title: Direct identification of rituximab main isoforms and subunit analysis by online selective comprehensive two-dimensional liquid chromatography-mass spectrometry.
abstract: In this proof-of-concept study, rituximab, which is a reference therapeutic monoclonal antibody (mAb), was characterized through the implementation of online, selective comprehensive two-dimensional liquid chromatography (sLC×LC) coupled with mass spectrometry (MS), using a middle-up approach. In this setup, cation exchange chromatography (CEX) and reverse-phase liquid chromatography (RPLC) were used as the first and second separation dimensions, respectively. As illustrated in this work, the combination of these two chromatographic modes allows a direct assignment of the identities of CEX peaks, using data from the TOF/MS detector, because RPLC is directly compatible with MS detection, whereas CEX is not. In addition, the resolving power of CEX is often considered to be limited; therefore, this 2D approach provides an improvement in peak capacity and resolution when high-performance second-dimension separations are used, instead of simply using the second-dimension separation as a desalting step. This was particularly relevant when separating rituximab fragments of medium size (25 kDa), whereas most of the resolution was provided by CEX in the case of intact rituximab samples. The analysis of a commercial rituximab sample shows that online sLC×LC-TOF-MS can be used to rapidly characterize mAb samples, yielding the identification of numerous variants, based on the analysis of intact, partially digested, and digested/reduced mAb samples.
authors.affiliation: †Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, United States.
authors.forename: Dwight R
authors.initials: DR
authors.lastname: Stoll
authors.affiliation: †Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Harmes
authors.affiliation: †Department of Chemistry, Gustavus Adolphus College, St. Peter, Minnesota 56082, United States.
authors.forename: John
authors.initials: J
authors.lastname: Danforth
authors.affiliation: ‡Center of Immunology Pierre Fabre, 5, Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France.
authors.forename: Elsa
authors.initials: E
authors.lastname: Wagner
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: §School of Pharmaceutical Sciences University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
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
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D009862:Online Systems; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D000069283:Rituximab / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D000069283:Rituximab