An Integrated NMR Approach for Evaluating Linker-Payload Conjugation with Monoclonal Antibodies

Antibody-drug conjugates (ADCs) are modern biopharmaceuticals that combine the therapeutic effects of small-molecule drugs with the outstanding selectivity of monoclonal antibodies (mAbs). Since their introduction in the biomedical field, research has focused on elucidating the structure, stability, and mode of action of ADCs. Nevertheless, standard characterization methods for ADCs heavily rely on disruptive techniques like mass spectrometry in a non-physiological environment. Here, we present an NMR approach combining ¹H-¹³C ALSOFAST-HMQC and T₂-edited ¹H CPMG experiments, which together provide information on: i. the fingerprint and higher-ordered structure (HOS) of mAbs and ADCs and ii. the properties of the bound linker-payload fragment. In this study, we chose Trastuzumab as a well-known mAb and a Remdesivir-derived fragment as a linker-payload model system to validate our approach.