1. An Online Four-Dimensional HIC×SEC-IM×MS Methodology for Proof-of-Concept Characterization of Antibody Drug Conjugates
- Author
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Oscar Hernandez-Alba, Florent Rouvière, Sabine Heinisch, Davy Guillarme, Olivier Colas, Sarah Cianférani, Valentina D'Atri, Alexandre Goyon, Anthony Ehkirch, Alain Beck, Morgan Sarrut, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, University of Geneva [Switzerland]-Université de Lausanne (UNIL), Chromatography & Hyphenated Techniques - Chromatographie et techniques couplées, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, This work was also supported by the 'Agence Nationale de la Recherche' (ANR) and the French Proteomic Infrastructure (ProFI, ANR-10-INBS-08-03). The authors thank GIS IBiSA and Region Alsace for financial support in purchasing a Synapt G2 HDMS instrument. A.E. acknowledges the 'Association Nationale de la Recherche et de la Technologie' (ANRT) and Syndivia for funding his Ph.D. fellowship. The authors would like to thank David Lascoux (Waters) for his kind support and valuable assistance. The authors acknowledge Liz Bevan and Tony Edge from Agilent Technologies, for providing the AdvanceBioSEC column. Finally, the authors wish to thank Waters for the loan of 2D-LC instrumentation used in this work., and ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010)
- Subjects
0301 basic medicine ,ddc:615 ,Chromatography ,Immunoconjugates ,Chemistry ,Hydrophilic interaction chromatography ,010401 analytical chemistry ,Mass spectrometry ,01 natural sciences ,Mass measurement ,Mass Spectrometry ,0104 chemical sciences ,Analytical Chemistry ,Characterization (materials science) ,03 medical and health sciences ,030104 developmental biology ,Proof of concept ,[CHIM.ANAL]Chemical Sciences/Analytical chemistry ,Chromatography, Gel ,[CHIM]Chemical Sciences ,Critical quality attributes ,Hydrophobic and Hydrophilic Interactions ,Conjugate - Abstract
International audience; There are currently two main techniques allowing the analytical characterization of interchain cysteine-linked antibody drug conjugates (ADCs) under native conditions, namely, hydrophobic interaction chromatography (HIC) and native mass spectrometry (MS). HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of average drug-to-antibody ratio (DAR) in quality control laboratories. Native MS offers structural insights into multiple ADC critical quality attributes, thanks to accurate mass measurement. However, both techniques can lead to misinterpretations or incomplete characterization when used as standalone methods. Online coupling of both techniques can thus potentially be of great interest, but the presence of large amounts of nonvolatile salts in HIC mobile phases makes it not easily directly compatible with native MS. Here, we present an innovative multidimensional analytical approach combining comprehensive online two-dimensional (2D)-chromatography that consists of HIC and size-exclusion chromatography (SEC), to ion mobility and mass spectrometry (IM-MS) for performing analytical characterization of ADCs under nondenaturing conditions. This setup enabled comprehensive and streamlined characterization of both native and forced degraded ADC samples. The proposed 4D methodology might be more generally adapted for online all-in-one HIC×SEC-IM×MS analysis of single proteins or analysis of protein complexes in nondenaturing conditions.
- Published
- 2017