Steinau an der Strasse, Germany
Steinau an der Strasse, Germany

Time filter

Source Type

Muller E.,TOSOH Bioscience GmbH | Josic D.,Rhode Island Hospital | Schroder T.,Atoll GmbH | Moosmann A.,University of Stuttgart
Journal of Chromatography A | Year: 2010

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5. kDa, 10. kDa and 30. kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1. mg. protein/ml and 10. mg. protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5. kDa PEGylated lysozyme. For both native lysozyme and 30. kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5. kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4. M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt. © 2010.


Muller E.,TOSOH Bioscience GmbH | Vajda J.,TOSOH Bioscience GmbH | Josic D.,Rhode Island Hospital | Schroder T.,Atoll GmbH | And 2 more authors.
Journal of Separation Science | Year: 2013

An essential part of the modulation of protein-binding capacity in hydrophobic interaction chromatography is the buffer-salt system. Besides using "single" electrolytes, multicomponent electrolyte mixtures may be used as an additional tool. Both the protein solubility and the binding capacity depend on the position of a salt in the so-called Hofmeister series. Specific interactions are observed for an individual protein-salt combination. For salt mixtures, selectivity, recovery, and binding capacity do not behave like for the single salts that are positioned in between the two mixed components in the Hofmeister series, as the continuous correlation would suggest. Thus, finding strategies for mixed salts could potentially lead to improved capacities in hydrophobic interaction chromatography. Mixtures of ammonium sulfate, sodium citrate, sodium sulfate, sodium chloride, sodium acetate, and glycine were used to investigate the binding capacities for lysozyme and a monoclonal antibody on various hydrophobic resins. Resin capacity for two investigated proteins increases when mixtures consisting of a chaotropic and a kosmotropic salt are applied. It seems to be related to the rather basic isoelectric points of the proteins. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Eppler S.,University of Ulm | Schroder T.,Atoll GmbH | Friedle J.,Atoll GmbH | Michl S.,Labor Dr. Merk and Kollegen GmbH | And 2 more authors.
Biosensors and Bioelectronics | Year: 2012

For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates.In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin - a protein of the protease family - was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach.The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin. © 2012 Elsevier B.V.


Trademark
Atoll GmbH | Date: 2010-06-29

Chromatography apparatus for laboratory use. chemistry services, namely, chemistry consultation, laboratory research in the field of chemistry, and packing and filling of chemical and biological media into chromatography apparatus for laboratory, scientific or research purposes.


Trademark
Atoll GmbH | Date: 2011-02-22

Chromatography apparatus for laboratory use. Biotechnology and chemistry laboratory research in the field of chromatography and its application.

Loading Atoll GmbH collaborators
Loading Atoll GmbH collaborators