-molecular type solvents, possess excellent physical-chemical properties, which make them useful in important separation applications in gas chromatography, liquid chromatography, and capillary electrophoresis. Among a plethora of potential uses of ionic liquids in separation science, capillary electrophoresis can utilize its resolution-enhancing effect in the analysis of proteins and carbohydrates, via the formation of intermolecular interactions, e.g., hydrophobic, hydrogen bonding, or electrostatic. ILs and polymeric ionic liquids (PIL) also represent an excellent choice as background electrolyte (BGE) additives for capillary coatings in CE, which is especially important in protein analysis. Another interesting utilization of ILs is the fabrication of monoliths for capillary electrochromatography in which instance the mechanism of retention is based on ion exclusion interactions. Carbohydrates can also be readily analyzed by CE with the help of ionic liquids without the need for an extra derivatization step. One of the future perspectives on the use of ILs is their utilization in the recently emerging biopharmaceutical industry exploiting the increased resolution of proteins and carbohydrates, two of the important components of glycoprotein therapeutics. In this paper, we address the so-far not-reviewed ionic liquid-mediated analysis of proteins and carbohydrates by capillary electrophoresis-based techniques also addressing their impact on the separation mechanism.

Capillary Zone Electrophoresis of 8-Aminopyrene-1,3,6-trisulfonic Acid Labeled Carbohydrates with Online Electrokinetic Sample Cleanup.

Capillary electrophoresis is one of the frequently used separation techniques for the analysis of complex carbohydrates. Since sugars lack chromophore or fluorophore groups, their capillary electrophoresis analysis usually requires tagging by a charged fluorophore. To speed up the derivatization reaction, a large excess of the labeling reagent is typically used; therefore, a purification step is necessary prior to CE analysis using the industry standard low-pH gel-buffer system. In addition to representing an extra sample preparation step with the associated labor and cost, the purification process also holds the risk of losing some of the sample components. In this paper we introduce an online electrokinetic sample cleanup process with electroosmotic flow (EOF)-assisted separation in a bare fused silica capillary using alkaline pH background electrolyte and normal polarity separation voltage. 8-Aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltooligosaccharides were analyzed first to understand the complex effect of the downstream EOF and the counter current electromigration of the sample components including the labeling dye. The use of 150 mM caproic acid-253 mM Tris (pH 8.1) running buffer facilitated the entrance of the sample components of interest into the separation capillary, while the excess labeling reagent was excluded and, therefore, did not interfere with the detection. The alkaline caproic acid-Tris running buffer was then applied to the N-glycome analysis of human serum samples, showing excellent separation performance, and more importantly, the extra sample purification step was not required.

Size separation of sodium dodecyl sulfate-proteins by capillary electrophoresis in dilute and ultra-dilute dextran solutions.

SDS capillary gel electrophoresis is a widely used in the biopharma and the biomedical fields for rapid size separation of proteins. However, very limited information is available on the use of dilute and ultra-dilute sieving matrices for SDS-protein analysis. Here, background electrolytes (BGEs) containing 1%-0% dextran were used in borate-based BGE to separate a protein sizing ladder (PSL) ≤225 kDa and the intact and subunit forms of a therapeutic monoclonal antibody (mAb). The separation performance for the PSL and mAb components differed significantly with decreasing dextran concentration. Ferguson and reptation plots were used to elucidate the separation mechanism. Highly diluted dextran solutions resulted in linear Ferguson plots for both solute types (cf. Ogston theory) in spite of this model assumes a rigid pore structure, thus cannot describe the separation mechanism in ultra-dilute polymer solutions with no reticulations. The saddle differences between the resolution of the PSL and the intact/subunit mAb forms in ultra-dilute dextran-borate matrices suggested the importance of shape selectivity, manifested by the adequate separation of the SDS covered intact as well as light and heavy chain subunits of the therapeutic mAb even at zero dextran concentration.

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.

Analysis of Peptides and Proteins by Native and SDS Capillary Gel Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry via a Closed-Circuit Coaxial Sheath Flow Reactor Interface.

A simple and widely applicable coaxial sheath flow reactor interface (CSFRI) is introduced for easy and robust connection of liquid-phase microseparation methods to mass spectrometric detection, especially for capillary gel electrophoresis analysis of proteins and peptides including SDS-protein complexes. The interface readily accommodated post-column reactions prior to MS detection. It was demonstrated that this novel closed-circuit connection allowed the utilization of non-MS friendly buffer components without significant ion suppression and supported stable electrospray. In SDS capillary agarose gel electrophoresis mode, addition of γ-cyclodextrin to the sheath liquid efficiently removed the SDS content of the sample and the background electrolyte in the flow reactor section by inclusion complexation, while maintaining good separation efficiency and decreasing ion suppression.

Microbead-based extracorporeal immuno-affinity virus capture: a feasibility study to address the SARS-CoV-2 pandemic.

In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Single-domain antibodies against the Wuhan (VHH-72) virus strain produced by recombinant DNA technology were immobilized on the surface of glass micro-beads, which were then utilized as stationary phase. For feasibility testing, the virus suspension was flown through the prototype immune-affinity device that captured the viruses and the filtered media left the column. The feasibility test of the proposed technology was performed in a Biosafety Level 4 classified laboratory using the Wuhan SARS-CoV-2 strain. The laboratory scale device actually captured 120,000 virus particles from the culture media circulation proving the feasibility of the suggested technology. This performance has an estimated capture ability of 15 million virus particles by using the therapeutic size column design, representing three times over-engineering with the assumption of 5 million genomic virus copies in an average viremic patient. Our results suggested that this new therapeutic virus capture device could significantly lower virus load thus preventing the development of more severe COVID-19 cases and consequently reducing mortality rate.

The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis.

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure-function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.

Electromigration Dispersion in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Proteins.

The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340-640 mM) caused electromigration dispersion shifts for both low (2%)- and high (10%)-dextran-concentration gels in all gel-buffer compositions. In case of the heavy-chain fragment, elevated borate concentrations resulted in decreasing tailing and increasing fronting with the use of higher- and lower-dextran-concentration gels, respectively. The light-chain fragment, on the other hand, exhibited increased fronting with increasing borate concentration for both dextran concentrations examined in this study. Increase of the glycerol ingredient level in the gel-buffer system caused the same effect as the increasing borate concentration in both dextran concentrations. The detected electromigration dispersion was considered as the result of the formation of monomeric and dimeric glycerol-borate complexes as co-ionic constituents, migrating slower than that of the unconjugated tetrahydroxyborate. In addition, complexation of the tetrahydroxyborate anion with the glucose building blocks of the dextran polymer decreased its mobility to practically zero, contributing to further decrease in the resultant effective mobility of the co-ionic species. We suggest that the observed fronting and/or tailing peak shapes of the monoclonal antibody fragments in SDS-CGE at increasing boric acid concentrations can be considered as the result of multiple effects including changes in pH, sieving matrix pore size, viscosity, and the mobility variation of the co-ionic borate adducts with the gel-buffer ingredients. While electromigration dispersion-mediated band broadening, in general, can be minimized via matching the effective mobility of the co-ionic species to the analyte molecules of interest, in case of borate cross-linked dextran gels, optimization of the boric acid concentration required special consideration of its gel cross-linking function. For the light- and heavy-chain fragments of the IgG analyte, best peak shapes were attained with the use of 10% dextran/340 mM boric acid and 10% dextran/640 mM boric acid-containing gel-buffer systems, respectively. Based on this observation, here we introduce the concept of borate-gradient-mediated transient mobility matching in SDS-CGE of proteins. This novel approach resulted in close to optimal peak shapes for the distantly migrating IgG subunits within a single run, as well as unraveled the long-sought possible solution to perform capillary pore-size-gradient gel electrophoresis.

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.

N-Glycosylation of monoclonal antibody therapeutics: A comprehensive review on significance and characterization.

N-glycosylation of therapeutic antibodies starts as a co-translational step followed by a set of post-translational modifications and is considered as one of the critical quality attributes because of its impact on biological functions as well as therapy outcome. In addition to detailed product characterization of these glycans by the manufacturers, their comprehensive analysis is also a regulatory requirement. However, the structural complexity and heterogeneity of these N-linked carbohydrates make their characterization quite challenging. In this review, we give a comprehensive overview of N-glycosylation diversity and its functional importance for monoclonal antibody therapeutic products. A descriptive coverage is also provided for the various strategies and techniques employed for oligosaccharide characterization, that include analysis of intact-glycoproteins, their sub-units, glycopeptides as well as released glycans through chromatographic, electrophoretic and spectroscopic techniques. To assist the readers, relevant examples from the literature are cited and critically discussed for each of the strategies and techniques. To conclude, a discussion on unique challenges associated with the analysis of this important post-translational modification is presented.

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.

Evaluation of an icIEF-MS system for comparable charge variant analysis of biotherapeutics with rapid peak identification by mass spectrometry.

Protein therapeutics are usually produced in heterogeneous forms during bioproduction and bioprocessing. Heterogeneity results from post-translational modifications that can yield charge variants and require characterization throughout product development and manufacturing. Isoelectric focusing (IEF) with UV detection is one of the most common methods to evaluate protein charge heterogeneity in the biopharmaceutical industry. To identify charge variant peaks, a new imaged microfluidic chip-based isoelectric focusing (icIEF) system coupled directly to mass spectrometry was recently reported. Bridging is required to demonstrate comparability between existing and new technology. As such, here we demonstrate the comparability of the pI value measurement and relative charge species distributions between the icIEF-MS system and the control data from a frequently utilized methodology in the biopharmaceutical industry for several blinded development-phase biopharmaceutical monoclonal antibodies across a wide pI range of 7.3-9.0. Hyphenation of the icIEF system with mass spectrometry enabled direct and detailed structural determination of a test molecule, with masses suggesting acidic and basic shifts are caused by sialic acid additions and the presence of unprocessed lysine residues. In addition, MS analysis further identified several low-abundance glycoforms. The icIEF-MS system provides sample quantification, characterization, and identification of mAb proteoforms without sacrificing icIEF quantification comparability or speed.

Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins.

Capillary sodium dodecyl sulfate gel electrophoresis has long been used for the analysis of proteins, mostly either with entangled polymer networks or translationally cross-linked gels. In this paper capillary agarose gel electrophoresis is introduced for the separation of low molecular weight immunoglobulin subunits. The light (LC~24 kDa) and heavy (HC~50 kDa) chain fragments of a monoclonal antibody therapeutic drug were used to optimize the sieving matrix composition of the agarose/Tris-borate-EDTA (TBE) systems. The agarose and boric acid contents were systematically varied between 0.2-1.0% and 320-640 mM, respectively. The influence of several physical parameters such as viscosity and electroosmotic flow were also investigated, the latter to shed light on its effect on the electrokinetic injection bias. Three dimensional Ferguson plots were utilized to better understand the sieving performance of the various agarose/TBE ratio gels, especially relying on their slope (retardation coefficient, KR) value differences. The best resolution between the LC and non-glycosylated HC IgG subunits was obtained by utilizing the molecular sieving effect of the 1% agarose/320 mM boric acid composition (ΔKR = 0.035). On the other hand, the 0.8% agarose/640 mM boric acid gel showed the highest separation power between the similar molecular weight, but different surface charge density non-glycosylated HC and HC fragments (ΔKR = 0.005). It is important to note that the agarose-based gel-buffer systems did not require any capillary regeneration steps between runs other than simple replenishment of the sieving matrix, significantly speeding up analysis cycle time.

Applications of capillary electrophoresis for biopharmaceutical product characterization.

Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization of biopharmaceutical products. It does not only offer fast separation, high resolution in miniaturized format, but equally importantly represents an orthogonal separation mechanism to high-performance liquid chromatography. Therefore, it is not surprising that CE-based methods can be found in all major pharmacopoeias and are recommended for the analysis of biopharmaceutical products during process development, characterization, quality control, and release testing. Different separation formats of CE, such as capillary gel electrophoresis, capillary isoelectric focusing, and capillary zone electrophoresis are widely used for size and charge heterogeneity characterization as well as purity and stability testing of therapeutic proteins. Hyphenation of CE with MS is emerging as a promising bioanalytical tool to assess the primary structure of therapeutic proteins along with any impurities. In this review, we confer the latest developments in capillary electrophoresis, used for the characterization of critical quality attributes of biopharmaceutical products covering the past 6 years (2015-2021). Monoclonal antibodies, due to their significant share in the market, have been given prioritized coverage.

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.

High sensitivity capillary electrophoresis with fluorescent detection for glycan mapping.

We present in this study a novel strategy to drastically improve the detection sensitivity and peak capacity for capillary electrophoresis with laser induced fluorescent detection (CE-LIF) of glucose oligomers and released glycans. This is based on a new approach exploiting a polymer-free background electrolyte (BGE) for CE-LIF of glycans. The best performance in terms of sample stacking and suppression of electroosmotic flow (EOF) was found for a BGE composed of triethanolamine/citric acid and triethanolamine/acetic acid at elevated ionic strengths (IS up to 200 mM). Compared to the conventional protocols for CE-LIF of glucose-oligosaccharides and released glycans, our polymer-free strategy offered up to 5-fold improvement of detection sensitivity and visualization of higher degree of polymerization (DP) of glucose oligomers (18 vs 15). To further improve the detection sensitivity, a new electrokinetic preconcentration strategy via large volume sample stacking with electroosmotic modulation without having recourse to neutrally coated capillaries is proposed, offering a 200-fold signal enhancement. This approach is based on variation of the buffer's IS, rather than pH adjustment as in conventional methods, for EOF modulation or quasi-total reduction. This strategy allows selecting with high flexibility the best pH conditions to perform efficient preconcentration and separation. The new approach was demonstrated to be applicable for the analysis of N-linked oligosaccharides released from a model glycoprotein (Human Immunoglobulin G) and applied to map N-glycans from human serum for congenital disorders of glycosylation (CDG) diagnosis.

Capillary Electrophoresis-Based N-Glycosylation Analysis in the Biomedical and Biopharmaceutical Fields.

Glycomics has a growing interest in the biopharmaceutical industry and biomedical research requiring new high-performance and high-sensitivity bioanalytical tools. Analysis of N-glycosylation is very important during the development of protein therapeutics and it also plays a key role in biomarker discovery. The most frequently used glycoanalytical methods are capillary electrophoresis, liquid chromatography, and mass spectrometry. In this chapter, the capillary electrophoresis-based N-linked carbohydrate analysis methods are conferred with emphasis on its use in the biopharmaceutical and biomedical fields.