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Dynamic Binding Capacities (dynamic + binding_capacity)

Distribution by Scientific Domains

Life Sciences	62%

Selected Abstracts

Dynamic binding capacity of plasmid DNA in histidine,agarose chromatography

BIOMEDICAL CHROMATOGRAPHY, Issue 9 2007
F. Sousa
Abstract The use of histidine,agarose chromatography in the purification of supercoiled (sc) plasmid DNA (pDNA) from Escherichia coli lysates has been reported recently. In the current work we describe a set of breakthrough experiments which were designed to study the effect of parameters such as flow-rate, temperature, concentration and conformation on the dynamic binding capacity of pDNA to the histidine support. One of the most striking results shows that the dynamic binding capacity for sc pDNA decreases linearly from 250.8 to 192.0 µg sc pDNA/mL when the temperature is varied from 5 to 24°C. This behaviour was attributed to temperature-induced, pre-denaturation conformational changes which promote the removal of negative superhelical turns in sc pDNA molecules and decrease the interaction of DNA bases with the histidine ligands. The capacity for sc pDNA was highly improved when using feeds with higher pDNA concentrations, a phenomenon which was attributed to the fact that pDNA molecules in more concentrated solutions are significantly compressed. A maximum capacity of 530.0 µg pDNA/mL gel was obtained when using a 125 µg/mL pDNA feed at 1 mL/min and 5°C, a figure which is comparable to the plasmid capacity values published for other chromatographic supports. Finally, a more than 2-fold increase in capacity was obtained when changing from open circular to sc pDNA solutions. Overall, the results obtained provide valuable information for the future development and implementation of histidine chromatography in the process scale purification of pDNA. Copyright © 2007 John Wiley & Sons, Ltd. [source]

Hydrophobic interaction chromatography in dual salt system increases protein binding capacity

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009
Anna M. Senczuk
Abstract Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein,resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity. Biotechnol. Bioeng. 2009;103: 930,935. © 2009 Wiley Periodicals, Inc. [source]

Purification of cell culture-derived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography

BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010
Michael W. Wolff
Abstract 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. [source]

Fractionation of ,-Lactoglobulin from whey by mixed matrix membrane ion exchange chromatography

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009
Syed M. Saufi
Abstract Mixed matrix membranes (MMMs), which incorporate adsorptive particles during membrane casting, can be prepared simply and have performances that are competitive with other membrane chromatography materials. The application of MMM chromatography for fractionation of ,-Lactoglobulin from bovine whey is described in this article. MMM chromatography was prepared using ethylene vinyl alcohol polymer and lewatit anion exchange resin to form a flat sheet membrane. The membrane was characterized in terms of structure and its static and dynamic binding capacities were measured. The optimum binding for ,-Lactoglobulin was found to be at pH 6.0 using 20 mM sodium phosphate buffer. The MMM had a static binding capacity of 120 mg/g membrane (36 mg/mL membrane) and 90 mg/g membrane (27 mg/mL membrane) for ,-Lactoglobulin and ,-Lactalbumin, respectively. In batch fractionation of whey, the MMM showed selective binding towards ,-Lactoglobulin compared to other proteins. The dynamic binding capacity of ,-Lactoglobulin in whey solution was about 80 mg/g membrane (24 mg ,-Lac/mL of MMM), which is promising for whey fractionation using this technology. This is the first reported application of MMM chromatography to a dairy feed stream. Biotechnol. Bioeng. 2009;103: 138,147. © 2008 Wiley Periodicals, Inc. [source]

An exclusion mechanism in ion exchange chromatography

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2006
C. Harinarayan
Abstract Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes. © 2006 Wiley Periodicals, Inc. [source]

Biporous polymeric beads fabricated by double emulsification for high-speed protein chromatography

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2007
Guo-Yong Sun
Abstract Rigid biporous beads (BiPB) were fabricated by double emulsification. An aqueous suspension of superfine calcium carbonate granules and organic solvent were used as porogenic agents to create superpores and micropores, respectively. The polymerization of monomers, glycidyl methacrylate, and ethylene glycol dimethacrylate was initiated with benzoin ethyl ether by ultraviolet irradiation. Modified with diethylamine (DEA), the BiPB were derivatized into an anion-exchange medium (which is denoted as DEA,BiPB). The DEA,BiPB with an average diameter of 46.3 ,m was characterized to possess two types of pores, that is, micropores (20,200 nm) and superpores (500,5300 nm). Flow hydrodynamic experiments showed that the DEA,BiPB column had a smaller backpressure than that of the conventional microporous beads column at a given flow rate. The static adsorption capacity of the DEA,BiPB was close to that of the DEA,MiPB for bovine serum albumin. However, frontal analysis demonstrated that the dynamic binding capacity of the DEA,BiPB column was two times higher than that of the DEA,MiPB at a flow rate of 1800 cm/h. Moreover, the purification of the molecular chaperone GroEL was carried out with the DEA,BiPB column at two flow rates (150 and 1500 cm/h). This showed that the GroEL purification was nearly the same at the two flow rates tested. These results indicate that the DEA,BiPB column is promising for high-speed protein chromatography. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 17,23, 2007 [source]

Dynamic binding capacity of plasmid DNA in histidine,agarose chromatography

Affinity Ligand Selection from a Library of Small Molecules: Assay Development, Screening, and Application

BIOTECHNOLOGY PROGRESS, Issue 1 2005
Lakshmi D. Saraswat
A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work. [source]