High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry

Abstract

Antibody-drug conjugates (ADCs) are one of the fastest growing classes of anticancer therapies. Combining the high targeting specificity of monoclonal antibodies (mAbs) with cytotoxic small molecule drugs, ADCs are complex molecular entities that are intrinsically heterogeneous. Primary sequence variants, varied drug-toantibody ratio (DAR) species, and conformational changes in the starting mAb structure upon drug conjugation must be monitored to ensure the safety and efficacy of ADCs. Herein, we have developed a high-throughput method for the analysis of cysteine-linked ADCs using trapped ion mobility spectrometry (TIMS) combined with top-down mass spectrometry (MS) on a Bruker timsTOF Pro. This method can analyze ADCs (∼150 kDa) by TIMS followed by a three-tiered top-down MS characterization strategy for multi-attribute analysis. First, the charge state distribution and DAR value of the ADC are monitored (MS1). Second, the intact mass of subunits dissociated from the ADC by lowenergy collision-induced dissociation (CID) is determined (MS2). Third, the primary sequence for the dissociated subunits is characterized by CID fragmentation using elevated collisional energies (MS3). We further automate this workflow by directly injecting the ADC and using MS segmentation to obtain all three tiers of MS information in a single 3-min run. Overall, this work highlights a multi-attribute top-down MS characterization method that possesses unparalleled speed for high-throughput characterization of ADCs.

Trapped Ion Mobility Spectrometry and Parallel Accumulation–Serial Fragmentation in Proteomics

“…However, when operated properlykeeping potentials low and not overfilling -TIMS can be a powerful tool for a broad range of native applications 59,60 . Further opportunities may arise from the combination of ion fragmentation in or before TIMS 61,62 , followed by a MS 3 analysis of the mobility-separated fragment ions with PASEF.…”

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

“…Recent work on newer generations of this instrument platform are capable of efficiently transferring higher m/z species and future work investigating their CIU capabilities are needed. 41…”

Ion Mobility Coupled to a Time-of-Flight Mass Analyzer Combined With Fragment Intensity Predictions Improves Identification of Classical Bioactive Peptides and Small Open Reading Frame-Encoded Peptides

“…To our knowledge, this is the first study where timsTOF is applied for neuropeptide and sORF discovery and only the second top-down MS study where non-digested peptide samples are identified using a timsTOF. The other study using top-down sample developed a new method for antibody-drug conjugate identification (Larson et al, 2021). To accommodate the fourdimensional data space (4D) of timsTOF data, mass over charge, retention time, intensity and ion mobility, specific modifications were newly implemented to commonly used search engines.…”

“…This can only be done when sufficient timsTOF datasets become available in the future. Together with a recent study to identify antibody-based drug products (Larson et al, 2021), this is only the second study where top-down strategies are analyzed on a timsTOF pro mass spectrometer. Alternatively, the spectral intensities could be predicted by a different algorithm, that is reported to be more robust toward different data characteristics, like PROSIT (Gessulat et al, 2019).…”