Development of a Native Nanoelectrospray Mass Spectrometry Method for Determination of the Drug-to-Antibody Ratio of Antibody–Drug Conjugates
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Abstract
Antibody-drug conjugates (ADCs), an increasingly important therapeutic modality for targeted cancer treatment, have been studied using many analytical methods. A class of ADCs that utilize the reduced interchain disulfide cysteine residues for drug attachment has attracted particular interest in drug development. One challenge in analytical characterization of this class of ADCs is that the intact mass information of the ADC molecule is not attainable using conventional reversed-phase liquid chromatography-mass spectrometry methods. In this paper, we report a mass spectrometry (MS) method engaging enzymatic digestion, nanoelectrospray ionization (nano-ESI), and native MS to achieve direct determination of the intact mass and, furthermore, to calculate the average drug-to-antibody ratio (DAR) of the cysteine-linked ADCs. The novel aspects of this method lie in the application of a nano-ESI technique and, more significantly, the utilization of limited enzymatic digestion with a cysteine protease as compared to the recently published method by Valliere-Douglass et al. In summary, this novel native nano-ESI MS method in combination with limited enzymatic digestion provides a sensitive method for direct DAR determination and possesses great potential in studying low-abundance ADC analytes such as those from animal or human in vivo investigations.
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