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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.

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.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (

With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently used methods to analyze biotherapeutic proteins in process control as well as in release and stability testing. In this work, the effect of the monomer (dextran) and cross-linker (borate) ratio was studied in SDS-CGE analysis of a therapeutic monoclonal antibody test item in its reduced and intact forms. A retention model was developed for better understanding of the separation selectivity between the non-glycosylated and glycosylated heavy chain fragments, exploiting the interaction between the dextran-borate adducts and the glycan moiety of the therapeutic antibody. The monomer cross-linker ratio played a significant role in the overall analysis times and affected the separation selectivity between the non-glycosylated and regular (glycosylated) heavy chain fragments; however, it had no effect on the separation of the regular and non-glycosylated intact forms of the monoclonal antibody. Introduction of three-dimensional selectivity plots offered an easy separation optimization option for the separation problem in hand.

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.

Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction, featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors, there are several quality control (QC) parameters that should be rigorously monitored to comply with clinical safety and efficacy. This review gives a short summary of the most frequently used AVV production and purification methods, focusing on the analytical techniques applied to determine the full/empty capsid ratio and the integrity of the encapsidated therapeutic DNA of the products.

Aims: 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.

Background: Type 2 diabetes (T2DM) and colorectal cancer (CRC) are both known to modulate gene expression patterns in peripheral blood leukocytes (PBLs). Objective : As T2DM has been shown to increase the incidence of CRC, we were prompted to check whether diabetes affects mRNA signatures in PBLs isolated from CRC patients. Methods : Twenty-two patients were recruited to the study and classified into four cohorts (healthy controls; T2DM; CRC; CRC and T2DM). Relative expression levels of 573 cell signaling gene transcripts were determined by reverse transcription real-time PCR assays run on low-density OpenArray platforms. Enrichment analysis was performed with the g:GOSt profiling tool to order differentially expressed genes into functional pathways. Results : 49 genes were found to be significantly up- or downregulated in tumorous diabetic individuals as compared to tumor-free diabetic controls, while 11 transcripts were differentially regulated in patients with CRC versus healthy, tumor-free and nondiabetic controls. Importantly, these gene sets were completely distinct, implying that diabetes exerts a profound influence on the transcription of signaling genes in CRC. The top 5 genes showing the most significant expression differences in both contexts were PCK2, MAPK9, CCND1, HMBS, TLR3 (p≤0.0040) and CREBBP, PPIA, NFKBIL1, MDM2 and SELPLG (p≤0.0121), respectively. Functional analysis revealed that most significantly affected pathways were cytokine, interleukin and PI3K/Akt/mTOR signaling cascades as well as mitotic regulation. Conclusions : We propose that differentially expressed genes listed above might be potential biomarkers of CRC and should be studied further on larger patient groups. Diabetes might promote colorectal carcinogenesis by impairing signaling pathways in PBLs.

Background: Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms, including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for the bulk release of plasmid products; thus, it should be analyzed accordingly. Methods and Results: The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduced a fast, sensitive, and robust plasmid analysis method using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). CGE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent DNA binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis. Conclusion: As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CGE-LIF to monitor plasmid topology and possible degradation.

Chronic obstructive pulmonary disease (COPD) and lung cancer are two major diseases of the lung with high rate of mortality, mostly among tobacco smokers. The glycosylation patterns of various plasma proteins show significant changes in COPD and subsequent hypoxia, inflammation and lung cancer, providing promising opportunities for screening aberrant glycan structures contribute to early detection of both diseases. Glycoproteins associated with COPD and lung cancer consist of highly sialylated N-glycans, which play an important role in inflammation whereby hypoxia leads to accumulation of sialyl Lewis A and X glycans. Although COPD is an inflammatory disease, it is an independent risk factor for lung cancer. Marked decrease in galactosylation of plasma immunoglobulin G (IgG) together with increased presence of sialic acids and more complex highly branched N-glycan structures are characteristic for COPD and lung cancer. Numerous glycan biomarkers have been discovered, and analysis of glycovariants associated with COPD and lung cancer has been carried out. In this paper we review fundamental glycosylation changes in COPD and lung cancer glycoproteins, focusing on IgG to provide an opportunity to distinguish between the two diseases at the glycoprotein level with diagnostic value.

Lung adenocarcinoma is one of the leading causes of mortality among cancer patients worldwide and Chronic Obstructive Pulmonary Disease (COPD) is also high in death statistics. In addition, patients with Chronic Obstructive Pulmonary Disease (COPD) have a high risk of developing primary lung cancer. Prevention, risk estimation and a non-invasive diagnostics are essential to decrease COPD and lung cancer mortality. Therefore, better and more accurate molecular diagnostic markers (biomarkers) are needed for the early differential diagnosis of these lung diseases to help clinicians make better therapeutic decisions. This review focuses on recently discovered adenocarcinoma and COPD biomarkers at the proteome and glycome level. In the first part, the protein markers are summarized, while the second part is focused on glycan markers. Their use to differentiate between chronic inflammation (COPD) and malignant (adenocarcinoma) diseases is discussed in detail.

Protein glycosylation is the most complex and prevalent post-translation modification in terms of the number of proteins modified and the diversity generated. To understand the functional roles of glycoproteins it is important to gain an insight into the repertoire of oligosaccharides present. The comparison and relative quantitation of glycoforms combined with site-specific identification and occupancy are necessary steps in this direction. Computational platforms have continued to mature assisting researchers with the interpretation of such glycomics and glycoproteomics data sets, but frequently support dedicated workflows and users rely on the manual interpretation of data to gain insights into the glycoproteome. The growth of site-specific knowledge has also led to the implementation of machine-learning algorithms to predict glycosylation which is now being integrated into glycoproteomics pipelines. This short review describes commercial and open-access databases and software with an emphasis on those that are actively maintained and designed to support current analytical workflows.

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, β-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.

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.

Background: A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. These highly variable and versatile post-translational modifications finetune protein functions by influencing sorting, folding, enzyme activity and subcellular localization. However, relatively little is known about regulatory factors of this procedure and about the accurate causative connection between glycosylation and disease. Objective: The aim of the present study was to investigate whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma. Methods: A total of 32 SNPs localized in genes related to N-glycosylation were selected for the association analysis. Polymorphisms with putative biological functions (missense or regulatory variants) were recruited. SNPs were genotyped by a TaqMan OpenArray platform. A single base extension-based method in combination with capillary gel electrophoresis was used for verification. Results: The TaqMan OpenArray approach provided accurate and reliable genotype data (global call rate: 94.9%, accuracy: 99.6%). No significant discrepancy was detected between the obtained and expected genotype frequency values (Hardy–Weinberg equilibrium) in the healthy control sample group in case of any SNP confirming reliable sampling and genotyping. Allele frequencies of the rs3944508 polymorphism localized in the 3’ UTR of the MGAT5 gene significantly differed between the sample groups compared. Conclusion: Our results suggest that the rs34944508 SNP might modulate the risk for lung cancer by influencing the expression of MGAT5. This enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) in beta 1-6 linkage to the alpha-linked mannose of biantennary N-linked oligosaccharides, thus, increasing branching that is the characteristic of invasive malignancies.

The temperature-dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) in the interval from 15 to 60 °C using borate cross-linked dextran sieving matrix. Arrhenius plots were generated to calculate the respective activation energy values for the various solute molecules. SDS-CGE analysis of the biotherapeutic protein test mixture revealed no correlation between the activation energy requirement of the different species and their molecular weights, emphasizing the importance of separation temperature optimization to obtain high resolution between the solute molecules of interest. In contrast, the molecular weight protein size ladder ranging from 10 to 225 kDa, built from the same polypeptide blocks with no post-translational and other modifications, showed predictable activation energy requirement. The electrophoretic mobility of the SDS-protein complexes was found to be the function of the reciprocal sixth root of the molecular weight (

Absztrakt: A krónikus obstruktív tüdőbetegség (COPD) világszerte előkelő helyet foglal el a morbiditási és mortalitási statisztikákban. A COPD megelőzhető és kezelhető betegség, kialakulásáért döntően a dohányzás tehető felelőssé. A prevenció kulcsfontosságú, de korlátozottan kivitelezhető, így a rizikó meghatározása és a korai noninvazív diagnosztika révén lehetne tovább csökkenteni a COPD miatti halálozást. A fejlődő diagnosztikus technikák ellenére az optimális szűrővizsgálat felfedezése még várat magára. Kellően szenzitív és specifikus biomarkerek felfedezése megfelelő eszközt adhat a klinikus kezébe a betegség korai diagnosztizálásához, a differenciáldiagnosztikához, a fenotipizáláshoz és a prognózis becsléséhez. Közleményünkben a COPD vonatkozásában a közelmúltban felfedezett potenciális fehérje és glikán biomarkereket foglaljuk össze. Orv Hetil. 2020; 161(4): 123–128.

A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

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.

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we report on the development and implementation of a high-throughput capillary electrophoresis based glycan analysis workflow for urinary PSA analysis. The technology utilizes selective, high yield single domain antibody based PSA capture, followed by preconcentration and capillary electrophoresis coupled with laser-induced fluorescence detection, resulting in high resolution N-glycan profiles. Urinary PSA glycan profiles were compared to a commercially available PSA standard revealing differences in their α2,3- and α2,6-sialylated isomers, proving the excellent selectivity of the suggested workflow. This is important as sialylation classification plays an important role in the differentiation between indolent, significant and aggressive forms of prostate cancer.

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.

Capillary electrophoresis-based glycan separation and structure identification is one of the most frequently used tools in almost all fields of biological-medical research and biopharmaceutical manufacturing. In this chapter, recent advances in database search assisted glycan structure identification techniques are discussed. The underlying theory together with the principles of the different approaches as well as practical aspects are described in detail targeting both academic and industrial audience. Methods, such as traditional glucose unit calculation, the so-called virtual ladder approach, exoglycosidase based glycan sequencing, as well as possible utilization options of the current databases are detailed emphasizing their fundamentals and applications.

The human serum N-glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the N-glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the N-glycosylation pattern of their serum samples was analyzed before and after the surgery using capillary electrophoresis separation with laser-induced fluorescent detection. The relative peak areas of 21 N-glycans were evaluated from the acquired electropherograms using machine learning-based data analysis. Individual glycans as well as their subclasses were taken into account during the course of evaluation. For the data analysis, both discrete (e.g., smoker or not) and continuous (e.g., age of the patient) clinical parameters were compared against the alterations in these 21 N-linked carbohydrate structures. The classification tree analysis resulted in a panel of N-glycans, which could be used to follow up on the effects of lung tumor surgical resection.

Simple Summary Globally, there were around 2.1 million lung cancer cases and 1.8 million deaths in 2018. Hungary—where this study was carried out—had the highest rate of lung cancer in the same year. We developed a new analytical method which can be readily used to follow up the tumor surgery by investigating the glycan (sugar) structures of proteins. As the results of such investigations are very complex, computer-assisted machine learning methods were utilized for data interpretation. Abstract The human serum N-glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the N-glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the N-glycosylation pattern of their serum samples was analyzed before and after the surgery using capillary electrophoresis separation with laser-induced fluorescent detection. The relative peak areas of 21 N-glycans were evaluated from the acquired electropherograms using machine learning-based data analysis. Individual glycans as well as their subclasses were taken into account during the course of evaluation. For the data analysis, both discrete (e.g., smoker or not) and continuous (e.g., age of the patient) clinical parameters were compared against the alterations in these 21 N-linked carbohydrate structures. The classification tree analysis resulted in a panel of N-glycans, which could be used to follow up on the effects of lung tumor surgical resection.

Human milk is a complex, dynamically changing biological fluid, which contains a large amount of non-conjugated carbohydrates, referred to as human milk oligosaccharides (HMOs). These HMOs are very important for the infants as they play important roles in the formation of the gut microbiome, the immune system and support brain development. HMOs show highly complex structural diversity due to numerous linkage possibilities of the building monosaccharides. In order to elucidate their structure-function relationship and to develop more effective infant formulas, cutting-edge analytical technologies are in great demand. In this paper, we review the current strategies for HMO analysis based on liquid phase separation methods. High performance liquid chromatography, capillary electrophoresis and their hyphenation with mass spectrometry are critically reviewed, emphasizing their advantages and disadvantages from practical point of views. Recent advances of the methods are categorized according to their application fields.

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 (mu(eff)) 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 mu(eff) 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 mu(eff) 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.

A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr4+ modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ultraviolet (UV) irradiation and then functionalized with Zr4+. The obtained monolith was characterized with scanning electron microscopy and mercury intrusion porosimetry. Large through-pores and a continuous skeleton with high permeability were observed. The N-glycans were labeled with the 1-aminopyrene-3, 6, 8-trisulfonic acid (APTS) and enriched by the Zr4+ modified monolith through IMAC interaction. This enrichment step was then coupled off-line to capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Successful preconcentration of the APTS labeled maltooligosaccharide ladder was achieved under optimized conditions. Enrichment factors obtained for the maltooligosaccharides ranged from 9 to 24 with RSDs from 2.0% to 9.2% (n = 3). Moreover, very good repeatabilities (

The utilization of N-glycan profiling recently gained high importance in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In this paper, the effect of human blood sample handling was studied prior to N-glycosylation profiling by capillary electrophoresis, coupled with high sensitivity fluorescence detection. Special attention was paid to the preservation of sialylated structures because of their important clinical - biological relevance. Our results suggested that it is adequate to refrigerate and store the collected total blood samples prior to analysis to obtain unbiased results. Furthermore, we report on the good practice of serum sample handling in order to prevent decomposition of the sialylated structures. Our findings may promote procedure standardization and easier clinical translation of diagnostic N-glycosylation profiling in molecular medicinal applications.

Prostate cancer has the highest malignancy rate diagnosed in men worldwide. Albeit, the gold standard serum prostate-specific antigen (PSA) assays reduced the mortality rate of the disease, the number of false positive diagnoses steeply increased. Therefore, there is an urgent need for complementary biomarkers to enhance the specificity and selectivity of current diagnostic methods. Information about PSA glycosylation can help to fulfill this gap as alterations of its carbohydrate moieties due to cancerous transformation may represent additional markers to distinguish malignant from benign tumors. However, development of suitable methods and instrumentations to investigate the N-glycosylation profile of PSA represents a challenge. In this paper, we critically review the current bioanalytical trends and strategies in the field of PSA glycobiomarker research focusing on separation based characterization methods.

Although knowledge on glycan biosynthesis and processing is continuously maturing, there are still a limited number of studies that examine biological functions of N-glycan structures in plants, which remain virtually unknown. Here, the statistical correlation between nutrient (nitrogen) deficiency symptoms of crops and changes in 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled complex type free oligosaccharides is reported. While deficiency symptoms are predicted by multispectral images and Kjeldahl digestion, APTS-labeled complex type free oligosaccharides are identified by their glucose unit (GU) values in tomato xylem sap, using capillary electrophoresis with laser induced fluorescence detection (CE-LIF). Given the limited number of structures obtained from plants, archived in the literature, in the future, it is intended to create an open access database of promising indicators, namely, glycan structures that are presumably responsible for the nutrient deficiency caused stress in plants (http://glycoplants.org).

The market segment of new biological drugs (monoclonal antibodies, fusion proteins, antibody-drug conjugates, and new modality protein therapeutics) is rapidly growing, especially after the patent expiration of the original biologics, initiating the emergence of biosimilars. N-glycosylation of therapeutic proteins has high importance on their stability, safety, immunogenicity, efficacy, and serum half-life. Therefore, Nglycosylation is considered to be one of the critical quality attributes. Consequently, it should be rigorously monitored during the development, manufacturing, and release of glycoprotein biologicals. In this review, first, the regulatory considerations for biosimilars are shortly summarized, followed by conferring the analytical techniques needed for monitoring and characterization of the N-glycosylation of biological drugs. Particular respect is paid to liquid phase separation techniques with high sensitivity and highresolution detection methods, including laser-induced fluorescence and mass spectrometry.

Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (

More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field.

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

The effect of air plasma exposure time on the surface energies and acid-base characteristics of polydimethylsiloxane (PDMS) particles was studied. Polymerized PDMS powder was radio frequency induced air plasma irradiated for 2–10 s with the power of 500 W. The efficiency of the plasma treatments was investigated by a new generation inverse gas chromatograph, a surface energy analyzer. The dispersive component of surface energy was determined by the Dorris-Gray method describing the van der Waals interactions, while the specific component of surface energy expressed the surface ability for Lewis acid-base interactions. It was demonstrated that the air plasma treatment did not affect the dispersive and acidic parts of the surface energy and the change of surface hydrophilicity was caused by the raise of the electron-donor ability of the PDMS surface, characterized by van Oss-Good-Chaudhury’s base number. The optimal plasma treatment time was found to be 5 s. Analysis of the specific surface energy and acid-base characteristics with respect to exposure time showed that partial to complete hydrophobic recovery occurred within 38 h.

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.

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.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.