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The Inducible System: Antigens
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
Recent progress in NMR (nuclear magnetic resonance) spectroscopy has allowed an even more precise resolution of antigen-antibody complexes. To resolve those parts of the antibody molecule that contact the antigen, the NMR analysis is combined with a technique called hydrogen-deuterium exchange. In this procedure, the antigen-antibody complex is suspended in heavy water [(D2O), in which the D stands for the hydrogen isotope deuterium]. The hydrogens of the antigen molecule are gradually replaced by deuterium atoms with the exception of those hydrogens that are in regions protected from hydrogen exchange by the bound antibody. NMR analysis of the antigen after the antigen is separated from the antibody reveals which of the amino acids were in contact with the antibody since they did not undergo hydrogen exchange.
Order Blubervirales: Core Protein
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Second, the hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) (Bereszczak et al. 2013) and the native mass spectrometry (MS) and gas phase electrophoretic mobility molecular analysis (GEMMA) (Bereszczak et al. 2014) were applied to resolve HBc-antibody complexes. These alternative methods were less time consuming than electron cryomicroscopy and still sufficient for the preliminary characterization of virus-antibody complexes.
3D Particles
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Using hydrogen/deuterium exchange-mass spectrometry (HDX-MS), Morton et al. (2010a) located regions of the MS2 coat dimer that exhibited conformational/dynamical changes, and hence changes in their HDX kinetics, upon binding to a genomic RNA stem-loop known to trigger the assembly initiation. These results demonstrated the potential utility of the HDX-MS to probe conformational and dynamical changes within the non-covalently bound protein-RNA complexes (Morton et al. 2010a).
Ab-Ligity: identifying sequence-dissimilar antibodies that bind to the same epitope
Published in mAbs, 2021
Wing Ki Wong, Sarah A. Robinson, Alexander Bujotzek, Guy Georges, Alan P. Lewis, Jiye Shi, James Snowden, Bruck Taddese, Charlotte M. Deane
The highest resolution method for studying antibody-antigen binding configurations is co-crystal complex structures. These give atomic level information but are expensive and difficult to obtain.2 Experimental mapping is often used as a surrogate because it is able to identify the binding regions of the antigen (“epitopes”) and antibody (“paratopes”; ref. 3). Competition assays exploit the cross-blocking effect of antibodies that displace one another if they bind to similar or neighboring epitopes.3,4 This method gives a coarse representation of which binders may share similar target sites, as minimal epitope overlap can be sufficient for a pair of antibodies to compete with each other.4 A more refined approach is hydrogen deuterium exchange (HDX). HDX assesses the solvent accessibility of the bound and unbound forms of the partner proteins, and highlights regions with the maximum changes upon binding (e.g., ref.5,6). The resolution is typically up to the range of peptides in the immediate proximity of the binding site. To achieve residue-level resolution, point mutations of the interacting proteins can be used to indicate key binding residues. Mutagenesis studies measure the binding kinetics upon mutation of specific residues, but structural integrity may be compromised by the mutations, leading to spurious results.7 All three of these experimental techniques provide an approximation of the binding regions, but are usually unable to provide a fine mapping of exact epitopes and paratopes.
Process optimization and protein engineering mitigated manufacturing challenges of a monoclonal antibody with liquid-liquid phase separation issue by disrupting inter-molecule electrostatic interactions
Published in mAbs, 2019
Qun Du, Melissa Damschroder, Timothy M. Pabst, Alan K. Hunter, William K. Wang, Haibin Luo
There is no effective a priori approach to predict whether a mAb will exhibit LLPS during downstream processing. Our study illuminates steps in the screening cascade to evaluate the potential for LLPS. Moving forward, we propose to evaluate the surface charge of a mAb destined for development, considering the number and position on the protein structure. For instance, when there are several charged residues in the CDRs (e.g., 10 in mAb-X vs. 4 in mAb-Y), the mAb’s behavior in solution at pH close to the pI at a low ionic strength (a condition favoring LLPS) should be evaluated. For this purpose, use of in silico graphics modeled on the protein structure resolved by crystal X-ray diffraction or NMR would be ideal. Others have reported antibody engineering guided by crystal structures that successfully reduced viscosity and self-association.6,24 When a crystal structure is not available, the combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and homology modelling is also an effective approach.16,24 Generally, HDX-MS is time consuming, and tight development timelines often do not allow such an in-depth study for a therapeutic mAb at an early development phase.
Hydrogen/deuterium exchange-mass spectrometry analysis of high concentration biotherapeutics: application to phase-separated antibody formulations
Published in mAbs, 2019
Yuwei Tian, Lihua Huang, Brandon T. Ruotolo, Ning Wang
Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a versatile tool for the assessment of protein conformations, dynamics, and interactions and is now increasingly applied to mAb analysis.15-19 However, traditional HDX-MS workflows are typically initiated through the exchange of labile backbone amide hydrogens by diluting protein samples into a D2O-containing buffer.19 Thus, the use of HDX-MS has been limited for analyzing protein samples at very high concentrations. Recently, HDX-MS workflows designed for the analysis of high concentration protein samples have been described.20,21 For example, a recently described HDX-MS methodology that relies upon reconstituting lyophilized mAb powders in a deuterated buffer was able to characterize mAb structures at 60 mg/mL.20 This approach identified protein–protein interfaces associated with a concentration-dependent reversible self-association. While lyophilization combined with HDX-MS can provide protein structure information in a dilution-free mode, the workflow introduces a reconstitution step and is limited to those buffers amenable to the lyophilization process. To overcome these limitations, a dialysis-coupled HDX-MS strategy was recently reported for mAb analysis, in which passive dialysis microcassettes are used for HDX labeling.21 While this approach successfully sampled high concentration (200 mg/mL) IgG4 formulations for comparison with low concentration (3 mg/mL) samples, the long timescales needed for dialysis likely render many known modes of protein motion inaccessible to the technology.