title: Separation of antibody drug conjugate species by RPLC: A generic method development approach.
abstract: This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet, 1, 1211 Geneva 4, Switzerland. Electronic address: szabolcs.fekete@unige.ch.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
authors.affiliation: Molnár-Institute for Applied Chromatography, Schneeglöckchen Strasse 47, 10407 Berlin, Germany.
authors.forename: Imre
authors.initials: I
authors.lastname: Molnár
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet, 1, 1211 Geneva 4, Switzerland.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000079963:Brentuximab Vedotin; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D018796:Immunoconjugates / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D008962:Models, Theoretical; D013696:Temperature
chemical_list: D000906:Antibodies; D018796:Immunoconjugates; D007074:Immunoglobulin G; D000079963:Brentuximab Vedotin; D003545:Cysteine
keywords: Antibody drug conjugate; Brentuximab vedotin; DryLab; Method development; Retention modelling; Reversed phase liquid chromatography