High-Resolution Accurate-Mass Mass Spectrometry Enabling In-Depth Characterization of in Vivo Biotransformations for Intact Antibody-Drug Conjugates

Abstract

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules and therefore have complex molecular structures. In vivo biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated in vivo biotransformations of multiple site-specific THIOMAB antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety in vivo.

Characterization ofin vivobiotransformations for trastuzumab emtansine by high-resolution accurate-mass mass spectrometry

“…Therefore, this approach is primarily applicable for ADCs with conjugation sites located in the Fab region. The second strategy was to analyze ADCs in the intact form using HR/AM Orbitrap MS, 16 which preserves the structural information of ADCs as much as possible with minimal treatment. This approach has broader applications, being suitable for ADCs with site-specific conjugations in both Fab and Fc regions, as well as for ADCs with random lysine conjugations.…”

“…The cysteine adducts have a greater abundance than the glutathione adduct ( Figure 3), which is consistent with a previous observation for a disulfide-linked PBD dimer TDC. 16 This may be attributed to the higher concentration of reduced cysteine in plasma. 17,18 Examination of the rest of the peaks within this mass range is discussed in the following section.…”

“…To address this issue, we have recently evaluated and implemented a high-resolution, accurate-mass (HR/AM) Orbitrap MS approach which enabled more comprehensive characterization of in vivo biotransformations for site-specific THIOMAB TM antibody-drug conjugates (TDCs). 16 Here, we used an affinity capture HR/AM Orbitrap MS strategy to investigate in vivo biotransformations of T-DM1, which is a more challenging molecule compared to TDCs due to its highly heterogeneous and complex structure. Three types of biotransformations and their corresponding antibody-conjugated catabolites were characterized for T-DM1 in circulation in mice bearing HER2-positive human gastric cancer xenografts.…”

LC–MS bioanalysis of intact proteins and peptides

“…Grafmuller et al used IAC combined with HRMS to reveal the DAR for in vivo monkey ADC samples (Grafmuller et al, ). He et al demonstrated that, with the higher resolving power of MS, small mass changes (deacetylation on the payload) at the level of intact ADCs can be resolved (He et al, ). Tumey et al later reviewed the in vivo biotransformation of ADC and stated that HRMS combined with IAC for intact protein analysis played critical roles in the characterization and semi‐quantitation of ADCs in biological matrices (Tumey et al, ).…”

Technologies and Strategies for Bioanalysis of Biopharmaceuticals

“…Over the last decade, liquid chromatography coupled with mass spectrometry (LC-MS), has emerged as an appealing technology for therapeutic protein bioanalysis, complementing traditional LBAs [18,19]. Nevertheless, there are also significant challenges in isolating, concentrating, detecting and quantifying specific proteins by LC-MS in the presence of the overwhelming abundance of endogenous proteins found in biological matrices.…”