Entity

Time filter

Source Type

Stuttgart Mühlhausen, Germany

Josic D.,Center for Cancer Research Development | Josic D.,Brown University | Josic D.,University of Rijeka | Breen L.,Center for Cancer Research Development | And 5 more authors.
Electrophoresis | Year: 2012

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Muller E.,Tosoh Bioscience GmbH | Josic D.,The Coro Center for Cancer Research and Development | 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. Source


Moosmann A.,University of Stuttgart | Christel J.,University of Stuttgart | Boettinger H.,University of Stuttgart | Mueller E.,Tosoh Bioscience GmbH
Journal of Chromatography A | Year: 2010

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30 kDa mono-PEGylated lysozyme. © 2009 Elsevier B.V. All rights reserved. Source


Moosmann A.,University of Stuttgart | Blath J.,University of Stuttgart | Lindner R.,University of Stuttgart | Muller E.,Tosoh Bioscience GmbH | Bottinger H.,University of Stuttgart
Bioconjugate Chemistry | Year: 2011

The mPEG-aldehyde PEGylation with two different PEG sizes and two proteins was experimentally determined with respect to yield, conversion, and selectivity. The kinetic behavior of these PEGylation reactions was simulated using a numerically solved set of differential equations. We show that the assumption of an inactivation of mPEG-aldehyde is crucial for the simulation of the overall PEGylation and that the inactivation is pH-dependent. We further demonstrate that ideal PEGylation parameters such as pH, temperature, reaction time, and protein concentration need to be chosen carefully depending on the protein and PEG size. In terms of selectivity and yield, we show that the reaction should be stopped before the highest mono-PEG concentration is reached. Moreover, room temperature and a slightly acidic pH of approximately 6 are good starting points. In conclusion, selectivity can be optimized choosing a shorter reaction time and a reduced reaction temperature. © 2011 American Chemical Society. Source


Muller E.,Tosoh Bioscience GmbH | Vajda J.,Tosoh Bioscience GmbH | Josic D.,Coro Center for Cancer Research and Development | 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. Source

Discover hidden collaborations