Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library

Chung, Wai Keen; Hou, Ying; Holstein, Melissa; Freed, Alexander S.; Makhatadze, George I.; Cramer, Steven M.

doi:10.1016/j.chroma.2009.08.005

Cited by 49 publications

(23 citation statements)

References 22 publications

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“…The chromatographic retention behavior of the charge Fab variants was obtained under linear gradient conditions in weak CEX (CM Sepharose Fast Flow) and multimodal (Capto MMC) chromatographic systems (Ligands presented in Table ). As shown in Figure B, the elution salt concentrations were significantly higher in the multimodal system as compared to the CEX material reflecting the stronger multimodal binding in agreement with previous results in the literature (Chung et al, ; Holstein et al, ). Furthermore, all charge variants exhibited decreased retention in both resin systems relative to the wild type.…”

Section: Resultssupporting

confidence: 91%

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Karkov

Krogh

Woo

et al. 2015

Biotech & Bioengineering

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In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

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Section: Resultssupporting

confidence: 91%

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Karkov

Krogh

Woo

et al. 2015

Biotech & Bioengineering

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“…We have recently employed a homologous library of cold shock protein B mutants to examine differences in protein binding behavior on an ionexchange (SP Sepharose FF) and MM chromatographic surface (Capto MMC) (15). Chromatographic experiments showed stronger retention for the majority of the proteins on Capto MMC as compared to SP Sepharose FF.…”

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confidence: 99%

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

Chung

Freed

Holstein

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarsegrained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems.ligand binding site | mixed mode chromatography | protein-ligand interactions | binding site mapping | pseudoaffinity T he development of efficient bioseparation processes for the production of high-purity biopharmaceuticals is one of the most pressing challenges facing the pharmaceutical and biotechnology industries today. In addition, high-resolution separations for complex bioanalytical applications are becoming increasingly important. Although it is generally accepted that nonspecific interactions can often complicate single mode chromatographic separations (e.g., ion-exchange, reversed-phase), these additional interactions can also result in unexpected selectivities (1, 2).Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials (3-9). Johansson et al. have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4, 5). Liu et al. have developed a silica-based MM resin capable of weak anion-exchange and reversed-phase interactions for the simultaneous separation of acidic, basic, and neutral pharmaceutical compounds (9). Small ligand pseudoaffinity chromatographic materials such as those used for hydrophobic charge induction chromatography have resulted in previously undescribed classes of MM ligands that offer unique selectivities due more to multiple low affinity MM interactions than to specific binding to certain classes of proteins (10). In addition, several libraries of MM ligands have been recently developed and employed on chro...

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“…The result of this equation is the energy contribution associated with the deformation of a bond from its equilibrium bond length. In addition, this descriptor has important role in some previous QSPR models [39]. The second and third significant descriptor (d 2 and d 3 ) involved in the Eq.…”

Section: Comparison Between Ann and Smlr Modelsmentioning

confidence: 99%

QSPR probing of Na+ complexation with 15-crown-5 ethers derivatives using artificial neural network and multiple linear regression

Daraei

Irandoust

Ghasemi

et al. 2011

J Incl Phenom Macrocycl Chem

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A quantitative structure-property relationship (QSPR) study is performed to develop a model, relating to Na ? complex stability constant (log K) and the structure of 74 derivatives of 1,4,7,10,13-pentaoxacyclo-pentadecane ethers (15C5). Stepwise Multiple Linear Regression (SMLR) and Artificial Neural Network (ANN) methods have been exploited as linear and nonlinear methods, respectively to build the QSPR model. MOPAC software embedded in ChemOffice 2004 package was used for the minimizing energy using semi-empirical AM1 method. The optimum structures have been applied to generate more than 50 descriptors using available servers in ChemOffice 2004. The five most important constitutional, steric, electronic, thermodynamic and molecular descriptors were selected using the common preselection method combined by SMLR method. SMLR and ANN models were constructed based on the five selected descriptors. Both proposed models efficiently predict log K of 15C5 complexes. However, the results of ANN were more effective respect to SMLR model. This phenomenon reveals that log K of 15C5 complexes have a deviation from linear behavior related to the selected descriptors.

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Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library

Cited by 49 publications

References 22 publications

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

QSPR probing of Na+ complexation with 15-crown-5 ethers derivatives using artificial neural network and multiple linear regression

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