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Researchers at Roche have recently described an elegant solution to address the persistent problem of rheumatoid interference (RF) in anti-drug antibody (ADA) immunoassays. IgMBRAZOR is a unique IgM-specific protease that digests human IgM at one specific site below the CH2 domain in the heavy chain, generating homogeneous F(ab’)2 and Fc fragments. By introducing IgMBRAZOR pretreatment…
Scientists at Boehringer Ingelheim and collaborators have developed a novel method to generate antibodies with defined asymmetrical Fc glycoforms and evaluate their Fc receptor (FcγRs) binding properties. Combining enzymatic digestion and redox-mediated Fc recombination, Fc fragments with specific glycan pairings could be generated and their Fc receptor binding analyzed by surface plasmon resonance (SPR) spectroscopy.
Scientists at New Jersey Institute of Technology, Johnson & Johnson and collaborators have demonstrated a novel approach for mass spectrometry (MS)-based characterization of hinge-mutated antibodies. Combining the recently introduced FabRICATOR Xtra LALA enzyme with ultrafast microdroplet digestion allows for fully automated, high-throughput and efficient antibody characterization, with the entire sample preparation and MS analysis completed…
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…
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…
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.…
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…
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.
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…
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…
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.
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…
