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Johann C.,Wyatt Technology Europe GmbH | Elsenberg S.,Wyatt Technology Europe GmbH | Roesch U.,Wyatt Technology Europe GmbH | Rambaldi D.C.,Wyatt Technology Europe GmbH | And 2 more authors.
Journal of Chromatography A | Year: 2011

A new system design and setup are proposed for the combined use of asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) within the same instrumentation. To this purpose, three innovations are presented: (a) a new flow control scheme where focusing flow rates are measured in real time allowing to adjust the flow rate ratio as desired; (b) a new HF5 channel design consisting of two sets of ferrule, gasket and cap nut used to mount the fiber inside a tube. This design provides a mechanism for effective and straightforward sealing of the fiber; (c) a new AF4 channel design with only two fluid connections on the upper plate. Only one pump is needed to deliver the necessary flow rates. In the focusing/relaxation step the two parts of the focusing flow and a bypass flow flushing the detectors are created with two splits of the flow from the pump. In the elution mode the cross-flow is measured and controlled with a flow controller device. This leads to reduced pressure pulsations in the channel and improves signal to noise ratio in the detectors. Experimental results of the separation of bovine serum albumin (BSA) and of a mix of four proteins demonstrate a significant improvement in the HF5 separation performance, in terms of efficiency, resolution, and run-to-run reproducibility compared to what has been reported in the literature. Separation performance in HF5 mode is shown to be comparable to the performance in AF4 mode using a channel with two connections in the upper plate. © 2010 Elsevier B.V.


Hagendorfer H.,Empa - Swiss Federal Laboratories for Materials Science and Technology | Hagendorfer H.,Ecole Polytechnique Federale de Lausanne | Kaegi R.,Eawag - Swiss Federal Institute of Aquatic Science and Technology | Traber J.,Eawag - Swiss Federal Institute of Aquatic Science and Technology | And 5 more authors.
Analytica Chimica Acta | Year: 2011

In this work we discuss about the method development, applicability and limitations of an asymmetric flow field flow fractionation (A4F) system in combination with a multi-detector setup consisting of UV/vis, light scattering, and inductively coupled plasma mass spectrometry (ICPMS). The overall aim was to obtain a size dependent-, element specific-, and quantitative method appropriate for the characterization of metallic engineered nanoparticle (ENP) dispersions. Thus, systematic investigations of crucial method parameters were performed by employing well characterized Au nanoparticles (Au-NPs) as a defined model system. For good separation performance, the A4F flow-, membrane-, and carrier conditions were optimized. To obtain reliable size information, the use of laser light scattering based detectors was evaluated, where an online dynamic light scattering (DLS) detector showed good results for the investigated Au-NP up to a size of 80 nm in hydrodynamic diameter. To adapt large sensitivity differences of the various detectors, as well as to guarantee long term stability and minimum contamination of the mass spectrometer a split-flow concept for coupling ICPMS was evaluated. To test for reliable quantification, the ICPMS signal response of ionic Au standards was compared to that of Au-NP. Using proper stabilization with surfactants, no difference for concentrations of 1-50 μg Au L -1 in the size range from 5 to 80 nm for citrate stabilized dispersions was observed. However, studies using different A4F channel membranes showed unspecific particle-membrane interaction resulting in retention time shifts and unspecific loss of nanoparticles, depending on the Au-NP system as well as membrane batch and type. Thus, reliable quantification and discrimination of ionic and particular species was performed using ICPMS in combination with ultracentrifugation instead of direct quantification with the A4F multi-detector setup. Figures of merit were obtained, by comparing the results from the multi detector approach outlined above, with results from batch-DLS and transmission electron microscopy (TEM). Furthermore, validation performed with certified NIST Au-NP showed excellent agreement. The developed methods show potential for characterization of other commonly used and important metallic engineered nanoparticles. © 2011 Elsevier B.V.


Schleeh T.,Luxembourg Institute of Science and Technology | Madau M.,Luxembourg Institute of Science and Technology | Roessner D.,Wyatt Technology Europe GmbH
Carbohydrate Polymers | Year: 2016

Biocompatibility and thickening properties predetermine alginates as ingredients in food, cosmetic and pharmaceutical products. Further chemical modifications are often desired for a product optimization. The introduction of hydrophobic groups can be realized by employing organic tetrabutylammonium alginate (TBA-Alg) solutions. The synthesis of alginic acid alkyl amides from TBA-Alg with 2-chloro-1-methylpyridinium iodide (CMPI) as a coupling agent, however, has so far not resulted in a high degree of amidation. The analysis of the coupling reaction revealed the formation of mannuronic acid γ-lactone structures, which required a conformation change from 1C4 to 4C1. The opening of the γ-lactone required a high excess of butylamine. In the case of CMPI, triethylamine had to be added prior to the coupling agent in order to suppress the assumed alginic acid formation. The degrees of amidation achieved were up to 0.8, and for propylphosphonic anhydride as the coupling agent up to 1. The molecular weights of the alginic acid butyl amide were ≥35 kDa. © 2015 Elsevier Ltd. All rights reserved.


Grant
Agency: European Commission | Branch: H2020 | Program: MSCA-ITN-ETN | Phase: MSCA-ITN-2015-ETN | Award Amount: 4.07M | Year: 2016

The revolution of biotechnology has led to the creation of various types of therapeutic biologics with the potential to provide treatment for new chronic and malignant diseases. Though the potential advantages of biologics lay in their high specificity and potency combined with few side effects, their formulation still remains a large challenge to pharmaceutical scientists. This is in part due to the complex, not-well understood relationship between the physicochemical properties of proteins and formulation conditions required for protein stability. A comprehensive understanding of the molecular mechanisms behind protein stabilization and solubility would provide the formulation scientists with knowledge of the interplay between formulation and stability that in turn could potentially make formulation development faster, cheaper and less labour intensive than the currently used broad screening approach. Understanding the susceptibilities of formulations to protein aggregation and denaturation can reduce the response time to for instance product failure. Few universities in Europe have formulation of biologics as a scientific subject. Consequently, the pharmaceutical industry is forced to train scientists - a challenge for larger companies, and an insurmountable task for smaller companies. Scientists in the field of structural biology, biophysics, protein formulation and stability have formed a consortium to systematically map physicochemical properties of biologics, formulation conditions and protein stability. The main objective of the consortium is to provide a new generation of innovative and entrepreneurial early-stage researchers that will develop methodologies, tools and databases to guide the formulation of robust biologics. The consortium will not only provide an excellent platform to train a new generation of formulation scientists, but also establish avenues for designing new formulation strategies and thereby securing the leading edge of EU expertise.


Lee J.Y.,Yonsei University | Choi D.,Yonsei University | Johan C.,Wyatt Technology Europe GmbH | Moon M.H.,Yonsei University
Journal of Chromatography A | Year: 2011

In this article, a simple experimental approach to improve lipoprotein separation and detection in flow field-flow fractionation (FlFFF) is detailed. Lipoproteins are globular particles composed of lipids and proteins in blood serum and their roles include transferring fats and cholesterols through blood vessels throughout the body. Especially, presence of small, dense low-density lipoproteins (LDL) is associated with cardiovascular risk. Two experimental approaches were explored in this study: an increase in the reproducibility of LDL particle separation by implementing a guard channel prior to an asymmetrical FlFFF (AFlFFF) channel in order to deplete small molecular weight serum proteins and reducing the required injection volume of a serum sample by implementing fluorescence detection. The guard channel was made of a simple hollow fiber module so that the serum sample can be washed with the help of radial flow prior to injection into the AFlFFF channel. The channel was tested with protein standards and serum samples to ensure precision of the retention time and the protein recovery rate. A fluorescent phospholipid dye was utilized to label lipoprotein particles before separation for fluorescence detection, which resulted in a reduction of the required injection volume of serum. © 2010 Elsevier B.V.


Schonfeld H.-J.,Schonfeld Protein Science Consulting | Roessner D.,Wyatt Technology Europe GmbH | Seelig J.,University of Basel
Journal of Physical Chemistry B | Year: 2016

Static and dynamic light scattering were employed to determine simultaneously the average relative molecular mass, Mr, and the average hydrodynamic radius, Rh, of protein molecules. The new method was applied to the association-dissociation equilibrium of apolipoprotein A-1 (Apo A-1) and its thermal unfolding. As a control, lysozyme was measured as a nonassociating protein. Apo A-1 forms oligomers as a function of concentration and temperature, and the equilibrium can be described by a cooperative association model, consisting of a nucleation step and a growth step. At concentrations of 1 and 2.7 mg/mL, the Apo A-1 solution contained mainly monomers and octamers, with intermediates occurring at very low concentrations. Oligomer formation was maximal at 22 °C and was characterized by a temperature-dependent association constant. The cooperative association model allows the quantitative analysis of both the average relative molecular mass, Mr, and the average hydrodynamic radius, Rh, with the same set of model parameters which, in turn, are also applicable to analytical ultracentrifugation experiments. The light scattering experiments were reversible as long as the Apo A-1 solution was not heated above 60 °C. © 2016 American Chemical Society.


Heim M.,University of Bayreuth | Ackerschott C.B.,TU Munich | Ackerschott C.B.,Wyatt Technology Europe GmbH | Scheibel T.,University of Bayreuth
Journal of Structural Biology | Year: 2010

The capture spiral of a spider's orb web is made of flagelliform silk, providing high elasticity and an outstanding toughness, perfectly suited for trapping prey. Flagelliform silk comprises mainly one single protein (FLAG) with an estimated molecular weight of 360. kDa. We engineered constructs mimicking distinct domains of FLAG (eFLAG) and produced them recombinantly to analyze the structure-function relationship of FLAG domains and assembly properties of FLAG. While in solution the small carboxy-terminal domain is structured, domains from the repetitive core region adopt a conformation typical for intrinsically unstructured proteins. To investigate the influence of the respective domains on solubility and assembly, we tested the aggregation behaviour of individual domains and domain ensembles in presence of conditions known to trigger silk assembly. Both, the length of the repetitive core domain as well as the presence of the carboxy-terminal non-repetitive domain showed impact on eFLAG aggregation. © 2009 Elsevier Inc.


Schleeh T.,Center De Recherche Public Gabriel Lippmann | Madau M.,Center De Recherche Public Gabriel Lippmann | Roessner D.,Wyatt Technology Europe GmbH
Carbohydrate Polymers | Year: 2014

The adaptation of alginates to food and pharmaceutical specifications is limited to aqueous chemistry due to the insolubility of sodium alginate (Na-Alg) and the insufficient solubility of tetrabutylammonium alginate (TBA-Alg) in organic solvents. In the present investigation, these restrictions were resolved by optimizing the solubility of TBA-Alg by improving its synthesis from Na-Alg via heterogeneous acidification with hydrochloric and formic acid, followed by neutralization with tetrabutylammonium hydroxide. The best acidification results were achieved with formic acid, because the reaction controlling solubility of the by-product in the acidic solvent was improved in comparison to hydrochloric acid. The solubility of TBA-Alg in polar aprotic organic solvents improved by increasing the degree of TBA substitution (DSTBA), decreasing the molecular weight of TBA-Alg and increasing the relative permittivity of the solvent. The best TBA-Algs, with DSTBA = 0.95 and relative high molecular weights, gave optically clear solutions with a turbidity of about 1 NTU. © 2014 Elsevier Ltd. All rights reserved.


Johann C.,Wyatt Technology Europe GmbH | Elsenberg S.,Superon GmbH | Schuch H.,Superon GmbH | Rosch U.,Superon GmbH
Analytical Chemistry | Year: 2015

A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (μ) as a function of size. The method provides an absolute value and does not require calibration. Results of μ for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine μ for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.5-5 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring μ, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field. © 2015 American Chemical Society.


Patent
Wyatt Technology Europe Gmbh | Date: 2011-09-22

A device for field flux fractioning comprises a sensor to determine the presence of particles in a liquid and a channel impermeable for particles and permeable for liquid. A pump conveys the liquid to first and second paths to the channel, where the second path connects in a first position to the pump outlet and in a second position to the sensor. An injection device injects a sample comprising particles into the liquid flowing through the first path. A distribution device distributes the flow volume conveyed by the pump in a first position at a predetermined ratio to the first and second paths. A control device comprises a valve and a measuring device to measure the flow volume. The valve controls the flow volume in the first path in consideration of the measuring device measurement and the pump conveys the liquid in a flow volume, which can be precisely dosed.

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