Coupling Multi-Angle Light Scattering to Reverse-Phase Ultra-High-Pressure Chromatography (RP-UPLC-MALS) for the characterization monoclonal antibodies

Multi-angle light scattering coupled with size-exclusion chromatography (SEC-MALS) is a standard approach for protein characterization. Recently MALS detection has been coupled with ion-exchange chromatography (IEX) which demonstrated the feasibility and high value of MALS in combination with non-sized-based fractionation methods. In this study we coupled reverse-phase ultra-high pressure liquid chromatography (RP-UPLC) with a low-dispersion MALS detector for the characterization of intact monoclonal antibody (mAbs) and their fragments. We confirmed a constant refractive index increment value for mAbs in RP gradients, in good agreement with the values in literature for other classes of proteins. We showed that the impurities eluting from a RP column can often be related to aggregated species and we confirmed that in most cases those oligomers are present also in SEC-MALS. Yet, in few cases small aggregates fractions in RP-UPLC are an artifact. In fact, proteins presenting thermal and physical stability not suitable for the harsh condition applied during the RP separation of mAbs (i.e. organic solvents at high temperature) can aggregate. Further, we applied RP-UPLC-MALS during a long term stability studies. The different principle of separation used in RP-UPLC-MALS provides an additional critical level of protein characterization compared to SEC-MALS and IEX-MALS

Sind südliche Arten in Nord- und Ostsee auf dem Vormarsch?

The long and warm summer 2003 resulted in water temperatures of about 2°C above the average. This promoted the immigration of southern species like the Anchovy, the Sardine, the Red Mullet and different Squid species. The possible conclusions of such invasions concerning the ordinary species of the area are rather contradictory: For the Western Baltic a positive correlation between the occurrence of Anchovy and a good Cod year-class is shown. For the southern North Sea, stomach investigations of Squid make a negative effect on fish eggs and larvae more likely

Scaling Down Large-Scale Thawing of Monoclonal Antibody Solutions: 3D Temperature Profiles, Changes in Concentration, and Density Gradients

PURPOSE Scale-down devices (SDD) are designed to simulate large-scale thawing of protein drug substance, but require only a fraction of the material. To evaluate the performance of a new SDD that aims to predict thawing in large-scale 2 L bottles, we characterised 3D temperature profiles and changes in concentration and density in comparison to 125~mL and 2 L bottles. Differences in diffusion between a monoclonal antibody (mAb) and histidine buffer after thawing were examined. METHODS Temperature profiles at six distinct positions were recorded with type T thermocouples. Size-exclusion chromatography allowed quantification of mAb and histidine. Polysorbate 80 was quantified using a fluorescent dye assay. In addition, the solution's density at different locations in bottles and the SDD was identified. RESULTS The temperature profiles in the SDD and the large-scale 2 L bottle during thawing were similar. Significant concentration gradients were detected in the 2 L bottle leading to marked density gradients. The SDD slightly overestimated the dilution in the top region and the maximum concentrations at the bottom. Fast diffusion resulted in rapid equilibration of histidine. CONCLUSION The innovative SDD allows a realistic characterisation and helps to understand thawing processes of mAb solutions in large-scale 2 L bottles. Only a fraction of material is needed to gain insights into the thawing behaviour that is associated with several possible detrimental limitations

Controlling the fatty acid hydroxylation regioselectivity of CYP152A1 (P450Bsb) by active site engineering

Regioselective hydroxylation on inactivated C-H bonds is among the dream reactions of organic chemists. Cytochrome P450 enzymes (CYPs) perform this reaction in general with high regio- and stereoselectivity (e.g. for steroids as substrates). Furthermore, enzyme engineering may allow to tune the regioselectivity of the enzyme. Regioselective in-chain hydroxylation of shorter or linear molecules (fatty acids), however, remains challenging even with this enzyme class, due to the high similarity of the substrate’s backbone carbons and their conformational flexibility. CYPs are well described for hydroxylating fatty acids selectively in the chemically more distinct a- or w-position. In contrast, selective in-chain hydroxylation of fatty acids lacks precedence. The peroxygenase CYP152A1 (P450Bsb) is a family member that displays fatty acid hydroxylation at both, the a- and b-position. Please click Additional Files below to see the full abstract

Structured analysis of broader GMO impacts inspired by technology assessment to inform policy decisions.

If genetically modified organisms (GMOs) are approved in the EU for experimental release or marketing authorization (placing on the market), a risk assessment (RA) is carried out beforehand to determine whether this may be associated with negative effects on human health, nature or the environment. Applications are reviewed by the European Food Safety Authority (EFSA) and the national Competent Authorities of the Member States. However, the potential ramifications of the GMOs that are systematically addressed in the current RA context are limited. Broader consideration can include environmental and health aspects beyond the scope of the statutory RA, as well as societal, ethical and cultural impacts. These other levels of impact may be considered during the comitology process of authorisation, but how this is done is typically not made explicit in a systematic way. However, with the dynamic developments of new kinds of GMOs, these considerations as well as transparency regarding the role of broader considerations in political decision-making become more and more relevant. Against this backdrop, we identified the requirements and suggest the main elements for such a broader assessment. We use insights from the field of Technology Assessment (TA) to explore the requirements for operationalising a rapid but still systematic, transparent and broad case-by-case GMO assessment compatible with the existing legislative framework.publishedVersio

The development of therapeutic proteins can be hindered by poor decision-making strategies in the early stage

In this study we address two major issues related to the current development process of therapeutic proteins and their characterization. First, due to limited samples amounts, the selection of lead molecules in the early stages is often based on the results from a limited physicochemical characterization. The latter can be based on measurements of only 2-3 parameters, e.g. protein melting temperature, protein aggregation temperature, and is usually performed in only one buffer, e.g. PBS. The hypothesis we present is that such approach can lead to the rejection of lead candidates that can still be manufacturable and can move on to clinical trials. The second matter we address are the often-reported correlations between protein physicochemical parameters in the literature. We propose that such correlations can be found only in a small sample population, e.g. one protein in different solution conditions or different proteins from the same class. However, we expect that such correlations would not be valid in a large population, including various protein structures and solution conditions. In order to address the above-mentioned issues, we created the PIPPI consortium (http://www.pippi.kemi.dtu.dk) and applied systematic approach to map the physicochemical properties of a wide range of proteins and extensively study their stability as a function of the solution conditions. We show that promising therapeutic protein lead candidate can appear as non-manufacturable when only limited physicochemical characterization is performed, e.g. a few methods are used and only a few solution conditions are tested. Therefore, the rejection rate during early-stage development can be improved by more thorough physicochemical characterization. Moreover, only weak linear correlations between biophysical properties of proteins are observed in a large populations. This suggests that the often-reported correlations between parameters describing the protein stability are not representative of a global population. Understanding the connections between various physiochemical parameters would require a systematic database which is currently in development by the PIPPI consortium

Bulletin der deutschen Slavistik 24.2018

Jahrbuch der Deutschen Slavistik mit Forumsbeiträgen zu Digital Humanities, Rubriken zu Personen, Publikationen, Preisen, Wirkungsorten, wissenschaftlichen Abhandlungen

Development, Manufacture and Characterization of C/C-SiC Components based on Filament Winding

In this contribution development, manufacture and characterization of C/C-SiC tubes based on different winding angles is described. Therefore, CFRP tubes were made by wet-winding of C-filaments using thermoset resins with high char yield and, in a second step were converted to C/C tubes via pyrolysis. Then, the porous tubes were infiltrated by liquid silicon providing C/C-SiC tubes which were characterized by their microstructure and mechanical properties. Mechanical testing was performed under tensile loading in axial and circumferential direction (burst test). In addition, a correlation of mechanical properties to the winding angle will be presented

Teilhabe oder Reproduktion sozialer Ungleichheit durch Crowdwork? Empirische Einblicke in Erfahrungen von Menschen mit Beeinträchtigungen und Menschen mit Sorgeverpflichtungen

Dieses Working Paper stellt die Ergebnisse des Forschungsprojekts "Teilhabe durch Crowdworking" (Universität Hamburg) dar, in dem die Partizipation von Menschen mit Behinderung und Menschen mit Sorgeverpflichtungen an ortsungebundener Plattformarbeit untersucht wurde. Einleitend werden die Problematiken von Crowdwork erörtert. Vor diesem Hintergrund wird ausgehend von quantitativen und qualitativen Befragungen die soziale Situation der Crowdworker*innen auf zwei Plattformen exemplarisch dargestellt. Anhand dieser Empirie analysieren wir spezifische Formen der Teilhabe der beiden Fokusgruppen. Auf dieser Basis setzen wir Crowdwork ins Verhältnis zu Forderungen nach Inklusion, Geschlechtergerechtigkeit und einer besseren Vereinbarkeit von Familie und Erwerbsarbeit. Wir diskutieren abschließend die Frage nach der Reproduktion sozialer Ungleichheit durch Crowdwork und verorten dies in sozialpolitischen Diskussionen über die (globale) Regulierung von Plattformarbeit