Hydrophobic Interaction Chromatography

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Csaba Horvath - One of the best experts on this subject based on the ideXlab platform.

  • retention thermodynamics in Hydrophobic Interaction Chromatography
    Industrial & Engineering Chemistry Research, 1996
    Co-Authors: Csaba Horvath
    Abstract:

    This paper is a review of the thermodynamics of retention in Hydrophobic Interaction Chromatography (HIC) with mildly Hydrophobic stationary phases and aqueous salt solutions usually employed in protein purification. Since the role of salt in HIC has been well documented, our focus was to investigate the temperature effect on the retention behavior in HIC and to compare the results with those obtained for other processes driven by the Hydrophobic effect. Using nonpolar dansyl amino acids as model compounds, retention data obtained on three stationary phases yielded nonlinear van't Hoff plots in the temperature range from 5 to 50 °C. Thermodynamic analysis of the data revealed significant heat capacity effects. The enthalpy and entropy changes were large and positive at low temperatures, decreased with increasing temperature, and became negative at high temperatures. The results parallel those of calorimetric studies on other processes based on the Hydrophobic effect, such as dissolution in water of nonpol...

  • temperature effects in Hydrophobic Interaction Chromatography
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Dietmar Haidacher, Anant Vailaya, Csaba Horvath
    Abstract:

    Abstract The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in Hydrophobic Interaction Chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the Hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.

  • Physicochemical Basis of Hydrophobic Interaction Chromatography
    Theoretical Advancement in Chromatography and Related Separation Techniques, 1992
    Co-Authors: Dietmar Haidacher, Csaba Horvath
    Abstract:

    In Hydrophobic Interaction Chromatography (HIC), which is widely used for protein separation, a mildly Hydrophobic stationary phase is employed with an aqueous salt solution as the mobile phase, and the magnitude of retention is modulated by the concentration of the salt. Retention is governed by the Hydrophobic effect, which is attributed to the strongly ordered structure and high cohesive energy of water. Various theoretical approaches are used to treat chromatographic retention in HIC. The most comprehensive is the solvophobic theory that was first adapted to provide a theoretical framework for reversed-phase Chromatography, a technique fundamentally similar to HIC. According to this approach the role of salt as the primary retention modulator in HIC is primarily due to the increase in the surface tension of the mobile phase with the salt concentration. However, when specific salt binding occurs the appropriate preferential Interaction parameters have to be considered. The effect of temperature on the retention in HIC is yet to be elucidated. Recent advances in exploring the temperature dependence of the Hydrophobic effect are expected to provide a framework for the interpretation of experimental data in HIC and to facilitate further understanding of the role of temperature in Hydrophobic Interactions.

Alois Jungbauer - One of the best experts on this subject based on the ideXlab platform.

  • Hydrophobic Interaction Chromatography as polishing step enables obtaining ultra-pure recombinant antibodies
    Journal of Biotechnology: X, 2020
    Co-Authors: Nico Lingg, Iara Rocha Antunes Pereira Bresolin, Alois Jungbauer
    Abstract:

    Abstract Hydrophobic Interaction Chromatography is a versatile method to polish antibodies. Here, we present a polishing procedure in order to obtain an ultra-pure preparation of antitumor necrosis factor (TNF) alpha IgG1. Hydrophobic Interaction Chromatography (HIC) was used with Toyopearl® Phenyl 650 M adsorbent in the presence of ammonium sulfate. Adsorption isotherms, breakthrough curves and chromatographic runs were carried out. The eluted antibody was recovered with 99.9 % purity and 96.2 % yield. In the main peak, aggregates, host cell proteins (HCP) and DNA content were below the limit of detection of the analytical methods used. Thus, the method proposed here shows potential to be employed in a downstream process when an ultra-pure antibody preparation is required.

  • Calorimetry for studying the adsorption of proteins in Hydrophobic Interaction Chromatography.
    Preparative Biochemistry & Biotechnology, 2019
    Co-Authors: Agnes Rodler, Rene Ueberbacher, Beate Beyer, Alois Jungbauer
    Abstract:

    Hydrophobic Interaction Chromatography is a very popular Chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this freq...

  • Hydrophobic Interaction Chromatography of proteins v quantitative assessment of conformational changes
    Journal of Chromatography A, 2008
    Co-Authors: Rene Ueberbacher, Emmerich Haimer, Rainer Hahn, Alois Jungbauer
    Abstract:

    Protein adsorption during Hydrophobic Interaction Chromatography (HIC) may induce conformational changes. We analyzed conformational changes in three model proteins, bovine serum albumin (BSA), β-lactoglobulin, and lysozyme by attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and pulse response experiments. Conformational changes occurred in the secondary structure of BSA, the tertiary structure of β-lactoglobulin, and no changes occurred in lysozyme under the adsorption conditions investigated. Protein unfolding varied substantially among proteins, caused incomplete isocratic elution in HIC, and was confirmed by in situ assessments. Lower temperatures and binding capacities significantly reduced protein unfolding; the activation energy for unfolding ranged from 47 to 125 kJ/mol.

  • Hydrophobic Interaction Chromatography of proteins iv kinetics of protein spreading
    Journal of Chromatography A, 2007
    Co-Authors: Emmerich Haimer, Anne Tscheliessnig, Rainer Hahn, Alois Jungbauer
    Abstract:

    Adsorption of proteins on surfaces of Hydrophobic Interaction Chromatography media is at least a two-stage process. Application of pure protein pulses (bovine serum albumin and β-lactoglobulin) to Hydrophobic Interaction Chromatography media yielded two chromatographic peaks at low salt concentrations. At these salt concentrations, the adsorption process is affected by a second reaction, which can be interpreted as protein spreading or partial unfolding of the protein. The kinetic constants of the spreading reaction were derived from pulse response experiments at different residence times and varying concentrations by applying a modified adsorption model considering conformational changes. The obtained parameters were used to calculate uptake and breakthrough curves for spreading proteins. Although these parameters were determined at low saturation of the column, predictions of overloaded situations could match the experimental runs satisfactorily. Our findings suggest that proteins which are sensitive to conformational changes should be loaded at high salt concentrations in order to accelerate the adsorption reaction and to obtain steeper breakthrough curves.

  • Hydrophobic Interaction Chromatography of proteins i comparison of selectivity
    Journal of Chromatography A, 2002
    Co-Authors: Christine Machold, Rainer Hahn, Karin Deinhofer, Alois Jungbauer
    Abstract:

    Currently, the selection of a Hydrophobic Interaction Chromatography (HIC) sorbent for protein separation purposes is entirely based on empirical means. An attempt was made to characterize different HIC sorbents from various manufacturers. The selectivity was determined by isocratic pulse experiments of a set of reference proteins and an algorithm was developed to classify the sorbents according to their selectivity and Hydrophobicity. The obtained semi-quantitative parameters take into account the dependence of salt on adsorption. The sorbent characteristics evaluated with the model proteins were compared to the separation of a real feedstock. A good agreement was achieved between the developed evaluation procedure and the separation behaviour of the real feed stock.

J. A. Queiroz - One of the best experts on this subject based on the ideXlab platform.

Andrea Mahn - One of the best experts on this subject based on the ideXlab platform.

  • Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering
    Biomedical Science Engineering and Technology, 2012
    Co-Authors: Andrea Mahn
    Abstract:

    Hydrophobic Interaction Chromatography (HIC) a powerful technique used for separation and purification of biomolecules. It was described for the first time by Shepard & Tiselius (1949), using the term “salting-out Chromatography”. Later, Shaltiel & Er-el (1973) introduced the term “Hydrophobic Chromatography”. Finally, Hjerten (1973) described this technique as “Hydrophobic Interaction Chromatography”, based on the retention of proteins on weakly Hydrophobic matrices in presence of salt. Owing of its high versatility and efficiency, HIC is widely used for the separation and purification of proteins in their native state (Porath et al., 1973), as well as for isolating protein complexes (Chaturvedi et al., 2000) and studying protein folding and unfolding (Bai et al., 1997). HIC has been applied in separating homologous proteins (Fausnaugh & Regnier, 1986), receptors (Zhang et al., 2008), antibodies (Kostareva et al., 2008), recombinant proteins (Lienqueo et al., 2003) and nucleic acids (Savard & Schneider, 2007). HIC shows similar capacity to ion exchange Chromatography (IEC) and a high level of resolution. Since it exploits a different principle than IEC and other separation techniques it can be used as an orthogonal method to achieve the purification of complex protein mixtures (Haimer et al., 2007). In this chapter, the theoretical principles underlying macromolecule retention in HIC are reviewed and discussed in sight of their application for predicting macromolecule behavior in HIC. Besides, novel applications of HIC are discussed regarding their suitability on Biomedical Engineering.

  • methods of calculating protein Hydrophobicity and their application in developing correlations to predict Hydrophobic Interaction Chromatography retention
    Journal of Chromatography A, 2009
    Co-Authors: Andrea Mahn, Elena M Lienqueo, Cristian J Salgado
    Abstract:

    Hydrophobic Interaction Chromatography (HIC) is a key technique for protein separation and purification. Different methodologies to estimate the Hydrophobicity of a protein are reviewed, which have been related to the chromatographic behavior of proteins in HIC. These methodologies consider either knowledge of the three-dimensional structure or the amino acid composition of proteins. Despite some restrictions; they have proven to be useful in predicting protein retention time in HIC.

  • current insights on protein behaviour in Hydrophobic Interaction Chromatography
    Journal of Chromatography B, 2007
    Co-Authors: Elena M Lienqueo, Andrea Mahn, Cristian J Salgado, Juan A. Asenjo
    Abstract:

    This paper gives a summary of different aspects for predicting protein behaviour in Hydrophobic Interaction Chromatography (HIC). First, a brief description of HIC, Hydrophobic Interactions, amino acid and protein Hydrophobicity is presented. After that, several factors affecting protein chromatographic behaviour in HIC are described. Finally, different approaches for predicting protein retention time in HIC are shown. Using all this information, it could be possible to carry out computational experiments by varying the different operating conditions for the purification of a target protein; and then selecting the best conditions in silico and designing a rational protein purification process involving an HIC step.

  • Predicting Protein Retention Time in Hydrophobic Interaction Chromatography
    Chemical Engineering & Technology, 2005
    Co-Authors: M.e. Lienqueo, Andrea Mahn
    Abstract:

    A review of Hydrophobic Interaction Chromatography (HIC) of proteins and theories for predicting protein retention time in HIC is given in this paper. A brief description of HIC, Hydrophobic Interactions and amino acid and protein Hydrophobicity is presented and different approaches and models for explaining the retention mechanism of proteins and predicting protein retention time in HIC are described. By using these models it is possible to test different operating conditions for the purification of a target protein and to select the best conditions.

  • Prediction of protein retention in Hydrophobic Interaction Chromatography.
    Biotechnology Advances, 2005
    Co-Authors: Andrea Mahn, Juan A. Asenjo
    Abstract:

    Hydrophobic Interaction Chromatography (HIC) is a powerful technique for protein separation. This review examines methodologies for predicting protein retention time in HIC involving elution with salt gradients. The methodologies discussed consider three-dimensional structure data of the protein and its surface Hydrophobicity. Despite their limitations, the methods discussed are useful in designing purification processes for proteins and easing the tedious experimental work that is currently required for developing purification protocols.

C.t. Tomaz - One of the best experts on this subject based on the ideXlab platform.

  • Chapter 6 – Hydrophobic Interaction Chromatography
    Liquid Chromatography, 2020
    Co-Authors: C.t. Tomaz, J. A. Queiroz
    Abstract:

    Hydrophobic Interaction Chromatography (HIC) is a powerful technique used for purification of biomolecules on both the analytical and preparative scale. This chapter presents an overview of the basic principles of HIC and the different proposed theories for the retention mechanisms as well as the main parameters to consider for the optimization of this chromatographic technique. Additionally, purification strategies and practical aspects are described, providing useful tools for HIC application. Recent selected examples of purification and analysis of different biomolecules by HIC, with particular emphasis on proteins, are also described.

  • Chapter 7 – Hydrophobic Interaction Chromatography*
    Liquid Chromatography, 2020
    Co-Authors: C.t. Tomaz
    Abstract:

    Hydrophobic Interaction Chromatography (HIC) is a high-resolution technique widely used for analysis and purification of biomolecules based on differences in their Hydrophobicity. In this chapter, the basic principles of HIC and theories for the retention mechanisms are addressed, as well as the main parameters to consider for the optimization of this chromatographic technique. Purification strategies and experimental considerations are described, providing useful tools for HIC application. Moreover, recent selected examples of purification and analysis of different biomolecules by HIC are also highlighted.

  • Fractionation of Trichoderma reesei cellulases by Hydrophobic Interaction Chromatography on phenyl-sepharose.
    Biotechnology Letters, 2004
    Co-Authors: C.t. Tomaz, J. A. Queiroz
    Abstract:

    Trichoderma reesei cellulase complex was fractionated using Hydrophobic Interaction Chromatography with a phenyl-Sepharose column. Using a linear gradient of ammonium sulphate in the eluent buffer, a selective separation of endoglucanases was obtained at 15 °C with a four-fold increase in specific activity.

  • Hydrophobic Interaction Chromatography of Trichoderma reesei cellulases on polypropylene glycol–sepharose
    Separation Science and Technology, 2002
    Co-Authors: C.t. Tomaz, Ana S. Rocha, J. A. Queiroz
    Abstract:

    The fractionation of Trichoderma reesei cellulases complex by Hydrophobic Interaction Chromatography was investigated using a mild Hydrophobic ligand—polypropylene glycol immobilized on Sepharose CL-6B. The influence of different salt type (sodium chloride, sodium sulfate, and ammonium sulfate) and their concentration in the mobile phase, on the chromatographic behavior of T. reesei cellulases was evaluated. A partial fractionation of β-glucosidase was obtained using 13 and 16% (w/v) sodium sulfate and ammonium sulfate in eluent buffer, respectively.

  • Hydrophobic Interaction Chromatography of proteins
    Journal of Biotechnology, 2001
    Co-Authors: J. A. Queiroz, C.t. Tomaz, J. M. S. Cabral
    Abstract:

    In this article, an overview of Hydrophobic Interaction Chromatography (HIC) of proteins is given. After a brief description of protein Hydrophobicity and Hydrophobic Interactions, we present the different proposed theories for the retention mechanism of proteins in HIC. Additionally, the main parameters to consider for the optimization of fractionation processes by HIC and the stationary phases available were described. Selected examples of protein fractionation by HIC are also presented.

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