62 research outputs found
Continuous‐Flow Systems for Ligand Binding and Enzyme Inhibition Assays Based on Mass Spectrometry
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Simultaneous Screening and Chemical Characterization of Bioactive Compounds Using LC-MS-Based Technologies (Affinity Chromatography)
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Analysis of sub-visible particles in complex injectable formulations
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<span style="font-size:12.0pt;font-family:"Times New Roman","serif";
mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-ansi-language:
NL;mso-fareast-language:NL;mso-bidi-language:AR-SA">Complex injectable formulations, such as protein therapeutics, controlled release systems and cell therapy products, are gaining a paramount position in the therapy of many life-threatening and chronic diseases. Most of these products have in common that sub-visible particles (SVP), i.e., particulate matter in the size range of about 1 – 100 µm, are critical
quality attributes. Most protein therapeutics are liquid or freeze-dried formulations in which the presence of SVP is unwanted; many injectable controlled release systems are based on particulate drug delivery systems in the sub-visible size range; and cell concentration and viability are important characteristics of cell therapy products. With the continuous
improvement of existing and emerging particle analysis techniques, the potentials of these tools in addressing current characterization challenges in the field of complex injectable formulations have to be investigated. Therefore, the aim of this thesis was to develop methods, based on a set of state-of-the-art particle analysis techniques, for characterization of pharmaceutically relevant sub-visible particles and to study the value of these methods in the characterization of complex injectable formulations. Drug Delivery Technolog
Direct Dynamic Protein-Affinity Selection Mass-Spectrometry
No full textA new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERα-LBD). In-solution incubation is performed of the analyte and the His-tagged ERα-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein–ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%
Novel monitoring strategies for xenoestrogens
No full textThe wide variety of known and unknown xenoestrogenic compounds requires intelligent solutions to detect polluted environmental samples and identify the structure and concentration of the relevant xenoestrogens. In this article, a summary is given of several methods that are at present employed in the monitoring of xenoestrogens. Screening methods based on biological assays for the class-selective detection of xenoestrogens are reviewed. Furthermore, the analysis of known (xeno-)estrogens is summarized. Novel interfaces between biological assays and chromatography that facilitate the identification and quantification of xenoestrogens complex samples are in also discussed.A.J. Oosterkamp acknowledges the
European Commission for financial support (EC
TMR-grant ERBFMBI-CT96-1640). B. Hock
thanks the BMBF for a research grant (OZWU9647lO).Peer reviewe
Recent developments in protein-ligand affinity mass spectrometry
No full textThis review provides an overview of direct and indirect technologies to screen protein-ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery. © The Author(s) 2010
Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures
No full textIn the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets-pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column online screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches. © The Author(s) 2010.
Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods.
No full textThis review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein-protein and protein-immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein-protein and protein-immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS. [Figure not available: see fulltext.] © 2011 The Author(s)
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