Articles in the Category ”References”
IgMBRAZOR™ in a Study on Structural Characterization of IgM
Scientists at Utrecht University and collaborators have described the use of online SEC combined with Orbitrap-based CDMS to perform real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, a novel protease which digests specifically at a single site in the hinge region of IgM. While IgMBRAZOR was shown to efficiently digest IgM, it was observed that digestion of one of the F(ab’)2 subunits occurred more slowly than the other four. It is suggested that interactions of the J-chain within the pentameric IgM structure may influence the accessibility of the IgMBRAZOR enzyme, thus, impacting the digestion kinetics. SEC-CDMS allows the characterization of heterogeneous and large proteins, enabling unique experimental designs such as monitoring of reaction kinetics from physiological buffers.
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TransGLYCIT™ in a Study on Antibody Characterization
Scientists from the Wistar Institute in Philadelphia and collaborators have studied the IgG N-glycan structures and inflammatory aging markers of people living with and without HIV to determine a potential structure-function relationship. In this study, TransGLYCIT was used to generate anti-HIV antibodies with defined N-glycan structures to study Fc-mediated immune activities in vitro.
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GlyCLICK® Enhances Development of Immuno-SPECT/Fluorescent Tracer for PD-L1 in Preclinical Models
Scientists at Université de Bourgogne and collaborators have described the development of a bimodal tracer targeting PD-L1 in human and murine models for preclinical studies. Using a [111In]-DOTA-aza-BODIPY bimodal probe following conjugation onto PD-L1 monoclonal antibodies (mAbs) through either site-specific or random methods, the ability of the bimodal antibodies to selectively identify PD-L1+ tumors were assessed using both fluorescence and SPECT imaging. There were significant benefits observed with the probe generated using the GlyCLICK site-specific conjugation method compared to a random lysine conjugation approach!
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Mechanistic Insights to Drive Rational Design of Masked Antibodies using FabALACTICA®
Scientists at the University of Cambridge, Astra Zeneca and collaborators undertook mechanistic studies to investigate antibody masking and better understand the parameters involved and their relative impact. FabALACTICA proved to be a valuable tool in this study, allowing the authors to generate antibody subunits. Using the Fab region, as opposed to the full-length antibody, allowed for both higher resolution SEC-MALS experiments to be performed, and supported structural determination of the complexes.
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FabULOUS™ in HIV Vaccine Research
The Fc N-glycosylation profile can be linked to non-neutralizing functions in a vaccine. In a recent study by Chen et al., FabULOUS digestion provided valuable insights in HIV vaccine research following N-glycosylation analysis of the Fc region in mouse antibodies.
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Localization of O-glycan Sites in CD24Fc by OpeRATOR® Digestion and LC-MS Analysis
Scientists from Merck & Co. and Glycotope describe the localization of 12 O-glycosylation sites in the heavily O-glycosylated fusion protein CD24Fc by digestion with the O-glycoprotease OpeRATOR in combination with LC-MS analysis. Two different workflows were developed, one based on fractionation in combination with LC-MS/MS CID and one based on LC-MS/MS EThcD. OpeRATOR digestion enabled the localization of O-glycan sites in both workflows and relative quantification of different glycoforms.
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FabRICATOR® in Correlative N-glycan Analysis of Cetuximab Produced in Different Expression Systems
A strategy for comparative characterization of cetuximab produced in a variety of expression cell lines is described in this paper from scientists at the National Institute for Bioprocessing Research and Training (NIBRT) in Dublin, Ireland. FabRICATOR (IdeS) is a critical tool in this study, allowing for site-specific glycan analysis to be performed following antibody digestion into F(ab’)2 and Fc/2 subunits.
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Unravelling Structural Conformers within a Therapeutic Multispecific mAb using FabALACTICA
Scientists at the University of Strasbourg and Sanofi describe the global structural characterisation of a trispecific antibody which exhibits two distinct structural isoforms when analyzed by size-exclusion chromatography (SEC). FabALACTICA proved to be a valuable tool in this study as above hinge digestion of this trispecific antibody allowed for middle-level characterisation of the isomeric Fab regions and facilitated cyclic-ion mobility (cIM) differentiation of the isomers, which was not possible in the intact molecule without prior SEC separation.
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FabRICATOR® in Middle-Up Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies
A middle-level approach to glycan characterisation is described in this paper by scientists at the University of Geneva and collaborators. FabRICATOR (IdeS), a critical tool in this study, is used to generate Fd’, LC and the glycan-containing Fc subunits, which can be chromatographically separated using HILIC and characterised using mass spectrometry.
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SialEXO® 23 in a Study of Osteoclast Fusion
In a recent paper from Johns Hopkins University School of Medicine in Baltimore, removal of sialic acids from toll-like receptor 2 using SialEXO 23 was found to inhibit osteoclast fusion. These new findings in osteoclast recognition signaling are important for understanding the pathogenesis of different skeletal disorders, also showcasing the versatility of Genovis SmartEnzymes!
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