A novel online four-dimensional SECxSEC-IMxMS methodology for characterization of monoclonal antibody size variants.
Fiche publication
Date publication
octobre 2018
Journal
Analytical chemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CIANFERANI Sarah
Tous les auteurs :
Ehkirch A, Goyon A, Hernandez-Alba O, Rouviere F, D'Atri V, Dreyfus C, Haeuw JF, Diemer H, Beck A, Heinisch S, Guillarme D, Cianférani S
Lien Pubmed
Résumé
The determination of size variants is a major critical quality attribute of therapeutic monoclonal antibody (mAbs), that may affect the drug product safety, potency and efficacy. Size variant characterization often relies on size exclusion chromatography (SEC) which could be hampered by difficult identification of peaks. On the other hand, mass spec-trometry (MS)-based techniques performed in non-denaturing conditions have proven to be valuable for mAb-related compound characterization. Based on the observation that limited SEC performance was observed in non-denaturing MS compatible ammonium acetate buffer compared to classical phosphate salts, a multidimensional analytical ap-proach was proposed. It combines comprehensive online two-dimensional chromatography (SECxSEC), with ion mo-bility and mass spectrometry (IM-MS) in non-denaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification and quantitation of ada-limumab size variants. Benefits of the SECxSEC-nativeIMxMS were further highlighted on forced degraded pembroli-zumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous iden-tification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results empha-size the power of comprehensive 2D LCxLC setups hyphenated to IMxMS in non-denaturing conditions with unprece-dented performance including: i) maintaining optimal SEC performance (under classical non volatile salt conditions), ii) performing online native MS identification, and iii) providing IM-MS conformational characterization of all sepa-rated size variants.
Référence
Anal. Chem.. 2018 Oct 29;: