Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins but can also alter the conformation or even biological functions. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. Innovative analytical strategies should be developed to map these PTMs as well as explore possible induced conformational changes. Cation-exchange chromatography (CEX) coupled with native mass spectrometry has already emerged as a valuable asset for the characterization of mAb charge variants. Nevertheless, questions regarding protein conformation cannot be explored using this approach. Thus, we have combined CEX separation with collision-induced unfolding (CIU) experiments to monitor the unfolding pattern of separated mAbs and thereby pick up subtle conformational differences without impairing the CEX resolution. Using this novel strategy, only four CEX-CIU runs had to be recorded for a complete CIU fingerprint either at the intact mAb level or after enzymatic digestion at the mAb subunit level. As a proof of concept, CEX-CIU was first used for an isobaric mAb mixture to highlight the possibility to acquire individual CIU fingerprints of CEX-separated species without compromising CEX separation performances. CEX-CIU was next successfully applied to conformational characterization of mAb glyco-variants, in order to derive glycoform-specific information on the gas-phase unfolding, and CIU patterns of Fc fragments, revealing increased resistance of sialylated glycoforms against gas-phase unfolding. Altogether, we demonstrated the possibilities and benefits of combining CEX with CIU for in-depth characterization of mAb glycoforms, paving the way for linking conformational changes and resistance to gas-phase unfolding charge variants.
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http://dx.doi.org/10.1021/acs.analchem.2c03163 | DOI Listing |
Immunohorizons
January 2025
Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
C3 glomerulopathy (C3G), a rare kidney disease caused by dysregulation of alternative pathway complement activation, is characterized by glomerular C3 deposition, proteinuria, crescentic glomerulonephritis, and renal failure. The anti-C5 monoclonal antibody (mAb) drug eculizumab has shown therapeutic effects in some but not all patients with C3G, and no approved therapy is currently available. Here, we developed and used a triple transgenic mouse model of fast progressing lethal C3G (FHm/mP-/-hFDKI/KI) to compare the therapeutic efficacy of a bifunctional anti-C5 mAb fused to a functional factor H (FH) fragment (short consensus repeat 1-5 [SCR1-5]) and the anti-C5 mAb itself.
View Article and Find Full Text PDFExpert Rev Clin Immunol
January 2025
Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China.
Introduction: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous disease. High proportions of patients with CRSwNP characterized by type 2 inflammation fail to gain adequate control with conventional medical and surgical approaches. The application of biologics in clinical practice and assessments of novel biologics in clinical trials are blooming in expectations to fulfill the unmet medical needs of patients with CRSwNP with type 2 inflammation.
View Article and Find Full Text PDFBiomedicines
December 2024
National Institute of Biologicals, Ministry of Health & Family Welfare, Govt. of India, A-32, Sector-62, Noida 201309, UP, India.
Trastuzumab is an effective therapeutic intervention for treating HER2-positive breast cancers. The cost-effectiveness, global demand, and patent expiration of trastuzumab have led to the inflow of its biosimilars in the global market. With the rise of biosimilars in the biopharmaceutical market, it has become crucial to ensure that the biosimilar is at par with the original monoclonal antibody (mAb)in terms of efficacy, safety, and quality.
View Article and Find Full Text PDFBiosensors (Basel)
January 2025
Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninsky Gory 1/3, 119991 Moscow, Russia.
2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the popular herbicides that is widely used in agriculture and can be found in food and water. A rapid and sensitive fluorescence polarization immunoassay (FPIA) was proposed for the detection of 2,4-D in juice and water. New tracers, 2,4-D-buthylenediamin fluoresceinthiocarbamyl (2,4-D-BDF) and 2,4-D-glycine aminofluorescein (2,4-D-GAF), were obtained and characterized.
View Article and Find Full Text PDFPharm Res
January 2025
Department of Chemical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
Purpose: Therapeutic monoclonal antibodies (mAbs) are prone to degradation via aggregation and fragmentation. In this study, forced degradation of trastuzumab (TmAb) was explored in saline and in-vitro models having HO and exposed to UV light (case study 1) both bleomycin (BML) formulation and ferrous ions (Fe) (case study 2) and sodium hypochlorite (NaOCl) (case study 3).
Methods: Size exclusion chromatography, dynamic light scattering, spectroscopic analysis, and fluorescence microscope image processing was carried out for characterizing TmAb degradation.
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