Development of a Native Nanoelectrospray Mass Spectrometry Method for Determination of the Drug-to-Antibody Ratio of Antibody–Drug Conjugates

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.

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

“…Moreover, by using native MS, non-covalently assembled proteins, such as ΔhingeIgG4 antibodies and cysteine-conjugated ADCs (brentuximab vedotin in our case), can be still be maintained and analyzed in their native quaternary state. This allows the assessment of the drug-antibody ratio (DAR) for cysteine-linked ADCs 29 , 35 …”

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures

“…With respect to detailed antibody-epitope interaction studies, a oneto-one scenario is regarded optimal for determining binding kinetics, such as on-rates, off-rates, and dissociation constants. Thus, a fast method to isolate immune complexes from complex mixtures is of great help because once an intact immune complex has been prepared, there are many well-developed methods by which detailed molecular studies can be performed (reviewed in (Ngounou Wetie et al, 2013)), such as surface plasmon resonance (Nelson et al, 1997;Müller et al, 1998;Sonksen et al, 1998) and related spectroscopy methods (Pierce et al, 1999;Velazquez-Campoy et al, 2004), native MS (Tito et al, 2001;Rose et al, 2011;Chen et al, 2013;Debaene et al, 2013;Thompson et al, 2013), and cross linking (Sutherland et al, 2008;Petrotchenko and Borchers, 2010;Schwarz et al, 2013). An example for "native" mass spectrometric analysis of an antibody-antigen epitope peptide complex is shown for illustration (Figure 7).…”

An integrated approach to analyze EG2-hFc monoclonal antibody N-glycosylation by MALDI-MS

“…Antibodies produced by recombinant methods and purified on protein A or other techniques can be characterized for molecular weight by SDS-PAGE, linear mode MALDI-MS, or electrospray ionization MS to assess the quality of the expressed product, and the latter has become a method of choice 27,41 for this purpose. SDS-PAGE, when operated under denaturing conditions, offers the advantage of physical separation of the different antibody components for further specific analysis (denaturation refers to reduction of disulfide bonds).…”