Hydrogen/Deuterium Exchange Mass Spectrometry (HDX MS) in the Studies of Architecture, Dynamics, and Interactions of Biopharmaceutical Products

Kaltashov, Igor A.; Bobst, Cedric E.; Abzalimov, Rinat R.

doi:10.1002/9781118180730.ch10

Cited by 5 publications

(4 citation statements)

References 73 publications

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“…Characterization of transiently expressed RVT1 revealed no post-translational modifications (PTMs) within its light chain and two PTMs within the heavy chain (conversion of the Nterminal Gln residue to pyroGlu and removal of the C-terminal Lys residue). Both PTMs are common among mAbs and are innocuous (neither the antibody stability nor its antigenbinding properties are compromised by these PTMs 37 ).…”

Section: Reverse Engineering Of the Anti-pf4 Vitt-associatedmentioning

confidence: 99%

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia

Ivanov,

Ivetic,

et al. 2023

J. Am. Chem. Soc.

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The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which�vaccine-induced immune thrombotic thrombocytopenia (VITT)�is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an Nglycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentrationdependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1) 3 (PF4) 2 , pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

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Section: Reverse Engineering Of the Anti-pf4 Vitt-associatedmentioning

confidence: 99%

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia

Ivanov,

Ivetic,

et al. 2023

J. Am. Chem. Soc.

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“…Detection of N-glycosylated peptides was carried out by using reporter ions corresponding to mono-and disaccharides in the fragment ion spectra. 50 Specifically, the following reporter ions were used: m/z 204.09 (HexNAc), m/z 274.10 (NeuAc-H 2 O), m/z 292.11 (NeuAc), and m/z 366.14 (Hex/HexNAc or HexNAc/Hex). In addition to identifying four glycopeptides representing the canonical N-glycosylation site within the C H 2 domain of the antibody (see Figure S4 in the Supporting Information), application of this filter allowed another glycopeptide to be identified (Figure 4A).…”

Section: Intact Mass Analysis Of Anti-pf4 Antibodies In the Clinical ...mentioning

confidence: 99%

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

Nguyen,

Le,

Ivanov

et al. 2024

Anal. Chem.

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Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviralvectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the V H domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1−IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

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“…Disulfide-containing proteins account for about a third of the human proteome, 28 and their fraction among the extracellular proteins is significantly larger; furthermore, nearly all multiunit therapeutic proteins contain external disulfide bridges. 29 Structural studies of noncovalent assemblies formed by interactions of such proteins with their physiological partners and/or therapeutic targets are often challenging due to their large size and heterogeneity. Native MS frequently fills this gap by allowing both composition and stoichiometry of these complexes to be determined; however, information about quaternary organization of such complexes proved difficult to obtain.…”

Section: ■ Conclusionmentioning

confidence: 99%

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis

Ivanov,

Cheung,

Kaltashov

2024

Anal. Chem.

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Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

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Hydrogen/Deuterium Exchange Mass Spectrometry (HDX MS) in the Studies of Architecture, Dynamics, and Interactions of Biopharmaceutical Products

Cited by 5 publications

References 73 publications

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis

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