title: Aqueous size-exclusion chromatographic separations of intact proteins under native conditions: Effect of pressure on selectivity and efficiency.
abstract: The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 μm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 μm particles are discussed.
authors.affiliation: Vrije Universiteit Brussel (VUB), Department of Chemical Engineering, Brussels, Belgium.
authors.forename: Jelle
authors.initials: J
authors.lastname: De Vos
authors.affiliation: DSM Biotechnology Center, part of DSM Food Specialties b.v, Delft, The Netherlands.
authors.forename: Erwin R
authors.initials: ER
authors.lastname: Kaal
authors.affiliation: Thermo Fisher Scientific, Germering, Germany.
authors.forename: Remco
authors.initials: R
authors.lastname: Swart
authors.affiliation: Vrije Universiteit Brussel (VUB), Department of Chemical Engineering, Brussels, Belgium.
authors.forename: Martyna
authors.initials: M
authors.lastname: Baca
authors.affiliation: Vrije Universiteit Brussel (VUB), Department of Analytical Chemistry and Pharmaceutical Technology, Brussels, Belgium.
authors.forename: Yvan Vander
authors.initials: YV
authors.lastname: Heyden
authors.affiliation: Vrije Universiteit Brussel (VUB), Department of Chemical Engineering, Brussels, Belgium.
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Eeltink
mesh_terms: D000418:Albumins / Q000737:chemistry; D000818:Animals; D002138:Calibration; D002417:Cattle; D002645:Chickens; D002850:Chromatography, Gel / Q000379:methods*; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D010047:Ovalbumin / Q000737:chemistry; D010316:Particle Size; D016062:Porosity; D011312:Pressure; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D013954:Thyroglobulin / Q000737:chemistry; D014867:Water / Q000737:chemistry*; D005719:gamma-Globulins / Q000737:chemistry
chemical_list: D000418:Albumins; D011506:Proteins; D005719:gamma-Globulins; D014867:Water; D010047:Ovalbumin; D013954:Thyroglobulin
keywords: Intact proteins; Kinetic performance limits; Kinetic plots; Poppe plots; Size-exclusion chromatography