title: Effect of mass overloading on binding and elution of unstable proteins in hydrophobic interaction chromatography.
abstract: Adsorption behavior of unstable proteins, i.e., bovine serum albumin and α-lactalbumin, has been studied on a hydrophobic interaction chromatography medium under mass overloading conditions at different kosmotropic salt concentrations in the mobile phase. A mechanistic model has been formulated and used to describe kinetics and thermodynamics of protein interactions with the adsorbent surface. The model assumed two-site binding adsorption and reversible protein unfolding, which allowed predicting the inhibition of protein unfolding at high column loadings. A simplified procedure for the determination of model parameters has been developed, which was based on the inverse method. The model was successfully used to reproduce the pattern of chromatographic elution as well as the course of breakthrough curves. The model formulation was supported by Nano Differential Scanning Fluorimetry measurements, which were exploited to determine the protein stability in the liquid and adsorbed phases at different column loadings and salt concentrations.
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Muca
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Marek
authors.affiliation: NanoTemper Technologies Sp.z.o.o. Bobrzyńskiego str. 14, 30-348, Kraków, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Żurawski
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Piątkowski
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland. Electronic address: dorota.antos@prz.edu.pl.
authors.forename: Dorota
authors.initials: D
authors.lastname: Antos
mesh_terms: D000327:Adsorption; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D005470:Fluorometry; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism*; D055550:Protein Stability; D058767:Protein Unfolding; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D012710:Serum Albumin, Bovine; D007768:Lactalbumin
keywords: HIC; Mathematical modeling; Protein unfolding; nanoDSF