Biosimilar, Biobetter, and Next Generation Antibody Characterization by Mass Spectrometry

Beck, Alain; Sanglier-Cianférani, Sarah; Dorsselaer, Alain Van

doi:10.1021/ac3002885

Cited by 226 publications

(199 citation statements)

References 54 publications

(97 reference statements)

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“…These structures represent glycosylation at both F C /2 domains corresponding to the expected G0/G0F, G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2, G1F/G2F, and G2F/G2F forms. The presence of asymmetric glycosylation, characterized by different glycan compositions for both F C /2 domains in monoclonal antibodies, has also been reported previously for mAbs, including Trastuzumab [337,340]. Deconvolution of the MS 1 spectra of dimeric Trastuzumab, presented in Figure 4-9G, shows the composition of dimeric proteoforms.…”

Section: Native Cze-ms Of a Monoclonal Antibodysupporting

confidence: 79%

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Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

Belov¹

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Section: Native Cze-ms Of a Monoclonal Antibodysupporting

confidence: 79%

“…In addition, several dimers (assumed to be non-covalently associated) were also observed (Figure 4-9G). It is important to note that dimers of Trastuzumab could not be observed in denaturing CZE-MS (or LC-MS) experiments conducted by us and others [337].…”

Section: Native Cze-ms Of a Monoclonal Antibodymentioning

confidence: 67%

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

Belov¹

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“…However, concerning MS-based methods [22][23][24][25], a large panel of methodologies were evaluated, except the CE-ESI-MS approach. Nevertheless, in 2008, Gennaro et al described the development of CE-ESI-MS technology with online LIF detection that allows identification of major and minor glycan species observed in the routine CE-LIF assay.…”

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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“…9 Biosimilar antibodies must have the same amino acid sequence as the reference marketed product. 10 However, because mAbs are large molecules with highly complex structures, the products are sensitive to changes in host cells, media, and manufacturing process. 11,12 Because even minor structural changes, including the glycosylation pattern, can affect the safety, purity or potency of the recombinant protein, it is important to characterize these differences.…”

Section: Introductionmentioning

confidence: 99%

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

Sorensen

Harmes

Stoll

et al. 2016

mAbs

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As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.

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Biosimilar, Biobetter, and Next Generation Antibody Characterization by Mass Spectrometry

Cited by 226 publications

References 54 publications

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

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

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

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