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Middle-level analysis is a universally accepted analytical strategy for rapid characterization of antibodies. The FabRICATOR® (IdeS)enzyme specifically digests IgG below the hinge, generating fragments that are suitable for high-resolution mass spectrometry. By immobilizing the FabRICATOR enzyme in an HPLC column format, a fully automated on-column digestion of IgG is possible. In a recent article…
Monoclonal antibodies (mAbs) are complex macromolecules that undergo a wide range of post-translational modifications that can result in charge heterogeneity. Ion exchange (IEX) chromatography is considered the gold standard for characterization of charge variants. This approach separates charge variants and allows for the collection of isoforms for protein identification by mass spectrometry (MS). However, the…
Bispecific monoclonal antibodies (BsAbs) are multi-functioning and complex biologics with the ability to recognize two different epitopes for improved therapeutic properties. Characterizing protein modifications such as glycation on biologics is vital to ensure consistency in stability and function. The structural complexity of BsAbs requires robust analytical methods, where conventional top-down and bottom-up strategies may lack…
Antibody drug conjugates (ADCs) consist of monoclonal antibodies chemically linked to a cytotoxic agent. The target specificity of the monoclonal antibody in combination with the potency of the cytotoxic drug make ADCs promising therapeutic agents. However, the molecules are often complex, making evaluation of the quality attributes for the ADC challenging. In order to…
Protein charge is a fundamental property that influences both the ability to interact with other molecules and the structure, solubility and stability of the protein. In this collaborative work by Boehringer Ingelheim Pharmaceuticals, Janssen BioTherapeutics and University of New Hampshire, the charge measurements of twelve monoclonal IgG and their F(ab’)2 and Fc fragments are…
Radioactively labelled antibodies are excellent immuno-PET tracers for evaluating in vivo distribution and performance of therapuetic agents. Site-specific conjugation at the antibody Fc glycan site by enzymatic remodeling allows for a uniform label distribution of such PET-tracers, compared to conjugates generated with conventional random labelling strategies. In an article by Kristensen et al. (2019), the…
The use of capillary electrophoresis for the analysis of therapeutic antibodies and other biopharmaceuticals is growing in popularity. A new article by researchers from CNRS in France showed how capillary zone electrophoresis-native mass spectrometry could be used for the quality control of intact therapeutic monoclonal antibodies. The authors show for the first time the use of a triple layer coated…
In biopharmaceutical product development and manufacturing, free thiol content is one of the product quality attributes of interest as its presence could impact structure, stability and function of the product. At Biogen, Yi Pu et al have optimized a label-free LC (UV) / MS method for free thiol quantification at a subunit level of IgG1…
Analysis of the glycosylation of therapeutic antibodies and other biopharmaceuticals is typically done by LC or LC/MS-based methods. However, each analytical technique has its strengths and weaknesses which makes the development of orthogonal methodologies crucial for in-depth characterization. In this study, researchers from the FDA present a middle-down NMR approach for studying glycosylation of therapeutic antibodies. Analogous to…
The Consortium for Top-down Proteomics is currently conducting a large inter lab study. They are developing methods for the analysis of intact mAbs and antibody subunits generated by digestion with either FabRICATOR or GingisKHAN. In this paper, van der Burgt et al. present a novel method for the analysis of mAbs based on MALDI in-source…
In an article by Valentina D’Atri et al. recently published in Analytical Chemistry (2019), the scientists developed a middle-up HILIC/HRMS workflow for detailed characterisation of the Fc fusion protein etanercept. The etanercept molecule consists of an IgG1 Fc domain fused to a tumour necrosis factor receptor (TNFR) and is used in the treatment of…
Scientists from the prestigious Johns Hopkins University School of Medicine have used OpeRATOR to develop a workflow to map O-glycosylated sites on proteins in very complex samples. O-glycoproteins are notoriously difficult to study due to the low abundance, high structural heterogeneity and low stability. Previous approaches using affinity enrichment or engineered cell culture systems either…
