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Protein Sci. 2015 Aug;24(8):1210-23. doi: 10.1002/pro.2666. Epub 2015 Mar 31.

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

Author information

1
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087, Strasbourg, France.
2
IPHC, CNRS, UMR7178, 67087, Strasbourg, France.
3
Centre d'Immunologie Pierre-Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164, Saint-Julien-en-Genevois, France.

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(®)). 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(®)), 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.

KEYWORDS:

Kadcyla®; T-DM1; antibody-drug conjugate (ADCs); drug to antibody ratio (DAR); ion mobility mass spectrometry; monoclonal antibody; native mass spectrometry

PMID:
25694334
PMCID:
PMC4534172
DOI:
10.1002/pro.2666
[Indexed for MEDLINE]
Free PMC Article

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