Accurate Determination of Drug-To-Antibody Ratio of Interchain Cysteine-Linked Antibody-Drug Conjugates by LC-HRMS


Accurate determination of the drug-to-antibody ratio (DAR) of interchain cysteine-linked antibody—drug conjugates (ADCs) is challenging. High-resolution mass spectrometry (HRMS) analysis of the ADCs at the intact or subunit level provides a feasible way to measure the DAR. However, the measured DAR is usually lower than the true DAR because of the variation in ionization efficiency between different DAR species. In this work, we developed a novel standard-free HRMS method involving isotope-labeled payload conjugation, protease digestion, and liquid chromatography—HRMS (LC-HRMS) analysis for accurate determination of the DAR of the interchain cysteine-linked ADCs with cleavable or non-cleavable linkers. Isotope-labeled payload conjugations eliminated the structural and chemical differences between different DAR species and ensured that the drugs or payload-containing peptides could be separated from each other in the mass spectrometer. A papain digestion strategy for ADCs with cleavable linkers showed a DAR of 3.79, with a relative standard deviation (RSD) of 0.48 (n = 3). Similarly, the trypsin and chymotrypsin digestion strategy that is applicable to ADCs with non-cleavable linkers showed a DAR of 3.77 and an RSD of 0.86 (n = 3). The DAR determined by this method was consistent with the DAR of the ADCs that was measured by the UV/Vis method. This method will be very useful to researchers working in the field of ADC discovery and development.



Research Center/Lab(s)

Center for Research in Energy and Environment (CREE)


This study was supported by Frontage Laboratories, Inc. and the Missouri University of Science and Technology.

Keywords and Phrases

Antibody-drug conjugates; Drug-to-antibody ratio; High-resolution mass spectrometry; Interchain cysteine; On-beads conjugation

International Standard Serial Number (ISSN)

1618-2642; 1618-2650

Document Type

Article - Journal

Document Version


File Type





© 2020 Springer, All rights reserved.

Publication Date

01 Feb 2020

PubMed ID