title: Comparison of various silica-based monoliths for the analysis of large biomolecules.
abstract: In the present study, three types of silica-based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide-pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first-generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3-5.8 kDa). In contrast, the wide-pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide-pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica-based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
authors.forename: Karine
authors.initials: K
authors.lastname: Vuignier
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
authors.forename: Pierre-Alain
authors.initials: PA
authors.lastname: Carrupt
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Veuthey
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
mesh_terms: D000327:Adsorption; D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation* / Q000379:methods; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D016062:Porosity; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D002411:Cation Exchange Resins; D010455:Peptides; D011506:Proteins; D012822:Silicon Dioxide
keywords: Monoclonal antibodies; Peptides; Protein adsorption; Proteins; Silica-based monolith; Wide-pore