The Experts below are selected from a list of 234 Experts worldwide ranked by ideXlab platform
Jianping Wu - One of the best experts on this subject based on the ideXlab platform.
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Purification of egg yolk phosvitin by anion Exchange Chromatography.
Journal of Chromatography A, 2011Co-Authors: Jianping WuAbstract:Abstract The objective of this study was to develop a simple method of phosvitin purification from hen egg yolk without using organic solvents. Egg yolk was diluted with equal volume of water and stirred for one hour at room temperature, followed by centrifugation to remove soluble proteins along with most of the yolk lipids in the supernatant. The granules were collected as the precipitate containing minimum amount of lipids (dry granules). The dry granules were dissolved in 0.05 M carbonate–bicarbonate buffer at pH 9.6, which yields a light yellowish solution used for anion Exchange Chromatography. Phosvitin fraction was collected from anion Exchange Chromatography as the last eluting peak with a purity of 92.6% and a yield of 35.4% of total phosvitin in the yolk or a recovery of 1.9% of total yolk dry matter, which are comparable to current methods employing organic solvents or Chromatography after salt fractionation and dialysis. This method developed is simple and fast without using organic solvents.
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co extraction of egg white proteins using ion Exchange Chromatography from ovomucin removed egg whites
Journal of Chromatography B, 2010Co-Authors: Dileep A Omana, Jiapei Wang, Jianping WuAbstract:Efficient isolation of egg white components is desired due to its potential uses. Existing methods mainly targeted on one specific protein; an attempt has been made in the study to co-extract all the valuable egg white components in a continuous process. Ovomucin was first isolated by our newly developed two-step method; the resultant supernatant obtained after ovomucin isolation was used as the starting material for ion-Exchange Chromatography. Anion-Exchange Chromatography of 100 mM supernatant yielded a flow-through fraction and three other fractions representing ovotransferrin, ovalbumin and flavoproteins. The flow-through fraction was further separated into ovoinhibitor, lysozyme, ovotransferrin and an unidentified fraction which represents 4% of total egg white proteins. Chromatographic separation of 500 mM supernatant resulted in fractions representing lysozyme, ovotransferrin and ovalbumin. This co-extraction protocol represents a global recovery of 71.0% proteins.
Brian D. Kelley - One of the best experts on this subject based on the ideXlab platform.
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exploration of overloaded cation Exchange Chromatography for monoclonal antibody purification
Journal of Chromatography A, 2011Co-Authors: Beth Mccooey, Tiago Duarte, Deanna E Myers, Terry Hudson, Ashraf Amanullah, Robert Van Reis, Brian D. KelleyAbstract:Abstract Cation Exchange Chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation Exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation Exchange membrane (Mustang S) and monolith (SO 3 Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation Exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity Chromatography, isocratic overloaded cation Exchange Chromatography using Poros 50HS and anion Exchange Chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity Chromatography, cation Exchange Chromatography using SPSFF in bind-elute mode and anion Exchange Chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.
Sam Guhan - One of the best experts on this subject based on the ideXlab platform.
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ph conductivity hybrid gradient cation Exchange Chromatography for process scale monoclonal antibody purification
Journal of Chromatography A, 2007Co-Authors: Joe X. Zhou, Tim Tressel, Shinta Dermawan, Felix Solamo, Greg Flynn, Rosalind Stenson, Sam GuhanAbstract:The commercial production of recombinant human monoclonal antibody therapeutics demands robust processes. In this article we describe the development of a pH-conductivity hybrid gradient for a cation-Exchange Chromatography step to obtain high binding capacity and consistent purification resolution in scale process. Operational parameters and their ranges were characterized with DOE statistical method. Aggregate, DNA and leached protein A removal were examined during development. The advantages and disadvantages of hybrid gradient elution compared to sodium chloride gradient elution were explored. As this step was designed as a good fit for the compatibility of the feed and operating pH/conductivity conditions for next step, the effects of elution by either changing sodium chloride concentration or changing pH of elution buffers on overall separation efficiency were compared. The operation condition was further confirmed in six 2000 L scale runs. The thorough evaluation demonstrated process reliability of hybrid gradient cation-Exchange Chromatography with high step purity and yield.
Dell Farnan - One of the best experts on this subject based on the ideXlab platform.
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validation of a ph gradient based ion Exchange Chromatography method for high resolution monoclonal antibody charge variant separations
Journal of Pharmaceutical and Biomedical Analysis, 2011Co-Authors: Tony G Moreno, Dell FarnanAbstract:Ion-Exchange Chromatography is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies. Despite good resolving power and robustness, ionic strength-based ion-Exchange separations are product-specific and time-consuming to develop. We have previously reported a novel pH-based separation of proteins by cation Exchange Chromatography that was multi-product, high-resolution, and robust against variations in sample matrix salt concentration and pH. In this study, a pH gradient-based separation method using cation Exchange Chromatography was evaluated in a mock validation. This method was shown to be robust for monoclonal antibodies and suitable for its intended purpose of charge heterogeneity analysis. Simple mixtures of defined buffer components were used to generate the pH gradients that separated closely related antibody species. Validation characteristics, such as precision and linearity, were evaluated. Robustness to changes in protein load, buffer pH and column oven temperature was demonstrated. The stability-indicating capability of this method was determined using thermally stressed antibody samples. In addition, intermediate precision was demonstrated using multiple instruments, multiple analysts, multiple column lots, and different column manufacturers. Finally, the precision for this method was compared to conventional ion-Exchange Chromatography and imaged capillary isoelectric focusing. These results demonstrate the superior precision and robustness of this multi-product method, which can be used for the high-throughput evaluation of in-process and final product samples.
Rainer Hahn - One of the best experts on this subject based on the ideXlab platform.
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Encyclopedia of Molecular Biology - Ion-Exchange Chromatography.
Methods in Enzymology, 2020Co-Authors: Alois Jungbauer, Rainer HahnAbstract:Ion-Exchange Chromatography is the most popular chromatographic method for separation of proteins. It is a versatile and generic tool and is suited for discovery of proteins, high-resolution purification, and industrial production of proteins. Separation conditions are within physiological range of salt and pH and in the most cases a native protein can be obtained. In this chapter, the guidance will be provided for binding and elution conditions and selection of stationary phases.
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Chapter 22 Ion-Exchange Chromatography
Methods in Enzymology, 2009Co-Authors: Alois Jungbauer, Rainer HahnAbstract:Ion-Exchange Chromatography is the most popular chromatographic method for separation of proteins. It is a versatile and generic tool and is suited for discovery of proteins, high-resolution purification, and industrial production of proteins. Separation conditions are within physiological range of salt and pH and in the most cases a native protein can be obtained. In this chapter, the guidance will be provided for binding and elution conditions and selection of stationary phases. © 2009 Elsevier Inc. All rights reserved.
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Ion Exchange Chromatography
Methods in Enzymology, 2009Co-Authors: Alois Jungbauer, Rainer HahnAbstract:Ion-Exchange Chromatography is the most popular chromatographic method for separation of proteins. It is a versatile and generic tool and is suited for discovery of proteins, high-resolution purification, and industrial production of proteins. Separation conditions are within physiological range of salt and pH and in the most cases a native protein can be obtained. In this chapter, the guidance will be provided for binding and elution conditions and selection of stationary phases
