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.
abstract: Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.
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: 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: Amélie
authors.initials: A
authors.lastname: Corgier
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: 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: Waters SAS, BP 608, 78056 St Quentin en Yvelines, France.
authors.forename: David
authors.initials: D
authors.lastname: Lascoux
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: 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 / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D057927:Hydrophobic and Hydrophilic Interactions; D018796:Immunoconjugates / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D018796:Immunoconjugates; D000079963:Brentuximab Vedotin
keywords: ADC; Adcetris(®); Antibody-drug conjugate; Brentuximab vedotin; HICxRPLC-QTOF-MS; On-line comprehensive 2D-LC