Stuttgart, Germany
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Vajda J.,Tosoh Bioscience GmbH | Weber D.,Karlsruhe Institute of Technology | Stefaniak S.,IDT | Hundt B.,IDT | And 2 more authors.
Journal of Chromatography A | Year: 2016

Different ions typically used in downstream processing of biologicals are evaluated for their potential in anion exchange chromatography of an industrially produced, pandemic influenza H1N1 virus. Capacity, selectivity and recovery are investigated based on single step elution parallel chromatography experiments. The inactivated H1N1 feedstream is produced in Madin-Darby Bovine Kidney cells. Interesting effects are found for sodium phosphate and sodium citrate. Both anions are triprotic kosmotropes. Anion exchange chromatography generally offers high scalability to satisfy sudden demands for vaccines, which may occur in case of an emerging influenza outbreak. Appropriate pH conditions for H1N1 adsorption are determined by Zeta potential measurements. The dynamic binding capacity of a salt tolerant polyamine-type resin is up to 6.4 times greater than the capacity of a grafted Q-type resin. Pseudo-affinity interactions of polyamines with the M2 protein of influenza may contribute to the obtained capacity increase. Both resins achieve greater capacity in sodium phosphate buffer compared to Tris/HCl. A recovery of 67% and DNA clearance close to 100% without DNAse treatment are achieved for the Q-type resin. Recovery of the virus from the salt tolerant resin requires the use of polyprotic acids in the elution buffer. 85% of the DNA and 60% of the proteins can be removed by the salt tolerant resin. The presence of sodium phosphate during anion exchange chromatography seems to support stability of the H1N1 particles in presence of hydrophobic cations. © 2016 The Authors.


Vajda J.,Tosoh Bioscience GmbH | Mueller E.,Tosoh Bioscience GmbH | Bahret E.,Furtwangen University of Applied Sciences
Biotechnology Journal | Year: 2014

In downstream processing of monoclonal antibodies, proper aggregate removal is crucial. Mixed mode ligands such as immobilized tryptophan have been developed to satisfy the need for efficient removal of antibody aggregates. However, method development for mixed mode applications is complicated, since protein binding and elution can be modulated by an increased set of parameters. In the current study, we investigate the effect of different dual salt mixtures on mixed mode chromatography using TOYOPEARL MX-Trp-650M resin, with respect to the dynamic binding capacity, resolution and monomer purity of two different humanized immunoglobulins. Binding capacities varying by more than 50% were observed for different salt mixtures. Furthermore, antibody monomer and aggregate resolution deviated by 30% for different salt mixtures and linear gradient elution. Similar trends were obtained using an immobilized carboxymethyl ligand for the same set of experiments, but the overall resolution was lower. Less kosmotropic salt systems emphasize the electrostatic binding of the relatively hydrophobic mAbs and reduce hydrophobic attraction to a selectivity-determining constraint. Kosmotropic salts such as citrate appear to cause dominating hydrophobic interactions in protein adsorption that hinder electrostatic protein-ligand interactions. This effect may depend on the ionic and hydrophobic site distribution of a protein. The data presented here are important for the further improvement of downstream processing of therapeutic monoclonal antibodies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Josic D.,Rhode Island Hospital | Josic D.,Brown University | Josic D.,University of Rijeka | Breen L.,Rhode Island Hospital | 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.


Muller E.,Tosoh Bioscience GmbH | Vajda J.,Tosoh Bioscience GmbH
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2016

Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130. mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100. mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9. mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected. © 2016 Elsevier B.V.


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.


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.


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.


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.


Vajda J.,Tosoh Bioscience GmbH | Roemling R.,Tosoh Bioscience GmbH
LC GC Europe | Year: 2012

Reversed-phase chromatography (RPC) is the most frequently used chromatographic mode when analysing small molecular weight compounds. Nevertheless, various applications for the separation of larger biomolecules, such as proteins, are also based on RPC. Conventional stationary phases with 80-140 Å pore sizes are not suited for the analysis of large intact proteins, as the large analytes are not able to access the surface area within the pores. A new silica-based wide-pore butyl/C4 phase - TSKgel Protein C4-300 - completes the existing range of TSKgel protein analysis columns.


PubMed | Tosoh Bioscience GmbH and Electronic BioSciences, Inc.
Type: | Journal: Journal of chromatography. A | Year: 2015

The growing importance of monoclonal antibodies and virus particles has led to a pressure for faster size exclusion chromatography. In recent years, numerous small particle columns for size exclusion chromatography of biologicals have been introduced. Small particles are a strategy to reduce analysis time. In the following study, opportunities of small particles in size exclusion chromatography of large biomolecules are investigated. Poppe plots reveal that the lower particle size limit depends on the size of the sample molecule. Hydrodynamic radii of monoclonal antibody monomer, aggregates and H1N1 as well as the diffusion coefficients were determined. Considering this sample compound dependency, kinetic plots referring to the resolution of a distinct compound pair instead of the plate number of a single analyte are more meaningful. Plate times were found to be equivalent with 4 and 2m particles for a monoclonal antibody aggregate separation at resolutions smaller than 1.8. Quantification of a H1N1 in clarified cell culture can be accomplished with 17m and 13m particles at equal plate times at resolutions smaller than 2.5. Virus polydispersity is likely to be affected by run times of several hours at room temperature and shear forces resulting from particles smaller than 10m. Comparatively high flow rates should be applied in size exclusion chromatography of the 100nm H1N1 virions.

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