Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates

Abstract

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.

Native size-exclusion chromatography-mass spectrometry: suitability for antibody–drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels

“…Since the initial publication of our work on the intact mass determination of interchain cysteine-linked ADCs, 25 additional publications have emerged describing similar nSEC-MS methods that have been deployed for unique and interesting purposes. In these studies, researchers have used nSEC-MS for assessing the composition of ADCs recovered from in vivo studies in rats, 30 to gain a better understanding of the gas-phase conformation of ADCs in ion-mobility separations, 31,32 and as a component of a suite of MS-based assays that can be used for comprehensive ADC characterization. 33 While the particulars of the nSEC-MS methods described above vary, they all share fundamental key attributes: a microbore SEC column is used to exchange the ADC into ammonium acetate, thus facilitating online native electrospray ionization (ESI) analysis.…”

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

“…This technology involves the collisional activation of protein ions prior to IM‐MS separation in order to initiate protein unfolding events in the gas‐phase. CIU is capable of differentiating IgG subclasses, detecting minor alterations in mAb glycoforms, assessing stability shifts associated with site‐specific ADCs, and evaluating the comparability of biosimilars …”

“…CIU is capable of differentiating IgG subclasses, detecting minor alterations in mAb glycoforms, assessing stability shifts associated with site-specific ADCs, and evaluating the comparability of biosimilars. [30][31][32][33][34][35][36][37] In this report, we present a CIU based workflow for the rapid characterization of a human IgG1 mAb conjugated with biotin via its native lysine residues, which we treat as a model ADC system. Despite the high degree of structural similarity across conjugation states revealed by native IM-MS, our CIU results indicate the presence of subtle structural changes in the mAbs upon biotin conjugation, as revealed by shifts in their overall stabilities and ground state CCSs.…”

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

“…Finally, collision induced unfolding (CIU) experiments (Figure ) were performed to compare the gas‐phase unfolding behavior of the three VHHs. This new approach has already been reported for the rapid characterization of intact monoclonal antibodies, fragments and antibody drug conjugates . Briefly, in CIU experiments, ions are progressively accelerated in the trap T‐wave of the mass spectrometer before IM separation in the IM cell.…”

“…This new approach has already been reported for the rapid characterization of intact monoclonal antibodies, 23-26 fragments 27 and antibody drug conjugates. 28,29 Briefly, in CIU experiments, ions are progressively accelerated in the trap T-wave of the mass spectrometer before IM separation in the IM cell. Drift times of the ions are reported as a function of collision energies.…”