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Title

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions

Authors
Sarrut, Morgane
Corgier, Amélie
Lascoux, David
Janin-Bussat, Marie-Claire
Beck, Alain
Heinisch, Sabine
Published in Journal of Chromatography. B. 2016, vol. 1032, p. 103-111
Abstract Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be char-acterized during development and for further quality control. The coupling of on-line HIC x RPLC tohigh resolution mass spectrometry can be considered as a very efficient analytical method, providingextensive information on ADC sample, within a reduced time scale. Our intention in this first paper is topresent the approach used to rationally optimize the numerous conditions that can affect the quality ofthe 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due tosolvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated thatadding salt in the sample solvent before HIC injection allows a significant peak shape improvement. Thegradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HICand bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolutionmass spectrometry is a useful method to obtain rapid and extensive structural information on the peaksobserved in the first HIC dimension, thereby leading, in a single step requiring 75 min, to the precisedetermination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drugload distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOFwill be discussed in the second part of this two-part series.
Keywords On-line comprehensive 2D-LCHICxRPLC-QTOF-MSAntibody-drug conjugateADCBrentuximab vedotinAdcetris®
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Structures
Research group Sciences analytiques
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SARRUT, Morgane et al. Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions. In: Journal of Chromatography. B, 2016, vol. 1032, p. 103-111. https://archive-ouverte.unige.ch/unige:91177

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Deposited on : 2017-01-18

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