Integrated SEC-MS Analysis of Monoclonal Antibody (mAb) and Antibody Drug Conjugates (ADCs) under Native Conditions

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Henry Shion, Dale Cooper-Shepherd, Laetitia Denbigh, Maria Basanta-Sanchez, Barbara Sullivan, Ying-Qing Yu, Weibin Chen
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Native mass spectrometer (native‐MS) has become a more common analytical tool in academic and industrial research labs utilized in many applications, such as studies of protein folding, protein‐ligand and protein‐protein interactions, protein complex architecture, small protein aggregation, antibodies, antibody derivatives,and antibody−an􀆟gen complexes and antibody drug conjugates (ADCs). Most of abovementioned native‐MS carried out using static infusion from glass nanoflow capillaries following extensive sample clean‐up. The effort of applying online native approaches is still far from ideal and routine. In this study, we describe the development of an analytical scale native SEC‐MS workflow for routine analysis of mAb and ADCs using a bench top QTOF MS instrument controlled by a workflow driven complaint software. The workflow includes automated data acquisition, processing, and reporting, with experiment results shown from antibodies, cysteine and lysine conjugated ADCs. Instrument parameters, such as LC flow rate, MS cone voltage, capillary voltage, collision energy, etc., were optimized for improved SEC separation and MS sensitivity and mass resolution of mAbs. The DAR values generated automatically from the SEC-MS experiments were in good agreement with the value obtained from a HIC method for the cysteine‐conjugated ADCs and from reversed phase LC‐MS experiment for the lysine‐conjugated ADCs. The optimized SEC‐MS analysis of ADCs was replicated with high precision on different systems operated by different scientists, which demonstrated the robustness of the method.

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