Influence of the sample-solvent on protein retention, mass transfer and unfolding kinetics in hydrophobic interaction chromatography (original) (raw)
Related papers
Journal of Chromatography A, 2007
A two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption has been developed to describe the effects of secondary protein unfolding on hydrophobic interaction chromatography (HIC). The model has been applied to a biotech protein and to -lactoglobulin on Phenyl Sepharose 6FF low sub HIC media. Thermodynamic property models for adsorption and protein stability with parameters obtained from experimental chromatographic data successfully describe observed chromatographic behavior over ranges of temperature and salt concentration, provide predictions of distribution among different conformers, and give a basis for calculating trends in retention strength and stability with changing conditions, that might prove useful in HIC process development.
A comprehensive study to protein retention in hydrophobic interaction chromatography
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2016
The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e., polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250mm long column packed with 5μm polyalkylamide functionalized silica particles and applying a 30min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as ...
Effect of mass overloading on binding and elution of unstable proteins in hydrophobic interaction chromatography, 2017
Adsorption behavior of unstable proteins, i.e., bovine serum albumin and ␣-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.
Journal of Chromatography A, 2009
The coupled effect of salt concentration and temperature on the retention behavior of proteins in hydrophobic interaction chromatography has been studied. The retention data of four model proteins, i.e., myoglobin, lysozyme, ␣-chymotrypsinogen and bovine serum albumin, have been acquired by isocratic experiments of chromatographic elution within the temperature range 5-25 • C at different ammonium sulphate concentrations in the mobile phase. The retention dependencies quantified as functions of the salt concentration and temperature have been exploited in designing the process of gradient elution. The propagation velocity of proteins under conditions of the step gradient of salt and temperature has been determined by use of the equilibrium theory. To evaluate kinetic effects accompanying the band propagation the transport-dispersive model has been employed. It has been shown that altering the propagation of the salt and temperature waves in a proper manner allows improving the separation efficiency. Moreover, manipulation of specific kinetics effects can also be exploited in protein separations.
Journal of Chromatography A, 1986
We have studied the conformational behavior of a-lactalbumin (a-LACT) in hydrophobic interaction chromatography (HIC). Retention characterization in terms of 2 (slope of plot of In k' v.r. In qg, where k' is the capacity factor and (PB is the volume fraction of mobile phase B) has been explored, and the relationship of Z to other slopes, such as S (slope of the plot of In k' vs. (PB) has been derived. The reasons for the sensitivity of Z to conformational change are discussed. The enhanced broadening of a-LACT in a temperature transition region of conformational change has been studied by spectral analysis using on-line photodiode array detection. The influence of Ca' + and Mg 2+ addition to the mobile phase is further explored. Since a-LACT is a calcium binding protein, addition of this metal leads to stabilization, i.e. higher column temperatures are required for conformational change. On the other hand, addition of Mg2 ' appears to destabilize the protein. We have explored the use of a more hydrophobic support, C2-(ethyl) ether phase, for the elution of a-LACT. In this case, two widely separated peaks are observed. By spectral analysis the first peak is shown to be native and the later eluted, broad second peak to be an unfolded mixture of species. As previously observed in reversed-phase liquid chromatography, the second peak grows at the expense of the first, as the column temperature is raised. The second peak also grows as the contact time of the protein with the surface increases. This behavior can be ascribed to the conformational change of cl-LACT in the column, the late eluted species under the second peak binding significantly more strongly to the phase than the native peak. Reinjection of the late eluted fraction reveals that reformation of the native species takes place in solution within 30 min. As before, addition of Ca2 + reduces the extent of unfolding under any specific condition. These results add further to our understanding and ability to control conformational changes in high-performance liquid chromatography. INTRODUCTION Over the past few years high-performance hydrophobic interaction chromato-
Journal of Chromatography A, 2020
The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.
New approaches for predicting protein retention time in hydrophobic interaction chromatography
Journal of Molecular Recognition, 2006
Hydrophobic interaction chromatography (HIC) is an important technique for the purification of proteins. In this paper, we review three different approaches for predicting protein retention time in HIC, based either on a protein's structure or on its amino-acidic composition, and we have extended one of these approaches. The first approach correlates the protein retention time in HIC with the protein average surface hydrophobicity. This methodology is based on the protein three-dimensional structure data and considers the hydrophobic contribution of the exposed amino acid residues as a weighted average. The second approach, which we have extended, is based on the high correlation level between the average surface hydrophobicity of a protein's hydrophobic interacting zone and its retention time in HIC. Finally, a third approach carries out a prediction of the average surface hydrophobicity of a protein, using only its aminoacidic composition, without knowing its three-dimensional structure. These models would make it possible to test different operating conditions for the purification of a target protein by computer simulations, and thus make it easier to select the optimal conditions, contributing to the rational design and optimization of the process.
Hydrophobic interaction high-performance liquid chromatography of proteins
Journal of Chromatography A, 1984
Protein separations by means of a new hydrophobic-interaction high-performance liquid chromatography column are described. A linear elution gradient was used, starting with 1.7 M ammonium sulfate and concluding with 0.1 M sodium phosphate (pH 7.0). All proteins except albumin and fl-lactoglobulin were eluted as sharp peaks. The broadening of some protein peaks may depend upon the influence of the eluent on the structure of those proteins. Variations in column temperature and additions of either methanol or urea to the mobile phase were studied. The results show that small adjustments in chromatographic conditions can greatly improve resolution when band spreading exists. In addition, this column can be used not only for the separation of proteins, but also to determine their relative hydrophobic character.
Journal of Chromatography A, 2003
Hydrophobic interaction chromatography (HIC) is widely used in the downstream processing of proteins. Resolution of HIC is very good, but sometimes not as high as expected. Resolution values could be increased if good operating conditions were selected. In this paper we present a methodology for selecting good operating conditions. First, it is necessary to predict 2 the dimensionless retention time (DRT) of each protein in the mixture. We use a correlation such as DRT 5 A 1 Bf 1 Cf , where f is the superficial hydrophobicity of the protein, which is calculated considering the hydrophobicity of the superficial amino acids using the Miyazawa-Jernigan scale. Considering that there was little interaction amongst proteins in a mixture at the concentrations investigated (2 g / l of each protein), the behaviour of the proteins in the mixture was considered to be similar to that of the individual proteins. Using simulations it was possible to test different operating conditions for the purification of a target protein from a mixture of proteins and it was possible to select ideal conditions. The methodology developed was also tested for the purification of a recombinant protein from a fermentation extract of yeast producing human superoxide dismutase and the results have been satisfactory.