title: Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatoraphy, part 2: Optimization of the phase system.
abstract: The goal of this second part was (i) to evaluate the performance of commercially available HIC columns and (ii) to develop a fast and automated "phase system" (i.e. stationary phase and salt type) optimization procedure for the analytical characterization of protein biopharmaceuticals. For this purpose, various therapeutic mAbs (denosumab, palivizumab, pertuzumab, rituximab and bevacizumab) and a cysteine linked ADC (brentuximab-vedotin) were selected as model substances. Several HIC column chemistries (butyl, ether and alkylamide) from different providers were evaluated in four different buffer systems (sodium acetate, sodium chloride, ammonium acetate and ammonium sulfate). As stationary phases, the historical TSK gel Butyl NPR phase and the brand new Thermo MAbPac HIC-10 were found to be the most versatile ones in terms of hydrophobicity, peak capacity and achievable selectivity. As salt types, ammonium sulfate and sodium acetate were found to be particularly well adapted for the analytical characterization of mAbs and ADCs, but it is important to keep in mind that a concentration 2 to 3-times higher of sodium acetate versus ammonium sulfate is required to achieve a similar retention in HIC. After selection of the most appropriate phase systems, the optimization of the separation can be carried out by computer assisted retention modeling in a high throughput manner.
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via S. Donato 19/2, 40127, Bologna, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Cusumano
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard 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: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: szabolcs.fekete@unige.ch.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002021:Buffers; D002853:Chromatography, Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D000645:Ammonium Sulfate
keywords: Antibody-drug-conjugate; Brentuximab-vedotin; Hydrophobic interaction chromatography; Method development; Monoclonal antibody