SEC-MS in denaturing conditions (dSEC-MS) for in-depth analysis of rebridged monoclonal antibody-based formats - Département Recherches Subatomiques
Article Dans Une Revue Talanta Année : 2024

SEC-MS in denaturing conditions (dSEC-MS) for in-depth analysis of rebridged monoclonal antibody-based formats

Résumé

Disulfide rebridging methods are emerging recently as new ways to specifically modify antibody-based entities and produce future conjugates. Briefly, the solvent-accessible disulfide bonds of antibodies or antigen-binding fragments (Fab) thereof are reduced under controlled conditions and further covalently attached with a rebridging agent allowing the incorporation of one payload per disulfide bond. There are many examples of successful rebridging cases providing homogeneous conjugates due to the use of symmetrical reagents, such as dibromomaleimides. However, partial rebridging due to the use of unsymmetrical ones, containing functional groups with different reactivity, usually leads to the development of heterogeneous species that cannot be identified by a simple sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) due to its lack of sensitivity, resolution and low mass accuracy. Mass spectrometry coupled to liquid chromatography (LC-MS) approaches have already been demonstrated as highly promising alternatives for the characterization of newly developed antibody-drug-conjugate (ADC) and monoclonal antibody (mAb)-based formats. We report here the in-depth characterization of covalently rebridged antibodies and Fab fragments in-development, using size-exclusion chromatography hyphenated to mass spectrometry in denaturing conditions (denaturing SEC-MS, dSEC-MS). DSEC-MS was used to monitor closely the rebridging reaction of a conjugated trastuzumab, in addition to conjugated Fab fragments, which allowed an unambiguous identification of the covalently rebridged products along with the unbound species. This all-in-one approach allowed a straightforward analysis of the studied samples with precise mass measurement; critical quality attributes (CQAs) assessment along with rebridging efficiency determination.

Domaines

Biotechnologies
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Dates et versions

hal-04547655 , version 1 (15-04-2024)

Identifiants

Citer

Rania Benazza, Ilias Koutsopetras, Valentine Vaur, Guilhem Chaubet, Oscar Hernandez Alba, et al.. SEC-MS in denaturing conditions (dSEC-MS) for in-depth analysis of rebridged monoclonal antibody-based formats. Talanta, 2024, 272, pp.125727. ⟨10.1016/j.talanta.2024.125727⟩. ⟨hal-04547655⟩
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