Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine‐linked antibody drug conjugate

Abstract

Antibody-drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla V R ). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin V R ), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1.

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

“…This phenomenon is known as spectral interference and can become a major challenge for data interpretation. 2 …”

“…This increased separation significantly reduces spectral interference. 2,9,10 Modern mass analyzers, such as Orbitrap MS systems, have enabled native MS to deliver increasingly higher resolution data. 1 , 2,11–15 Achieving baseline resolution of intact protein isoforms, such as monoclonal antibody (mAb) glycoforms, allows heterogeneous intact protein samples to be efficiently measured without sample pre-treatment (e.g., reduction or deglycosylation), and offers a true intact mass measurement of complex biologics.…”

Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

“…Native MS has been employed for some protein therapeutic studies after significant advancements in instrumentation, namely high resolution mass spectrometers that allow for their accurate mass determination. These native MS studies often focus on glycoform identification and subunit stoichiometry [23]. Two MS-based methods have emerged for protein HOS characterization: hydrogen/deuterium exchange MS (HDX-MS) and ion mobility-MS (IM-MS) [24].…”

“…Several studies of large proteins, protein complexes, and protein therapeutics have incorporated IM-MS or CIU IM-MS to determine CCS values [23,[30][31][32][34][35][36][37][38]; however, the suitability of this technique to compare and differentiate protein therapeutics has not yet been evaluated. In this work, we evaluated the suitability of IM-MS and CIU IM-MS measurements for analysis of protein therapeutic systems.…”

New Insights in Tissue Distribution, Metabolism, and Excretion of [3H]-Labeled Antibody Maytansinoid Conjugates in Female Tumor-Bearing Nude Rats

“…The bioanalytical and distribution, metabolism, and excretion (DME) properties of antibody maytansinoid-based ADCs have been recently reviewed (Erickson and Lambert, 2012;Kaur et al, 2013;Deslandes, 2014;Han and Zhao, 2014;Thudium et al, 2013;Marcoux et al, 2015;Beck et al, 2016;Kraynov et al, 2016) The fate of antibody maytansinoid conjugates was investigated in naive or tumor-bearing mice and naive rats with iodine labels on the antibody or a tritium label on emtansine (DM1) (Xie and Blättler, 2006;Shen et al, 2012a;Saad et al, 2015). The measured tissue exposures were low compared with blood and the main components in serum were attributed to ADC.…”