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Anal Biochem. 2015 Sep 15;485:34-42. doi: 10.1016/j.ab.2015.06.001. Epub 2015 Jun 9.

A rapid approach for characterization of thiol-conjugated antibody-drug conjugates and calculation of drug-antibody ratio by liquid chromatography mass spectrometry.

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Covance Laboratories, Harrogate, North Yorkshire HG3 1PY, UK. Electronic address:
Covance Laboratories, Harrogate, North Yorkshire HG3 1PY, UK.
ADC Biotechnology, St Asaph, Denbighshire LL17 0JD, UK.


We present the demonstration of a rapid "middle-up" liquid chromatography mass spectrometry (LC-MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody-drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison-a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug-antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.


ADC; Antibody; Characterization; DAR; LC–MS; Mass spectrometry

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