title: Systematic evaluation of mobile phase additives for the LC-MS characterization of therapeutic proteins.
abstract: Trifluoroacetic acid (TFA) is commonly used as mobile phase additive for the analysis of proteins in reversed phase liquid chromatography (RPLC). Due to its interesting features, it provides symmetrical and narrow peak shapes for proteins, but decreases mass spectrometric sensitivity through ion-pairing and spray destabilizing. Since RPLC-MS is an important technique for the characterization of proteins, some alternative MS-compatible mobile phases may be required. The aim of this study was to evaluate various acidic and basic mobile phase additives for the LC-MS analysis of therapeutic proteins possessing molecular weight between 5 and 150kDa. At the end, 10mM formate buffer pH 3 was found to be the most promising alternative, since it provided acceptable peak shapes in most cases, together with an average improvement of MS sensitivity by 5-times, compared to TFA.
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szt. Gellért tér 4, 1111 Budapest, Hungary. Electronic address: bbobaly@mail.bme.hu.
authors.forename: Balázs
authors.initials: B
authors.lastname: Bobály
authors.affiliation: Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France. Electronic address: alain.beck@pierre-fabre.com.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szt. Gellért tér 4, 1111 Budapest, Hungary. Electronic address: fekete@mail.bme.hu.
authors.forename: Jenő
authors.initials: J
authors.lastname: Fekete
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: davy.guillarme@unige.ch.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: szabolcs.fekete@unige.ch.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
mesh_terms: D002853:Chromatography, Liquid; D005561:Formates / Q000737:chemistry; D013058:Mass Spectrometry; D011506:Proteins / Q000032:analysis*; D014269:Trifluoroacetic Acid / Q000737:chemistry
chemical_list: D005561:Formates; D011506:Proteins; C030544:formic acid; D014269:Trifluoroacetic Acid
keywords: Formate buffer; Monoclonal antibody; RPLC–MS; TFA; Therapeutic proteins