title: Optimization of non-linear gradient in hydrophobic interaction chromatography for the analytical characterization of antibody-drug conjugates.
abstract: The goal of this work was to evaluate the potential of non-linear gradients in hydrophobic interaction chromatography (HIC), to improve the separation between the different homologous species (drug-to-antibody, DAR) of commercial antibody-drug conjugates (ADC). The selectivities between Brentuximab Vedotin species were measured using three different gradient profiles, namely linear, power function based and logarithmic ones. The logarithmic gradient provides the most equidistant retention distribution for the DAR species and offers the best overall separation of cysteine linked ADC in HIC. Another important advantage of the logarithmic gradient, is its peak focusing effect for the DAR0 species, which is particularly useful to improve the quantitation limit of DAR0. Finally, the logarithmic behavior of DAR species of ADC in HIC was modelled using two different approaches, based on i) the linear solvent strength theory (LSS) and two scouting linear gradients and ii) a new derived equation and two logarithmic scouting gradients. In both cases, the retention predictions were excellent and systematically below 3% compared to the experimental values.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
authors.forename: Balázs
authors.initials: B
authors.lastname: Bobály
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
authors.forename: Giuseppe Marco
authors.initials: GM
authors.lastname: Randazzo
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
authors.forename: Serge
authors.initials: S
authors.lastname: Rudaz
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
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
mesh_terms: D000079963:Brentuximab Vedotin; D002845:Chromatography / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D018796:Immunoconjugates / Q000032:analysis* / Q000737:chemistry; D008962:Models, Theoretical; D013997:Time Factors
chemical_list: D018796:Immunoconjugates; D000079963:Brentuximab Vedotin; D003545:Cysteine
keywords: Antibody-drug-conjugate; Hydrophobic interaction chromatography; Method development; Non-linear gradient; Retention modelling