Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS

Camperi, Julien; Combès, Audrey; Fournier, Thierry; Pichon, Valérie; Delaunay, Nathalie

doi:10.1007/s00216-020-02684-8

Cited by 23 publications

(13 citation statements)

References 28 publications

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“…Currently, HILIC separation of intact glycoproteins is less common in comparison to the use of HILIC for glycopeptide or released glycan separation, however HILIC has shown great promise in the separation of glycoproteins that vary in glycan occupancy and the amount of neutral glycans. 28,30,55 Given the importance of protein glycosylation in modulating immune responses 12,13 and many other biological processes, we envision HILIC coupled to MS will have great potential in the characterization of heterogeneous glycoprotein products.…”

Section: Discussionmentioning

confidence: 99%

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

Wilson

Bilbao

Wang

et al. 2022

Preprint

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SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid-chromatography coupled to mass spectrometry (LCMS) has been widely used to characterize post-translational modifications in proteins including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogenous glycans at the intact protein level. Herein, we evaluated several online separation techniques: 1. C2 reverse-phase liquid chromatography (RPLC), 2. capillary zone electrophoresis (CZE), and 3. acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down MS. Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation, N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogenous glycoproteins for facile comparison of biosimilars in quality control applications.

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Section: Discussionmentioning

confidence: 99%

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

Wilson

Bilbao

Wang

et al. 2022

Preprint

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“…D'Atri et al described a middle-up approach to compare the glycosylation patterns of originators and biosimilars mAbs by using HILIC coupled to HRMS (108). The HILIC mode was the most suitable for the elucidation of glycosylation patterns, due to the high resolving power and compatibility with MS (87,120,121). Similar strategies were successfully applied for the characterization of an ADC(122), Brentuximab Vedotin, and a recombinant Fc fusion protein, Etanercept (107).…”

Section: Middle-up Analysis 31 Conventional Middle-down/up Analysismentioning

confidence: 99%

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

Camperi

Goyon

Guillarme

et al. 2021

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“…10 Direct infusion electrospray ionization (ESI) MS can be used for intact glycoform analysis of systems with relatively low levels of glycosylation (such as monoclonal antibodies built on IgG templates), 10 although it can certainly benefit from incorporating an online separation step prior to MS analysis. 11 Hydrophilic interaction chromatography (HILIC) remains the most popular option for the glycoform separation prior to the MS step; 12,13 however, it requires the use of mobile phases with a high organic solvent content, inevitably leading to the glycoprotein denaturation. Apart from being problematic for proteins having low tolerance to organic co-solvents, this also creates challenges for the analysis of large oligomeric proteins in which variation of the number of polypeptide subunits within the protein molecule is another source of intrinsic heterogeneity (in addition to glycosylation).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

Yang¹,

Pawlowski²,

Carrick³

et al. 2021

Preprint

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Intact-mass measurements are becoming an increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS measurements provides an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein, but also the mass range populated by various proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation, with the glycan load of phenotype 2-1 being notably lighter. More detailed characterization of their glycosylation patterns is enabled by the recently introduced crosspath reactive chromatography (XP-RC) with on-line MS detection, a technique that combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains, and provides a wealth of information on glycosylation patterns of the heavy chains. The haptoglobin 1-1 glycans are mature fully sialylated biantennary structures that exhibit high degrees of fucosylation. In contrast, phenotype 2-1 contains a significant fraction of incomplete biantennary structures and exhibit significantly lower levels of sialylation and fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of haptoglobin analysis by limited charge reduction, suggesting that the latter can be employed in situations when a fast assessment of a protein heterogeneity is needed (e.g., comparability studies of biopharmaceutical products). <br>

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Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS

Cited by 23 publications

References 28 publications

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

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