Capillary electrophoresiselectrospray ionization mass. In addition to high resolution glycosylation profiling, sugar residue and. Fast glycan labeling and analysis kit for the ma m application guide fast glycan labeling and analysis kit. Advances in techniques used for n glycan structural identification. One approach to this analysis is by using capillary zone electrophoresis cze with laser induced fluorescence lif to monitor the abundance of individual monosaccharides from released and hydrolyzed n glycan chains on the mab biotherapeutic. Analysis of glycans derived from glycoconjugates by. One approach to this analysis is by using capillary zone electrophoresis cze with laser induced fluorescence lif to monitor the abundance of individual monosaccharides from released and. Highly sensitive methods using liquid chromatography and capillary electrophoresis for quantitative analysis of glycoprotein glycans shigeo suzuki faculty of pharmaceutical sciences, kinki university. An alternative platform to analyze nglycan sequence using multiplexed capillary gel electrophoresis was developed based on a dna. Expanding the capillary electrophoresisbased glucose unit database of the gucal app gabor jarvas. Carbohydrate sequencing by exoglycosidase arrays is described in conjunction with capillary electrophoresis ce to identify linkage and positional isomers.
Analysis of nglycans is assisted by the existence of several enzymes that can release nglycans. All 9 important glycoanalysis steps are thoroughly discussed. A series of detailed optimal workflows and best practices would help to harmonize analytical procedures between and. Advantages and disadvantages of capillary electrophoresis. Automated nglycosylation sequencing of biopharmaceuticals. Exoglycosidase digestion, when combined with highperformance separation techniques such as chromatography or electrophoresis, is a rapid and costeffective method for oligosaccharide. Of these, the most wellknown is peptidenglycosidase f pngase f, an amidase that releases intact complex bianennary, highmannose and hybridtype nglycans by cleaving between the innermost glcnac of the nglycan and asparagine, unless the glcnac is. There is a need for a method for glycan profiling by capillary electrophoresis and a capillary electrophoresis instrument for performing the method that reduces costs, with simplicity in operation.
Phospholipase c plc belongs to a class of enzymes that cleave phospholipids to. Ce is a 12 versatile analytical platform, which can be operated in several separation modes, simply. We want to perform glycan analysis using tissue samples human and mouse. Ce analysis was conducted after each digestion step obtaining in. Capillary electrophoresis with laserinduced fluorescence celif is one of the typical techniques for nglycan analysis due to its high separation efficiency.
Electrophoresis running buffer systems using ammonium acetate and. Performance liquid chromatography and capillary gel electrophoresis with fluorescent labeling. Precision of glycan detection and separation among 24 capillaries figure 2 aptslabeled mixed glycans prepared from fetuin and human serum igg were run from 24 capillaries. Pdf microfluidic capillary electrophoresismass spectrometry for.
High throughput and high resolution glycan analysis by capillary electrophoresis on applied biosystems dna sequencers results fig. Ce is a 12 versatile analytical platform, which can be operated in several separation modes, simply by altering separation conditions 14 during operation. Analysis of nglycans from a monoclonal antibody by capillary. Capillary electrophoresis separations of glycans ncbi nih. Innovations in glycan analysis on the pa 800 plus, lcms and. The narrow tubes used in capillary electrophoresis help to give the technique good resolution. Liquid chromatography uplc and capillary electrophoresis ce analytical platforms. Preassembled cartridge k f capillary cartridge k f and capillaries, barefused i 50 m i. Glycoform characterization of erythropoietin combining. Analysis of nglycans from a monoclonal antibody by capillary electrophoresis and mass spectrometry application note biopharma abstract glycosylation is one of the most important posttranslational. This chapter highlights recent developments in the analysis of proteins glycosylated of the amino groups n of asparagine residues i. High resolution labchip gxii, touch ht perkinelmer.
The fully automated platform is demonstrated to result in high quality of glycan profiles analyzed on both uplc. Miyako nakano, daisuke higo, etsuo arai, takatoshi nakagawa, kazuaki kakehi, naoyuki taniguchi, akihiro kondo, capillary electrophoresiselectrospray ionization mass spectrometry for. Nglycan analysis of biotherapeutic proteins biopharm. The method provides high resolution capable of separating carbohydrates by chargeto. The advantages of using capillary tubes are that lateral diffusion effects are reduced and temperature differences. Dec 12, 2019 expanding the capillary electrophoresisbased glucose unit database of the gucal app. Abstract a capillary zone electrophoresislaserinduced fluorescence detection czelif method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin. Monoclonal antibody charge heterogeneity analysis by capillary isoelectric focusing on the agilent 7100 capillary electrophoresis system application note biopharma abstract due to the importance of. The analysis of isomeric glycan structures has been attained using several separation methods, including high.
Capillary electrophoresis fundamentals capillary zone electrophoresis separations are wellsuited to. Mass spectrometry method for complex glycan analysis using a flowthrough microvial interface roxana g. In 1991, the first glycan analysis by celif was demonstrated, using 34carboxybenzoyl2. Profiling of nlinked glycans from 100 cells by capillary. Sample preparation scaleup for deep nglycomic analysis of. Pdf chapter 20 analysis of glycoproteins and their. Application of capillary electrophoresis in glycoprotein. Capillary electrophoresis separations of glycans chemical. Capillary electrophoresis glucose unit glycan software structure assignment doi 10.
Capillary electrophoresis laser induced fluorescence high resolution and high speed glycan analysis for microheterogeneity determination rapid sample preparation and analysis of monoclonal antibody nglycans by magnetic bead technology and celif. Here, we describe the application of a microfluidicsbased cems system for analysis of released glycans, glycopeptides and monosaccharides. Highresolution mass spectrometry ms and highperformance liquid chromatography hplc the most commonly applied. Expanding the capillary electrophoresisbased glucose unit. Glycan sequencing using exoglycosidases sigmaaldrich. Capillary electrophoresis ce is a widely used technique in biosciences. Capillary electrophoresis ce is a versatile analytical method used to characterize glycoproteins.
Highly sensitive methods using liquid chromatography and capillary electrophoresis for quantitative analysis of glycoprotein glycans shigeo suzuki faculty of pharmaceutical sciences, kinki university, 341 kowakae, higashiosaka, osaka 5778502, japan abstract. High throughput and high resolution glycan analysis by capillary electrophoresis on applied biosystems dna sequencers development of a rapid and robust n glycan profiling method for therapeutic antibodies with uhplc and multi capillary ce n glycan analysis by orthogonal methods. Innovations in glycan analysis on the pa 800 plus, lcms and cesims. A single analytical platform for glycan analysis, charge. Pdf application of capillary electrophoresis in glycoprotein analysis. Capillary electrophoresis for total glycosaminoglycan. Fast glycan labeling and analysis kit designed to rapidly label glycoprotein released nglycans with apts followed by effective removal of the excess dye using magnetic bead based cleanup. Capillary electrophoresis methods have been developed that are capable of separating glycans with the same monosaccharide sequence but different positional isomers, as well as determining whether monosaccharides composing a glycan are alpha or beta linked. Analysis of the capillary electrophoresis separation results to identify the type of glycans in the. Recent developments in liquid chromatography and capillary. We consider to purchase a capillary electrophoresis machine pace mdq, however we are not sure if it is user friendly. When a sample is introduced in a tube and an electric field applied the components move at different rates leading to separation. Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. Fast glycan labeling and analysis kit fast glycan labeling and analysis kit.
A single analytical platform, the capillary electrophoresisbased sciex pa 800. Highthroughput glycosylation analysis of therapeutic. The method provides high resolution capable of separating carbohydrates by chargetosize ratio. Their diagnostic and biopharmaceutical importance, biomedical chromatography on. Analysis of nglycans from a monoclonal antibody by. High throughput and high resolution glycan analysis by.
Chromatography and capillary gel electrophoresis with fluorescent. Based glycan structural assignment method for capillary electrophoresis analysis. Ce analysis was conducted after each digestion step obtaining in this way. Now, the most important uses of glycan profiles obtained from chromatography is to monitor physiological states, and for the quality control of glycoproteinaceous pharmaceuticals. Automated nglycosylation sequencing of biopharmaceuticals by. Read glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis electrospray time. Selection of glycoanalysis technologies, methods, and strategy there is a wide array of technologies that can be applied to glycan analysis see tables iiii.
Chapter 20 analysis of glycoproteins and their glycopeptide and glycan fragments by electrophoresis and capillary electrophoresis. Guttman, largescale carbohydrate analysis by capillary array electrophoresis. W some versions of the fast glycan labeling and analysis include a preassembled cartridge. Glycan analysis is an important field of bioanalytics, especially in clinical and biopharmaceutical research as well as in production quality control of glycoprotein therapeutics. Abstract glycans are a class of macromolecules essential for all forms of. We have used several modes of ce separation such as cesds gel, imaged capillary isoelectric focusing icief, and capillary zone electrophoresis cze to study therapeutic glycoprotein products. Nacetylhexosaminidase 1 l, and after 32 min incubation at 60 c the reaction mixture were injected and analyzed by celif. Temperature gradient capillary electrophoresis was introduced to enhance separation selectivities for branched glycans of biotherapeutic interest. Principle applications are heavily focused on nglycans. In this paper we introduce an automated carbohydrate sequencing. Ce designed for rapid glycan analysis glyq is a small, simple, user friendly capillary electrophoresis ce system with an easilyreplaceable gel cartridge. Analysis of glycans derived from glycoconjugates by capillary.
Capillary electrophoresis separations of glycans request pdf. The labeled glycans are then separated by high resolution capillary electrophoresis with laser induced flourescence detection using the hrncho separation. Capillary electrophoresis in the nglycosylation analysis of. Glycan analysis ce with laserinduced fluorescence detection. Cesds gel is applied to characterize the glycan occupancy and number of glycosylation sites, and icief is used to study the charge heterogeneities due to sialic. Oct 25, 2008 miyako nakano, daisuke higo, etsuo arai, takatoshi nakagawa, kazuaki kakehi, naoyuki taniguchi, akihiro kondo, capillary electrophoresiselectrospray ionization mass spectrometry for rapid and sensitive nglycan analysis of glycoproteins as 9fluorenylmethyl derivatives, glycobiology, volume 19, issue 2, february 2009, pages 5143, s. The glycanassure system is an integrated glycan analysis platform with three components. Application of capillary electrophoresis in glycoprotein analysis. Capillary electrophoresis in the nglycosylation analysis. View the article pdf and any associated supplements and figures for a period. As it is applied to carbohydrate analyses, the separation technique has been interchangeably referred to as ce or capillary gel electrophoresis.
Common sample preparation methods in glycan analysis. Analysis of nglycans released from proteins of therapeutic. Innovations in glycan analysis on the pa 800 plus, lcms. Aug 31, 2018 capillary electrophoresis ce with laserinduced fluorescence lif detector has been a powerful tool for oligosaccharide analysis due to its high sensitivity and resolution. Read strategies for analysis and structure characterization of glycansproteoglycans by capillary electrophoresis. Analysis of nglycans from a monoclonal antibody by capillary electrophoresis and mass spectrometry application note biopharma abstract glycosylation is one of the most important posttranslational modi. The labeled glycans are then separated by high resolution capillary electrophoresis with laser induced flourescence detection using the hrncho separation matrix. A mixture of afucosylated, fucosylated, and high mannose oligosaccharides was separated in the range of 15 to 45 c at 5 c temperature intervals. The high resolution labchip is designed to be compatible with the glycan screening assay and low molecular weight protein assay on the labchip gxii microfluidic capillary electrophoresis cesds platform. Separation of the labeled glycans by capillary electrophoresis with c detectionk 6. Guttman, structure analysis of highmannose type oligosaccharides by ulcms and capillary electrophoresis, electrophoresis, 25 2004 20032009. Based glycan structural assignment method for capillary electrophoresis. In 1991, the first glycan analysis by celif was demonstrated, using 34carboxybenzoyl2quinolinecarboxaldehyde cbqca fluorescencelabeling reagent, for amino sugars, and glycoproteinderived nglycans 25, 52, 53. The emergence of capillary electrophoresis ce with the various modes.
Capillary electrophoresis and capillary electrophoresismass. Comparative ce analysis of apts labeled released glycans from polyclonal human igg using pct and atmospheric ndeglycosylation with 1. This paper describes the application of hplc and capillary electrophoresis ce for the analysis of oligosaccharide chains. In nglycosylation analysis of biopharmaceuticals, analytical need depends on the phase of the manufacturing process. The following are examples of the commonly used techniques in glycan analysis. Fast glycan labeling and analysis kit application guide. Strategies for analysis and structure characterization of. Carbohydrate sequencing by exoglycosidase arrays isomers is described 10 in conjunction with capillary electrophoresis ce to identify linkage and positional. Sep 12, 2018 capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. All important glycoanalysis steps are thoroughly discussed.
High fidelity glycan sequencing using a combination of. Precision of glycan detection and separation among 24 capillaries. Online capillary electrophoresislaserinduced fluorescence. Glycan analysis is an important field of bioanalytics. Dec 16, 2011 analysis of glycans derived from glycoconjugates by capillary electrophoresis. Dna fingerprinting after the dna has been amplified, it can be separated by ce. Her research work focused on glycan analyses using ce coupled with ms. Characterization of nlinked glycosylation in a monoclonal. Principle applications are heavily focused on nglycans, which are highly relevant to biological therapeutics and biomarker research. Simple capillary electrophoresis mass spectrometry method. Capillary electrophoresis laserinduced fluorescence celif. This quantitative approach was applied to nine biotherapeutic monoclonal antibodies.
Highresolution glycan analysis by temperature gradient. Profiling of nlinked glycans from 100 cells by capillary electrophoresis with largevolume dual preconcentration by isotachophoresis and. N glycans are typically released by peptide n glycosidase pngase f, fluorescently labeled with 8aminopyrene1,3,6trisulfonic acid apts, and detected. High fidelity glycan sequencing using a combination of capillary electrophoresis and exoglycosidase digestion andras guttman senior manager applications, separations sciex, brea, ca 92822 the rapidly increasing usage of glycoproteins as biopharmaceutical products has created a demand for fast. Monoclonal antibody charge heterogeneity analysis by. Capillary electrophoresis fundamentals capillary zone electrophoresis separations are wellsuited to biomolecular separations because the method is based on transport in an electric. Capillary electrophoresis all capillary electrophoresis separations were performed on a. Capillary electrophoresis and capillary electrophoresis. An integrated application for capillary electrophoresis based glycan analysis recent emergence in the use of monoclonal antibody therapeutics and other glycoprotein. Highly sensitive methods using liquid chromatography and. Monoclonal antibody charge heterogeneity analysis by capillary isoelectric focusing on the agilent 7100 capillary electrophoresis system application note biopharma abstract due to the importance of monoclonal antibodies mab as therapeutic agents, there is a growing demand for high resolution analytical methods to characterize these complex. On the cart page you will be able to print your cart.
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