Articles in the Category ”References”
Evaluating the Impact of Antibody Fragments on Aggregation using FabALACTICA®
Scientists at Biogen have developed an approach to better understand monoclonal antibody (mAb) aggregation by characterizing fragments that may form during the production process. Fab/c fragments – common impurities generated through hinge digestion and loss of a Fab subunit – can offer valuable insights into aggregation mechanisms. To support this work, FabALACTICA, which specifically digests human IgG1 above the hinge, was used to generate structurally relevant Fab/c fragments. Its ability to produce consistent and defined mAb fragments makes FabALACTICA a powerful tool for studying mAb aggregation through this approach.
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Multi-Attribute mAb Variant Characterization with CE-MS and FabRICATOR® Digestion
Scientists at Aalen University and their collaborator present a detailed methodology for the rapid and thorough characterization of monoclonal antibody (mAb) variants using capillary electrophoresis-mass spectrometry (CE-MS) at the subunit level. mAbs are an important class of therapeutic molecules for treating various diseases, including cancer and autoimmune disorders. mAbs are large, complex molecules and can undergo a range of post-translational modifications (PTMs), which may affect their biological activity, stability, and therapeutic efficacy. Comprehensive characterization of these modifications is crucial to ensuring the safety and quality of therapeutic mAbs.
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GlyCLICK®-generated ADC Suppresses Tumor Growth in Pancreatic Cancer Models
Scientists at The Finsen Laboratory and collaborators have described the potential for a novel antibody-drug conjugate (ADC) to treat pancreatic ductal adenocarcinoma (PDAC). The ADC candidate is conjugated with a highly potent anthracycline payload (PNU-159682) using GlyCLICK conjugation technology, and targets uPAR (urokinase plasminogen activator receptor), a protein overexpressed in PDAC and its surrounding stroma. GlyCLICK facilitated the site-specific attachment of the payload to the antibody’s Fc-glycans, ensuring a precise drug-to-antibody ratio (DAR) of 2. This study highlights how GlyCLICK’s precision and efficiency significantly enhanced the development, efficacy, and safety of the ADC molecule.
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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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