Prediction of pH-Dependent Chromatographic Behavior in Ion-Exchange Systems

Yang, Ting; Sundling, Matthew; Freed, Alexander S.; Breneman, Curt M.; Cramer, Steven M.

doi:10.1021/ac071101j

Cited by 27 publications

(20 citation statements)

References 33 publications

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“…Yang et al used predictive quantitative structure–property relationship models to calculate a set of pH‐dependent descriptors, which were used for prediction of the SMA model parameters. The calculated pH‐dependent descriptors represent charge and electrostatic potential properties on the protein surfaces, but protein crystal structures and sequence information is needed for their calculation .…”

Section: Introductionmentioning

confidence: 99%

Modeling of salt and pH gradient elution in ion-exchange chromatography

2014

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The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.

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Section: Introductionmentioning

confidence: 99%

Modeling of salt and pH gradient elution in ion-exchange chromatography

2014

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“…The pH-dependent and hydrophobic surface descriptors were developed by Breneman et al and are described elsewhere (Yang et al, 2007). Briefly, the pH-dependent descriptors include atomic partial charges and protein electrostatic potentials which take into account the effect of the environmental pH.…”

Section: Descriptor Calculationsmentioning

confidence: 99%

“…where [A À ] and [HA] are the concentrations of deprotonated and protonated residues (Yang et al, 2007). The pH value was equal to the pH value of buffer solution (pH 5).…”

Section: Descriptor Calculationsmentioning

confidence: 99%

“…where q i is the atomic charge, and d i is the atom's distance to EP query position d (Yang et al, 2007). The hydrophobic surface descriptors were calculated using four different hydrophobicity scales including log P, Cowan-Whittaker, Miyazawa-Jernigen and Hearn Scales (Cowan and Whittaker, 1990;Miyazawa and Jernigan, 1985;Wilce et al, 1995).…”

Section: Descriptor Calculationsmentioning

confidence: 99%

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Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein library

Chung¹,

Hou²,

Freed³

et al. 2008

Biotech & Bioengineering

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A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure-property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems.

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“…Recently, pH-gradient ionexchange chromatography has also been proposed [9]. Computational approaches were also undertaken to predict protein separations onto ion exchangers [10]. However, the mentioned studies and others found in the open literature have focused on the separation of a discrete number of model proteins.…”

Section: Introductionmentioning

confidence: 99%

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Cabrera¹,

Zhelyazkova²,

Galvis³

et al. 2008

J of Separation Science

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Original PaperTailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline TM strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/ conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.

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Prediction of pH-Dependent Chromatographic Behavior in Ion-Exchange Systems

Cited by 27 publications

References 33 publications

Modeling of salt and pH gradient elution in ion-exchange chromatography

Modeling of salt and pH gradient elution in ion-exchange chromatography

Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein library

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

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