title: Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals.
abstract: A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used to explore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various, highly-relevant samples were tested, including different insulins, interferon α-2b and trastuzumab. This work shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs, using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elution order and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while the kinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquely able to resolve several important glycoforms at the middle-up level of analysis (fragments of 25-100kDa). Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX. Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers several additional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) a reduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC to limit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility to couple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Periat
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: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.
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: Jean-Luc
authors.initials: JL
authors.lastname: Veuthey
authors.affiliation: Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, Saint-Julien-en-Genevois, 74160, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Lauber
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address: davy.guillarme@unige.ch.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
mesh_terms: D000327:Adsorption; D000577:Amides / Q000737:chemistry; D000818:Animals; D001688:Biological Products / Q000737:chemistry*; D001702:Biopharmaceutics; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D061385:Insulins / Q000737:chemistry; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry; D008024:Ligands; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013696:Temperature; D000068878:Trastuzumab / Q000737:chemistry
chemical_list: D000577:Amides; D001688:Biological Products; D061385:Insulins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008024:Ligands; D011506:Proteins; D011994:Recombinant Proteins; D012822:Silicon Dioxide; D000068878:Trastuzumab
keywords: HILIC; Hydrophilic interaction chromatography; Insulins; Interferon α-2b; Trastuzumab