The Experts below are selected from a list of 68796 Experts worldwide ranked by ideXlab platform
Udo Reichl - One of the best experts on this subject based on the ideXlab platform.
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purification of cell culture derived modified vaccinia ankara virus by pseudo affinity membrane adsorbers and Hydrophobic Interaction chromatography
Biotechnology and Bioengineering, 2010Co-Authors: Michael W Wolff, Udo Reichl, C Siewert, S P Hansen, R FaberAbstract:A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and Hydrophobic Interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the Hydrophobic Interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%.
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purification of cell culture derived modified vaccinia ankara virus by pseudo affinity membrane adsorbers and Hydrophobic Interaction chromatography
Biotechnology and Bioengineering, 2010Co-Authors: Michael W Wolff, Udo Reichl, C Siewert, S P Hansen, R FaberAbstract:A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and Hydrophobic Interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the Hydrophobic Interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312–320. © 2010 Wiley Periodicals, Inc.
Juan A Asenjo - One of the best experts on this subject based on the ideXlab platform.
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Hydrophobic Interaction chromatography for purification of monopegylated rnase a
Journal of Chromatography A, 2012Co-Authors: María Elena Lienqueo, Karla Mayolodeloisa, Barbara A Andrews, Marco Ritopalomares, Juan A AsenjoAbstract:Abstract The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein Hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20 kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of Hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1 M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ∼85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.
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current insights on protein behaviour in Hydrophobic Interaction chromatography
Journal of Chromatography B, 2007Co-Authors: Elena M Lienqueo, Cristian J Salgado, Andrea Mahn, Juan A AsenjoAbstract: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.
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New approaches for predicting protein retention time in Hydrophobic Interaction chromatography.
Journal of molecular recognition : JMR, 2006Co-Authors: María Elena Lienqueo, Andrea Mahn, G. Navarro, J. C. Salgado, Tomas Perez-acle, Ivan Rapaport, Juan A AsenjoAbstract: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 amino-acidic 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.
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Prediction of protein retention in Hydrophobic Interaction chromatography.
Biotechnology Advances, 2005Co-Authors: Andrea Mahn, Juan A AsenjoAbstract: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.
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effect of surface Hydrophobicity distribution on retention of ribonucleases in Hydrophobic Interaction chromatography
Journal of Chromatography A, 2004Co-Authors: Andrea Mahn, María Elena Lienqueo, Juan A AsenjoAbstract:Abstract The effect of surface Hydrophobicity distribution of proteins on retention in Hydrophobic Interaction chromatography (HIC) was investigated. Average surface Hydrophobicity as well as Hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface Hydrophobicity but different surface Hydrophobicity distribution. It was shown experimentally that surface Hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter “Hydrophobic contact area,” which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the Hydrophobic patches can therefore have an important effect on protein retention in HIC, and the Hydrophobic contact area adequately describes this.
Michael W Wolff - One of the best experts on this subject based on the ideXlab platform.
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purification of cell culture derived modified vaccinia ankara virus by pseudo affinity membrane adsorbers and Hydrophobic Interaction chromatography
Biotechnology and Bioengineering, 2010Co-Authors: Michael W Wolff, Udo Reichl, C Siewert, S P Hansen, R FaberAbstract:A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and Hydrophobic Interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the Hydrophobic Interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%.
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purification of cell culture derived modified vaccinia ankara virus by pseudo affinity membrane adsorbers and Hydrophobic Interaction chromatography
Biotechnology and Bioengineering, 2010Co-Authors: Michael W Wolff, Udo Reichl, C Siewert, S P Hansen, R FaberAbstract:A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and Hydrophobic Interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the Hydrophobic Interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312–320. © 2010 Wiley Periodicals, Inc.
Andrea Mahn - One of the best experts on this subject based on the ideXlab platform.
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methods of calculating protein Hydrophobicity and their application in developing correlations to predict Hydrophobic Interaction chromatography retention
Journal of Chromatography A, 2009Co-Authors: Andrea Mahn, Elena M Lienqueo, Cristian J SalgadoAbstract: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.
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current insights on protein behaviour in Hydrophobic Interaction chromatography
Journal of Chromatography B, 2007Co-Authors: Elena M Lienqueo, Cristian J Salgado, Andrea Mahn, Juan A AsenjoAbstract: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.
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New approaches for predicting protein retention time in Hydrophobic Interaction chromatography.
Journal of molecular recognition : JMR, 2006Co-Authors: María Elena Lienqueo, Andrea Mahn, G. Navarro, J. C. Salgado, Tomas Perez-acle, Ivan Rapaport, Juan A AsenjoAbstract: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 amino-acidic 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.
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Predicting Protein Retention Time in Hydrophobic Interaction Chromatography
Chemical Engineering & Technology, 2005Co-Authors: María Elena Lienqueo, Andrea MahnAbstract: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.
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Prediction of protein retention in Hydrophobic Interaction chromatography.
Biotechnology Advances, 2005Co-Authors: Andrea Mahn, Juan A AsenjoAbstract: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.
Raja Ghosh - One of the best experts on this subject based on the ideXlab platform.
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purification and analysis of mono pegylated hsa by Hydrophobic Interaction membrane chromatography
IEEE Journal of Solid-state Circuits, 2013Co-Authors: Xiaojiao Shang, William M Wittbold, Raja GhoshAbstract:: We discuss the purification of mono-PEGylated HSA by Hydrophobic Interaction membrane chromatography. The Hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly Hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono-, di-, and tri-), mono-PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS-PAGE, dynamic light scattering, and SEC combined with multi-angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono-PEGylated HSA. A scaled-down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.
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paper peg based membranes for Hydrophobic Interaction chromatography purification of monoclonal antibody
Biotechnology and Bioengineering, 2008Co-Authors: Deqiang Yu, Xiaonong Chen, Robert Pelton, Raja GhoshAbstract:: This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out Hydrophobic Interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become Hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.
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purification of humanized monoclonal antibody by Hydrophobic Interaction membrane chromatography
Journal of Chromatography A, 2006Co-Authors: Raja Ghosh, Lu WangAbstract:Abstract Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by Hydrophobic Interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained.
