Sheathless CE‐MS as a tool for monitoring exchange efficiency and stability of bispecific antibodies

Gstöttner, Christoph; Vergoossen, Dana L.E.; Huijbers, Maartje G.; Domínguez‐Vega, Elena

doi:10.1002/elps.202000166

Cited by 12 publications

(7 citation statements)

References 12 publications

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“…The exchange efficiency and residual amount of the bivalent (monospecific) anti-MuSK antibodies were determined with capillary electrophoresis (CE) hyphenated with mass spectrometry (MS) (27). The monovalent (bispecific) IgG4 was separated from its two bivalent parents with CE, permitting reliable determination of their relative amounts down to 0.5% ( Fig.…”

Section: Significancementioning

confidence: 99%

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

Vergoossen

Plomp

Gstöttner

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed “Fab-arm exchange” in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.

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Section: Significancementioning

confidence: 99%

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

Vergoossen

Plomp

Gstöttner

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…Type Surface charge References polyarginine dynamic cationic [254] linear polyacrylamide (LPA) permanent (covalent) neutral [122,231] polyvinyl alcohol (PVA) permanent (covalent) neutral [235][236][237] hydroxypropyl methylcellulose (HPMC) permanent (covalent) neutral [236] N-acryloylamidoethoxyethanol permanent (covalent) neutral [255] PS1 neutral coating permanent (covalent) neutral [239,253,238] linear carbohydrate polymer (LCP) permanent (covalent) neutral [248] polyethyleneimine (PEI) permanent (covalent) cationic [229,230,233,234] (3-aminopropyl) diisopropylethoxysilane permanent (covalent) cationic [256] omega-iodoalkylammonium salts (M7C4I) permanent (covalent) cationic [232] polybrene (PB) permanent (adsorbed) cationic [122] PB-dextran sulfate-PB permanent (adsorbed) cationic [122,144] successive multiple ionic polymer layer (SMIL) permanent (adsorbed) cationic [122,125,126] anionic polymer permanent (adsorbed) anionic [257] coating procedures, easy coating regeneration, and minimal dependence on surface chemistry. The preparation of covalent coatings is usually more laborious and timeconsuming.…”

Section: Coatingmentioning

confidence: 99%

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Maráková

Opetová

Tomašovský

2023

J of Separation Science

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Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis‐mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in‐depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis‐mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis‐mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018–March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

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“…With the assistance of hinge region cleavages using enzymes and disulfide‐bond reduction, the system separated six different subunits of the BsAbs. More recently, the authors further successfully applied the CZE‐MS system to monitor exchange efficiency and stability of in‐house produced BsAbs (Gstöttner et al, 2021).…”

Section: Ce‐ms For Multilevel Proteomics Applicationsmentioning

confidence: 99%

Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

Chen

McCool

Yang

et al. 2021

Mass Spectrometry Reviews

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Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels.Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges.capillary isoelectric focusing-mass spectrometry, capillary zone electrophoresis-mass spectrometry, multilevel proteomics, protein complex, proteoform | INTRODUCTIONProteomics aims to characterize proteins in cells comprehensively as a function of biological conditions, including but not limited to their expression, posttranslational modifications (PTMs), interactions, locations, and turnover (Aebersold & Mann, 2016;Zhang et al., 2013). Mass spectrometry (MS)-based proteomics has become crucial for pursuing better understandings of molecular mechanisms of fundamental cellular processes and disease development (Aebersold & Mann, 2016). There are three main strategies: bottom-up proteomics (BUP), top-down proteomics (TDP), and native proteomics.BUP is the most mature and widely used strategy. It identifies proteins through the MS and tandem MS (MS/MS) measurements of peptides derived from the enzymatic

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Sheathless CE‐MS as a tool for monitoring exchange efficiency and stability of bispecific antibodies

Cited by 12 publications

References 12 publications

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)

Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

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