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. II- Identification of sub-units for the characterization of even and odd load drug species.
abstract: This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris(®)). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC-MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC-MS in one single injection. It was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1, 3 and 5) and their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches.
authors.affiliation: Univ Lyon, CNRS, Université Claude Bernard Lyon 1, Ens de Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 VILLEURBANNE, France.
authors.forename: Morgan
authors.initials: M
authors.lastname: Sarrut
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
authors.affiliation: Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Janin-Bussat
authors.affiliation: Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Colas
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
authors.affiliation: Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Univ Lyon, CNRS, Université Claude Bernard Lyon 1, Ens de Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 VILLEURBANNE, France. Electronic address: sabine.heinisch@univ-lyon1.fr.
authors.forename: Sabine
authors.initials: S
authors.lastname: Heinisch
mesh_terms: D000079963:Brentuximab Vedotin; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003545:Cysteine / Q000032:analysis; D057927:Hydrophobic and Hydrophilic Interactions; D018796:Immunoconjugates / Q000737:chemistry*; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D018796:Immunoconjugates; D000079963:Brentuximab Vedotin; D003545:Cysteine
keywords: ADCs; Antibody-drug conjugate; HICxRPLC-QTOF-MS; Odd DARs; On-line comprehensive 2DLC; Quadrupole time-of-flight