Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies

Kamoda, Satoru; Ishikawa, Rei; Kakehi, Kazuaki

doi:10.1016/j.chroma.2006.08.028

Cited by 111 publications

(103 citation statements)

References 35 publications

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“…Since the release of a light chain is easier than that of the heavy chains (which are held together by two disulfide bonds in hinge region), the light chain is predominant LMW fragment in the sample. The shoulders of the peaks of the heavy chain and half antibody (marked with *) are probably their non-glycosylated forms (as suggested by [8]) or the results of incomplete SDS binding [6] and/or unfolding of the mAb [6]. With the CGE the LMW components could be well separated from each other and from the monomer.…”

Section: Comparison Of Cze and Cge Using Sds-sieving Matrix For Hetermentioning

confidence: 72%

“…Earlier the CGE was also applied to separate the size variants (fragments, aggregates) of mAb [6][7][8]. In our experiments the Rtx was diluted with the sample matrix buffer just before the injection.…”

Section: Comparison Of Cze and Cge Using Sds-sieving Matrix For Hetermentioning

confidence: 99%

“…While the size heterogeneity of the monoclonal antibodies was studied by size-exclusion chromatography [3,4], peptide mapping [4], LC-MS [5] or gel electrophoresis [6][7][8], the charge heterogeneity was mostly studied by cation-exchange chromatography [3,4]. The different methods of capillary electrophoresis (CZE, CGE, CIEF) are useful in the complex analysis of the mAbs.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

G¹

2014

J Chromatogr Sep Tech

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This paper focuses on the applicability of capillary zone electrophoresis (CZE) using uncoated fused silica capillaries for the determination of heterogeneity of rituximab (MabThera, Roche) and for the study of its solution and thermal stability. The best resolution for the charge variants of the main component was obtained with a buffer electrolyte containing 800 mM 6-amino caproic acid, 2 mM triethylene tetramine and 0.05% hydroxypropyl methylcellulose at pH = 5.2. It was found that the pH and the components of the buffer used for the electrophoretic separation and for the dilution of the sample prior to the analysis are important to the stability of the rituximab. We demonstrated the rituximab is stable in the pharmaceutical product MabThera due to the stabilizing additives, but the dilution of the MabThera caused a slow formation of acidic variants, while the amount of the basic variants did not change. After incubation of the diluted rituximab at higher temperature several charge variants could be determined by CZE.

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Section: Comparison Of Cze and Cge Using Sds-sieving Matrix For Hetermentioning

confidence: 72%

Section: Comparison Of Cze and Cge Using Sds-sieving Matrix For Hetermentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

G¹

2014

J Chromatogr Sep Tech

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“…These results show that peak A is a glycan with Neu5Gc residues at the non-reducing end. By contrast, no peaks disappeared with pre-injection of anti-Neu5Gc antibody in rituximab [61].…”

Section: Specific Detection Of N-glycolylneuraminic Acid Andmentioning

confidence: 93%

“…This antibody contains minute amount of high-mannose and hybrid type oligosaccharides [55]. A typical electropherogram for the analysis of APTS-labeled oligosaccharides from rituximab is shown in Fig.…”

Section: Pface Analyses Of Apts-labeled Oligosaccharides From Rituximabmentioning

confidence: 99%

Highly Sensitive Methods Using Liquid Chromatography and Capillary Electrophoresis for Quantitative Analysis of Glycoprotein Glycans

Suzuki

2014

Chromatography

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This review summarizes our recent works concerned on the analysis of glycoprotein glycans. Glycoprotein glycans are highly complicated and have variations that number into the thousands. Therefore the method for their analyses should be carefully chosen according to the complexity and quantity of glycans in samples. To shorten the time for the preparation of labeled glycans from glycoprotein, we proposed two methods: PNGase F/NBD-F method enables the preparation of fluorescently labeled glycans from glycoproteins about 2 hr. A combination of a neutrally coated capillary and borate buffer eliminates the purification steps for the removal of excess reagents from the glycan samples for capillary electrophoresis. Partial filling affinity capillary electrophoresis using glycan-recognizing proteins such as lectins, glycosidases and immunoglobulins, was used for the structural analysis of glycans. The method was applied to the specific detection of NeuGc and α-Gal containing glycans known as the heterogenic antigens. Sensitivity of the detection of glycoprotein glycans separated by microchip-based electrophoresis was enhanced by in situ fabricated sufonate-type polyacrylamide gel. This method enhances the sensitivity by a factor of 10 5 . In situ fabrication of lectin impregnated gels enables specific entrapment and analysis of glycans. Conversion reaction from fluorescently labeled glycans (1-amino-1-alditols) to free oligosaccharides will be helpful for the functional analysis of glycans.

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Analytical Characterization of Proteins and Peptides

Wang¹,

Hosken²

2013

Pharmaceutical Sciences Encyclopedia

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Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies

Cited by 111 publications

References 35 publications

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

Highly Sensitive Methods Using Liquid Chromatography and Capillary Electrophoresis for Quantitative Analysis of Glycoprotein Glycans

Analytical Characterization of Proteins and Peptides

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