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. 2019 Nov;23(6):395-403.
doi: 10.29252/ibj.23.6.395. Epub 2019 May 20.

MALDI-MS: a Rapid and Reliable Method for Drug-to-Antibody Ratio Determination of Antibody-Drug Conjugates

Mehri Abedi  1 , Reza Ahangari Cohan  2 , Fereidoun Mahboudi  1 , Mehdi Shafiee Ardestani  3 , Fatemeh Davami  1
Affiliations

MALDI-MS: a Rapid and Reliable Method for Drug-to-Antibody Ratio Determination of Antibody-Drug Conjugates

Mehri Abedi et al. Iran Biomed J. 2019 Nov.

Abstract

Background: It is believed that the loading value of anticancer drug conjugated to the monoclonal antibody, called drug-to-antibody ratio (DAR), is the main quality feature of antibody-drug conjugates.

Methods: In this study, matrix assisted laser desorption/ionization mass spectrometry was used to determine the average molecular weight of trastuzumab and its three conjugated forms. The differences in the measured masses for each conjugate and unconjugated trastuzumab were compared to the expected mass change through the conjugation of one mole of related drug-linker, in order to measure DAR.

Results: There was a consistency between the loading results of mass spectrometry and the measurements of UV spectrometry in most cases.

Conclusion: According to our findings, the MALDI-MS method for determining the loading values can be used rapidly and reliably to estimate the covalently bound drugs conjugated to antibodies when ESI-TOF-MS is unavailable.

Keywords: Antibody-drug conjugate; Mass spectrometry; Trastuzumab.

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Figures

Fig. 1
Fig. 1
Two-step conjugation of DM1 drug to trastuzumab via three different linkers. The first step of the conjugation involves reacting the N-hydroxysuccinimide ester of each linker with available lysine residues on the antibody and formation of a stable amide bond with the primary amine of lysine residues. In the second step, the maleimide moiety of attached linkers reacts with sulfhydryl group on DM1 drug to form stable thioether bonds. The process resulted in the formation of a mixture of conjugates with diferent DARs
Fig. 2
Fig. 2
SDS-PAGE analysis of conjugates on 12% poly-acrylamide, trastuzumab, and kadcyla®, which were used as negative and positive controls, respectively
Fig. 3
Fig. 3
Peptide mapping of conjugates. Comparison of mirror plots of tryptic digests of trastuzumab with (A) TSMD, (B) TSPD2, and (C) TSPD12 at 214 nm
Fig. 4
Fig. 4
UV spectroscopy and mass spectrometry of conjugates. (A) UV spectra analysis revealed an increase in absorbance at the wavelength of 252 nm of conjugates. MADLI -TOF/TOF intact mass spectrum of (B) Trastuzumab, (C) Kadcyla®, (D) TSMD, (E) TSPD2, and (F) TSPD12
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