Antibody drug conjugates or ADCs are macromolecules which include cytotoxic drugs covalently attached via a conditionally stable linker to monoclonal antibodies (mAbs). With the approval of a number of ADCs and a large number in preclinical and clinical development, antibody-drug conjugates are among the most promising next generation of empowered mAbs.

Complexity
compared to naked mAbs, ADCs have an increased level of complexity as the heterogeneity of conjugation cumulates with the inherent microvariability of the biomolecule. Hence, there is an increasing underlying need in the development and optimization of ADC’s to improve the analytical and bioanalytical characterization by assessing three main quality attributes: drug distribution, amount of naked antibody, and the average drug to antibody ratio or DAR.

In an article published in the October 21, 2014 edition of Analytical Chemistry, François Debaene, from the BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg , 25 rue Becquerel, 67087 Strasbourg, France and her colleagues compared the analytical potential of native mass spectrometry (MS) and native ion mobility mass spectrometry or IM-MS to the hydrophobic interaction chromatography (HIC), the reference method for quality control of interchain cysteinyl-linked ADCs.

Proof of Concept
Brentuximab Vedotin (Adcetris®; Seattle Genetics) was selected for a proof of principle study.

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High resolution native mass spectrometry provided accurate mass measurement (<30 ppm) of intact ADCs together with average DAR and drug distribution, confirming the unique ability of native mass spectrometry for simultaneous detection of mixtures of covalent and noncovalent products.

Native native ion mobility mass spectrometry was next used for the first time to characterize an ADC. This method evidenced ADC multiple drug loading, collisional cross sections measurement of each payload species attesting slight conformational changes. A semiquantitative interpretation of native ion mobility mass spectrometry data was developed to directly extrapolate average DAR and DAR distribution.

Finally, hydrophobic interaction chromatography fractions were collected and analyzed by both native mass spectrometry and native ion mobility mass spectrometry, assessing the interpretation of each HIC peak.

The researchers conclude that their results illustrate how native mass spectrometry andnative ion mobility mass spectrometry can rapidly assess ADC structural heterogeneity and how easily these methods can be implemented into mass spectrometry workflows for in-depth analytical characterization of antibody-drug conjugates.

Published in: Analytical Chemistry