Mass spectrometry for glycosylation analysis of biopharmaceuticals

Dotz, Viktoria; Haselberg, Rob; Shubhakar, Archana; Kozak, Radoslaw P.; Falck, David; Rombouts, Yoann; Reusch, Dietmar; Somsen, Govert W.; Fernandes, Daryl L.; Wuhrer, Manfred

doi:10.1016/j.trac.2015.04.024

Cited by 73 publications

(50 citation statements)

References 78 publications

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“…The complexity and heterogeneity of the glycosylation pattern is mainly due to mAbs production in living expression systems [14][15][16] and requires a number of orthogonal analytical techniques to be fully characterized. Several analytical methods have been described for the glyco-variants characterization at different levels (from released glycans to intact protein level) including separative techniques (liquid chromatography (LC), capillary electrophoresis (CE)) often coupled to spectrometric, amperometric and mass spectrometric detection [17][18][19][20][21]. Recently, Reusch's group published two major articles dealing with the analysis of Fc-glycosylation profiles, and comparing several separation methods hyphenated or not with mass spectrometry (MS) detection [20,21].…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

Giorgetti

D’Atri

Canonge

et al. 2018

Talanta

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A B S T R A C TCharacterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.

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Section: Introductionmentioning

confidence: 99%

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

Giorgetti

D’Atri

Canonge

et al. 2018

Talanta

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“…Moreover, N-glycans have a common five-membered trimannosyl chitobiose core, Man␣1-6(Man␣1-3)Man␤1-4GlcNAc␤1-4GlcNAc␤1-Asn-X-Ser/Thr. The highly complex nature of N-glycosylation analysis has given rise to a proliferation of different methods (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Currently, N-glycosylation is examined at the level of intact proteins, protein fragments, peptides, glycans, or monosaccharides.…”

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confidence: 99%

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Leoz

Duewer

Fung³

et al. 2020

Molecular & Cellular Proteomics

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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.

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“…1,2 IMS applications at present include the detection of chemical warfare agents, 3–5 illicit drugs, 6–8 and explosives 9–13 and more recently have been combined with mass spectrometry (i.e., ion mobility (IM)-MS) for the detection, separation, and characterization of biomolecules, e.g., in metabolomics, 14–18 glyco-mics, 19–22 and proteomics applications. 23–36 A constraint of contemporary IMS technology is its limited ability to resolve species with similar mobilities (<2%) in a mixture, particularly in conjunction with achieving high sensitivity.…”

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Ultra-High Resolution Ion Mobility Separations Utilizing Traveling Waves in a 13 m Serpentine Path Length Structures for Lossless Ion Manipulations Module

et al. 2016

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We report the development and initial evaluation of a 13 m path length Structures for Lossless Manipulations (SLIM) module for achieving high resolution separations using traveling waves (TW) with ion mobility (IM) spectrometry. The TW SLIM module was fabricated using two mirror-image printed circuit boards with appropriately configured RF, DC, and TW electrodes and positioned with a 2.75 mm intersurface gap. Ions were effectively confined in field-generated conduits between the surfaces by RF-generated pseudopotential fields and moved losslessly through a serpentine path including 44 “U” turns using TWs. The ion mobility resolution was characterized at different pressures, gaps between the SLIM surfaces, and TW and RF parameters. After initial optimization, the SLIM IM-MS module provided about 5-fold higher resolution separations than present commercially available drift tube or traveling wave IM-MS platforms. Peak capacity and peak generation rates achieved were 246 and 370 s-1, respectively, at a TW speed of 148 m/s. The high resolution achieved in the TW SLIM IM-MS enabled, e.g., isomeric sugars (lacto-N-fucopentaose I and lacto-N-fucopentaose II) to be baseline resolved, and peptides from an albumin tryptic digest were much better resolved than with existing commercial IM-MS platforms. The present work also provides a foundation for the development of much higher resolution SLIM devices based upon both considerably longer path lengths and multipass designs.

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Mass spectrometry for glycosylation analysis of biopharmaceuticals

Cited by 73 publications

References 78 publications

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Ultra-High Resolution Ion Mobility Separations Utilizing Traveling Waves in a 13 m Serpentine Path Length Structures for Lossless Ion Manipulations Module

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