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Capillary gel electrophoresis is a widely used method for rapid separation of fluorophore labeled carbohydrates. Even though, many publications conferred about this popular technique, no report yet investigated the possible sample losses during the purification process of the fluorophore labeling reaction mixture. In the present work, normal polarity capillary zone electrophoresis separation mode was applied to take advantage of the opposite migration directions of the electroosmotic flow and the negatively charged sample components using Tris-hexanoic acid running buffer at basic pH. For purification free oligosaccharide analysis, the separation parameters were designed in such a way that the triple charged labeling reagent of aminopyrenetrisulfonate (APTS) could not enter the separation capillary in contrary to the labeled sample components of interest, therefore, the APTS did not have to be removed before analysis. The method was used to show electrophoretic profile differences possibly caused by the cleanup process that was immediately apparent by comparing the electropherograms of the purified and non-purified APTS labeled maltooligosaccharides. Furthermore, qualitative and quantitative N-glycosylation profile alterations were revealed during CZE separation of the fluorophore labeling reaction mixtures before and after purification along with the analysis of the consecutively used washing solutions for the well characterized standard glycoproteins of IgG, ribonuclease B and fetuin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aims: Application of capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions., Background: Altered N-glycosylation of body fluid glycoproteins can be an effective indicator of most inflammatory processes. Immunoglobulin A (IgA) is the second highest abundant immunoglobulin and has a major role in the immune-defense against potential pathogen attacks. While IgA is abundant in serum, secretory immunoglobulin A (sIgA) is one of the most prevalent proteins in mucosal surfaces, such as in saliva., Objective: Our aim was to investigate the changes of IgA glycosylation in serum and saliva as a response to an administered cytostatic treatment in patients with malignant hematological disorders., Methods: Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) was used to analyze the N-glycosylation profiles of Z(IgA1) partitioned immunoglobulin A in pooled serum and saliva of 10 control subjects and 8 patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions., Results: Eight of 31 and four of 38 N-glycans in serum and saliva, respectively, showed significant (p<0.05) differences upon comparison to the control group. Thirteen glycans were present in the saliva but not in the serum, on the other hand, six structures were found in the serum samples not present in the saliva., Conclusion: The developed Z(IgA1) partitioning and the high resolution CE-LIF based glyocoanalytical methods provided an efficient and sensitive workflow to detect and monitor IgA glycosylation alterations in serum and saliva with the scope for widespread molecular medicinal use., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Formalin-fixed, paraffin-embedded (FFPE) samples are generally used for histology-study, however, they also possess important molecular diagnostics information. While it has been reported that the N-glycan moieties of glycoproteins is not affected by the FFPE process, no information is available about the effect of the elapsed time between sampling and fixation on the resulting N-glycosylation profile. In this study, lung, brain, heart, spleen, liver, kidney, and intestine mouse tissue specimens were used for N-glycan profiling analysis and the elapsed sampling time effect was investigated with the lung tissue. N-glycan extraction from the tissue samples was performed by glycoprotein retrieval from the FFPE specimens using radioimmunoprecipitation assay (RIPA) buffer followed PNGase F digestion. The released oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis-laser induced fluorescent detection (CE-LIF). N-glycosylation profiles of freshly collected lung-tissue samples (zero time point), as well as 1 and 2 h after sampling were compared by carbohydrate profiling and exoglycosidase treatment based deep glycomic analysis. It was found that up to two hours of room temperature storage of tissue specimens apparently did not cause changes in the N-glycosylation profiles of complex carbohydrates, but resulted in considerable decrease in the amount of linear glucose oligomers and high mannose type glycans present in the samples., (© The Authors. Electrophoresis Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Comprehensive analysis of the N-linked carbohydrates of glycoproteins is gaining high recent interest in both the biopharmaceutical and biomedical fields. In addition to high resolution glycosylation profiling, sugar residue and linkage specific enzymes are also routinely used for exoglycosidase digestion based carbohydrate sequencing. This latter one, albeit introduced decades ago, still mostly practiced by following tedious and time consuming manual processes. In this paper we introduce an automated carbohydrate sequencing approach using the appropriate exoglycosidase enzymes in conjunction with the utilization of some of the features of a capillary electrophoresis (CE) instrument to speed up the process. The enzymatic reactions were accomplished within the temperature controlled sample storage compartment of a capillary electrophoresis unit and the separation capillary was also utilized for accurate delivery of the exoglycosidase enzymes. CE analysis was conducted after each digestion step obtaining in this way the sequence information of N-glycans in 60 and 128 minutes using the semi- and the fully-automated methods, respectively.

Multiple myeloma (MM) is an immedicable malignancy of the human plasma cells producing abnormal antibodies (also referred to as paraproteins) leading to kidney problems and hyperviscosity syndrome. In this paper, we report on the N-glycosylation analysis of paraproteins from total human serum as well as the fragment crystallizable region (F c ) and fragment antigen binding (F ab ) κ/λ light chain fractions of papain digested immunoglobulins from multiple myeloma patients. CE-LIF detection was used for the analysis of the N-glycans after endoglycosidase (PNGase F) mediated sugar release and fluorophore labeling (APTS). While characteristic N-glycosylation pattern differences were found between normal control and untreated, treated and remission stage multiple myeloma patient samples at the global serum level, less distinctive changes were observed at the immunoglobulin level. Principal component analysis adequately differentiated the four groups (control and three patient groups) on the basis of total serum N-glycosylation analysis. 12 N-glycan features showed statistically significant differences (p <0.05) among various stages of the disease in comparison to the control at the serum level, while only six features were identified with similar significance at the immunoglobulin level, including the analysis of the partitioned F c fragment as well as the F ab κ and F ab λ chains., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical field, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profiling of glycoproteins. One of the newest exciting developments of the field is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fluorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fluorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.

Despite the ever growing use of capillary electrophoresis in biomedical research and the biopharmaceutical industry, the development of data interpretation methods is lagging behind. In this paper we report the design and implementation of a coinjected triple-internal standard method to alleviate the need of an accompanying run of the maltooligosaccharide ladder for glucose unit (GU) calculation. Based on the migration times of the coinjected standards of maltose, maltotriose, and maltopentadecaose (bracketing the peaks of interest), a data processing approach was designed and developed to set up a virtual ladder that was used for GU calculation. The data processing was tested in terms of the calculated GU values of human IgG glycans, and the resulting relative standard deviation was ≤1.07%. This approach readily supports high-throughput capillary electrophoresis systems by significantly speeding up the processing time for glycan structural assignment.

N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead-based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead-based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead-based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences., (© 2015 Society for Laboratory Automation and Screening.)

Temperature dependent differential migration shifts were studied in capillary electrophoresis between linear (maltooligosaccharides) and branched (sialylated, neutral and core fucosylated biantennary IgG glycans) carbohydrates. Background electrolytes without as well as with low and high molecular weight additives (ethylene glycol, linear polyacrylamide and poly(ethylene oxide)) were investigated for this phenomena in the temperature range of 20-50 °C. Glucose unit (GU) value shifts were observed with increasing temperature for the all IgG glycans both in additive-free and additive-containing background electrolytes, emphasizing the importance of tight temperature control during glycosylation analysis by capillary electrophoresis. The activation energy concept was applied to understand the structure specific electrophoretic migration of the different sugar molecules. Activation energy values were derived from the slopes of the Arrhenius plots of logarithmic mobility vs reciprocal absolute temperature and compared for the linear and branched sugars as well as for the various background electrolyte additives.

Recent emergence in the use of monoclonal antibody therapeutics and other glycoprotein biopharmaceuticals requires high-throughput, robust, and automated techniques for their glycosylation analysis. Capillary electrophoresis is one of the high-performance methods of choice; however, while the necessary instrumentation is well developed, the related bioinformatics tools are lacked behind. In this paper, we introduce an integrated toolset dubbed as GUcal, to automatically calculate the glucose unit (GU) values for all sample components of interest in an electropherogram with a concomitant database search for structural assignment. The database comprises CE GUs and suggested structures of N-glycans released from human IgG. The app is freely available online (www.lendulet.uni-pannon.hu/gucal) and readily facilitates CE-based glycan analysis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on solid surfaces in a spatially discrete pattern, are sensitive and versatile tools for the analysis of glycosylation changes in complex biological samples. Glycoarrays are also suitable for monitoring multiple molecular interactions with biomolecules where sugars are involved, offering a large variety of bioassay options. In this paper we review the most important glycan microarray types currently used with their main applications, and discuss some of the future challenges the technology faces.

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Glycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis. We here describe a short and robust assay to prepare 8-aminopyrene-1,3,6-trisulphonic acid-labeled N-glycans from serum samples. It only involves the subsequent addition of reagents and incubation in a PCR thermocycler. Moreover, this assay allows the detection of these glycans, which are only present in minute amounts in serum, on high-throughput microfluidics CE platforms including the MCE-202 MultiNA, 2100 Bioanalyzer, and eGene system. Using this clinical glycomics assay, we could reliably measure GlycoHepatoTest, a panel of biomarkers allowing the follow-up of chronic liver disease patients from the early stage onward.

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use.