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In a study by Vanderschaeghe et al. (2018), a new assay to measure IgG galactosylation in serum has been developed. The setup includes hydrolysis of IgG Fc glycans using the IgG-specific endoglycosidase IgGZERO® (EndoS). A reduced level of IgG galactosylation in serum is a promising biomarker to evaluate the severity, determine the treatment and…
A new study from Janssen by Mo et al, demonstrates the use of intact mass spectrometry to determine the levels of glycation on therapeutic antibodies. To perform the assay, the authors used IgGZERO for rapid removal of the Fc-glycans. Glycation occurs when reducing sugars such as glucose, galactose or fructose, reacts with protein amino…
In a collaboration headed by Davy Guillarme at University of Geneva, scientists have explored the characterization of subunits derived from antibody drug conjugates (ADCs) using hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (D’Atri et al. 2018). The scientists used brentuximab vedotin (BV, Adcetris®), an approved ADC for treatment of Hodgkin lymphoma (HL) and systemic anaplastic large…
Researchers at the Free University of Berlin have performed a comparability study of the Fc-fusion protein etanercept and a biosimilar using FabRICATOR® and subunit analysis. The etanercept molecule consists of an IgG1 Fc domain fused to a tumor necrosis factor alpha receptor (TNFaR) and is used for autoimmune diseases such as rheumatoid arthritis. The originator etanercept…
Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al. 2017). In the paper, the combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge…
The Monoclonal Antibody Project initiated by The Consortium for Top Down Proteomics (CTDP) aims to assess top-down mass spectrometry approaches for antibody characterization. It is a multicentre study, where the participants use several workflows (for example top-down sequencing, middle-down sequencing and intact mass analysis) to investigate the detailed structure and modifications of three monoclonal antibodies.…
"GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies." In this paper, the scientists at Roche Innovation Center Munich, Moelleken et al, discuss the use of GingisKHAN in antibody lead selection screening and affinity measurements. The binding strength of…
In a collaboration between Universite de Strasbourg, Pierre-Fabre, and Catalent Biologics a multilevel mass spectrometry analysis of a site-specific conjugated ADC was recently published in the journal mAbs. As a first step in the analysis, FabRICATOR (IdeS) was used to perform middle-up analysis and confirm the drug to antibody ratio, DAR. The scientists write on p.7:…
A team of scientist from Quality Assistance in collaboration with Alain Beck from Pierre Fabre, have published a detailed article highlighting the analytical strategies for both N- and O-linked glycan analysis of biotherapeutics using LC/MS. The workflows for glycan analysis on antibodies includes FabRICATOR digestion and study of the Fc/2 fragment for identification of the Fc glycoforms.…
Biosimilars are gaining in popularity as patents of innovator drugs are expiring. The analytical strategies to characterize and assess the similarity between the innovator product and the biosimilar often involve liquid chromatography and mass spectrometry (LC/MS). In a recent paper from the Freie Universität Berlin, Montacir et al. studied Rituximab and follow-on molecules using FabRICATOR digestion, reduction, and…
Oxidation of methionine residues in the Fc region of a therapeutic antibody may affect the binding of the antibody to Protein A and FcRn leading to difficulties in purification or increased clearence in vivo. For the variable regions of the antibody, oxidation may affect antigen binding or lead to increased immunogenicity. For these reasons, the…
Antibodies are important tools in several scientific research areas, and sometimes it is necessary to cleave antibodies to generate antigen binding (Fab) fragments. The Fab fragments can be used within e.g. imaging, removal of effector functions, infection biology, binding studies, and mass spectrometry. In a recently published book (“Bacterial Pathogenesis, Methods and Protocols”, Humana Press,…