Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

Lippold, Steffen; Nicolardi, Simone; Domínguez‐Vega, Elena; Heidenreich, Anna-Katharina; Vidarsson, Gestur; Reusch, Dietmar; Haberger, Markus; Wuhrer, Manfred; Falck, David

doi:10.1080/19420862.2019.1636602

Cited by 44 publications

(50 citation statements)

References 30 publications

(63 reference statements)

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“…[4] In recent years,H PLC-mass spectrometry (MS) analysis of intact proteins has increasingly been used in both academic and industrial environments. [5] Qualitative and quantitative profiling of glycosylation variants (glycoforms) by intact protein mass determination is complementary to released glycan analysis:I tr etains the context of posttranslational modifications (PTMs) and provides information on pairing of protein subunits,a so bserved in dimeric antibodies. [6] This is especially relevant in the context of regulatory issues if glycoform "fingerprinting" is to be considered as aC QA in the future.…”

Section: Introductionmentioning

confidence: 99%

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

et al. 2020

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N‐glycosylation may affect the safety and efficacy of biopharmaceuticals and is thus monitored during manufacturing. Mass spectrometry of the intact protein is increasingly used to reveal co‐existing glycosylation variants. However, quantification of N‐glycoforms via this approach may be biased by single hexose residues as introduced by glycation or O‐glycosylation. Herein, we describe a simple strategy to reveal actual N‐glycoform abundances of therapeutic antibodies, involving experimental determination of glycation levels followed by computational elimination of the “hexosylation bias”. We show that actual N‐glycoform abundances may significantly deviate from initially determined values. Indeed, glycation may even obscure considerable differences in N‐glycosylation patterns of drug product batches. Our observations may thus have implications for biopharmaceutical quality control. Moreover, we solve an instance of the problem of isobaricity, which is fundamental to mass spectrometry.

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Section: Introductionmentioning

confidence: 99%

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

et al. 2020

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“…The absence of fucose on the core Fc‐glycan has been shown to increase binding affinity for FcγRIIIa, thereby increasing the capacity of the antibody for NK cell activation . Similarly, galactosylation is known to enhance FcγRIIIa binding, albeit to a lesser extent than afucosylation, and this is particularly evident in the case of afucosylated IgG . We therefore asked whether fucosylation of RSV‐specific antibodies correlated with the antibody‐induced NK cell activation observed in our assay.…”

Section: Resultsmentioning

confidence: 95%

“…25,38 Similarly, galactosylation is known to enhance FccRIIIa binding, albeit to a lesser extent than afucosylation, and this is particularly evident in the case of afucosylated IgG. 25,39 We therefore asked whether fucosylation of RSV-specific antibodies correlated with the antibody-induced NK cell activation observed in our assay. Indeed, although not very strong, we observed a significant negative correlation between fucosylation and the induction of NK cell IFN-c production ( Figure 6a) and CD107a surface expression ( Figure 6b).…”

Section: Fucosylation Of Rsv-specific Antibodies Correlates With Nk Cmentioning

confidence: 97%

Natural killer cell activation by respiratory syncytial virus‐specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc‐glycosylation

et al. 2020

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Objectives Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody‐mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection. Since antibodies also mediate natural killer (NK) cell activation, we investigated whether this functionality correlates with RSV disease. Methods We performed an observational case–control study including infants hospitalised for RSV infection, hernia surgery or RSV‐negative respiratory viral infections. We determined RSV antigen‐specific antibody levels in plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc‐glycosylation of the RSV‐specific antibodies by mass spectrometry. Results We found that RSV‐specific maternal antibodies activate NK cells in vitro. While concentrations of RSV‐specific antibodies did not differ between cases and controls, antibodies from infants hospitalised for severe respiratory infections (RSV and/or other) induced significantly less NK cell interferon‐γ production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc‐fucosylation of RSV‐specific antibodies, but their glycosylation status did not significantly differ between cases and controls. Conclusion Our results suggest that Fc‐dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of using these properties to evaluate and improve the efficacy of novel vaccines.

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“…The absence of fucose in the core Fc-glycan has been shown to increase binding affinity for FcγRIIIa, thereby increasing the capacity of the antibody for NK cell activation (23, 36). Similarly, galactosylation is known to enhance FcγRIIIa binding, albeit to a lesser extent than afucosylation, and this is particularly evident in the case of afucosylated IgG (23, 37). We therefore asked whether fucosylation of RSV-specific antibodies correlated with the antibody-induced NK cell activation observed in our assay.…”

Section: Resultsmentioning

confidence: 99%

Natural killer cell activation by respiratory syncytial virus-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation

Erp

Lakerveld

Graaf

et al. 2019

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AbstractBackgroundRespiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune system and maternal antibodies. However, the mechanisms by which antibodies can protect against RSV disease are incompletely understood, as both antibody levels and neutralization capacity correlate poorly with protection. We therefore asked whether antibody-mediated natural killer (NK) cell activation correlates with RSV disease.MethodsWe performed an observational case-control study including infants hospitalized for RSV infection (n=43, cases), hernia surgery (n=16, controls), or RSV-negative viral respiratory tract infections (n=18, controls). First, we determined RSV antigen-specific antibody levels in infant plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc-glycosylation of the RSV-specific antibodies by mass spectrometry.ResultsWe found that RSV-specific maternal antibodies potently activate NK cells in vitro. While the concentrations of RSV-specific antibodies did not differ between cases and controls, antibodies from infants hospitalized for severe lower respiratory tract infections (RSV and/or other) induced significantly less NK cell interferon gamma production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc-fucosylation of RSV-specific antibodies, but their glycosylation status did not significantly differ between cases and controls.ConclusionsOur results suggest that Fc-dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of harnessing these activities to develop an effective vaccine.

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Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

Cited by 44 publications

References 30 publications

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

Natural killer cell activation by respiratory syncytial virus‐specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc‐glycosylation

Natural killer cell activation by respiratory syncytial virus-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation

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