2,346 research outputs found

    Characterization and Bioanalysis of Protein-Based Biopharmaceuticals, Peptides and Amino Acids by Liquid Chromatography and Mass Spectrometry

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    Biopharmazeutika sind zu einer essenziellen Klasse von Therapeutika geworden und werden für verschiedene medizinische Indikationen wie Diabetes, Krebs, entzündliche Erkrankungen und Infektionskrankheiten eingesetzt. Monoklonale Antikörper (mAbs) haben innerhalb der Biopharmazeutika den größten Anteil bezogen auf die Zulassungszahlen. Den Vorteilen bezüglich hoher Spezifität und Effektivität stehen jedoch Nachteile durch hohe Kosten und erhöhter Komplexität gegenüber. Die Komplexität ergibt sich einerseits aufgrund des hohen Molekulargewichts und anderseits aufgrund der strukturellen Heterogenität, wodurch die analytische Charakterisierung und Qualitätskontrolle von mAbs und anderer Biopharmazeutika zu einer Herausforderung wird. Neben diesen protein-basierten Biopharmazeutika ist auch die Aufklärung der absoluten Konfiguration von therapeutischen und natürlichen (Lipo)peptiden von besonderem Interesse für die Wirkstoffforschung. Zur Bewältigung dieser Herausforderungen wurden in der hier präsentierten Arbeit flüssigchromatographische (LC) und massenspektrometrische (MS) Methoden für die umfassende Analyse eingesetzt. Die erste Publikation dieser Dissertation bezog sich auf die Analyse von Ladungsvarianten von mAbs, welche wichtige Qualitätsmerkmale darstellen und die Sicherheit und Wirksamkeit des Arzneimittels beeinflussen können. Zur Charakterisierung der Ladungsvarianten wurden die mAbs auf Ebene des intakten Proteins als auch auf Fragmentebene nach begrenztem Verdau und Reduzierung der Disulfidbrücken mittels starker Kationenaustauschflüssigkeitschromatographie (SCX) analysiert. Die SCX-Methode wurde systematisch mittels statistischer Versuchsplanung (DoE) dahingehend optimiert, die höchstmögliche Anzahl an Ladungsvarianten zu trennen. Die mobile Phase der optimierten SCX-Methode enthielt jedoch eine hohe Konzentration an nicht-flüchtigen Salzen, wodurch sie nicht mit MS Detektion kompatibel ist, welche wiederum entscheidend für die Identifikation der Ladungsvarianten ist. Um dieser Herausforderung zu begegnen, wurde erfolgreich eine online zweidimensionale flüssigchromatographische (2D-LC) Methode entwickelt, bei der SCX in der ersten Trenndimension und Umkehrphasenflüssigchromatographie (RP-LC) in der zweiten Trenndimension zur Entsalzung vor der MS Detektion verwendet wurde. Die Entwicklung einer extrem kurzen (≤ 1 min) RP-LC Methode war unabdingbar zur Etablierung einer umfassenden 2D-LC Methode. Dazu wurde eine Säulenvergleichsstudie mit monolithischen und oberflächlich porösen Partikelsäulen (SPP-Säulen) durchgeführt und die Trenneffizienz sowie die Analysengeschwindigkeit untersucht. Eine noch umfassendere Säulenvergleichsstudie mit Fokus auf das kinetische Leistungsvermögen wurde in der zweiten Arbeit dieser Dissertation durchgeführt. Eine Auswahl von 13 RP-Proteintrennsäulen inklusive monolithischer, SPP und vollporöser Partikelsäulen (FPP-Säulen) wurde hinsichtlich ihrer Fähigkeit, Peaks in der kürzest möglichen Zeit zu trennen, untersucht. Es konnte gezeigt werden, dass SPP-Säulen mit einer Porengröße von etwa 400 Å und einer dünnen, porösen Schicht die beste Performance insbesondere für größere Proteinen besitzen. Proteine selbst können auch potenzielle Ziele für Arzneistoffe sein, wie z.B. das Tumorsuppressorprotein p53, welches in der dritten Publikation dieser Arbeit untersucht wurde. Intakte Protein LC-MS wurde erfolgreich verwendet, um die Bindungseffizienz und -spezifität des kovalenten Inhibitors an p53 nachzuweisen. Aminosäuren sind die Bausteine von Proteinen und Peptiden und die Mehrheit dieser Aminosäuren sind chiral. Die biologische Aktivität ist in der Regel abhängig von der absoluten Konfiguration der Aminosäuren, wodurch die enantiomerenselektive Analyse von höchster Wichtigkeit für die Strukturaufklärung und zur Qualitätskontrolle ist. Daher war die Entwicklung schneller und umfassender Trennmethoden zur Analyse von Aminosäuren, deren Enantiomeren, Diastereomeren und konstitutionellen Isomeren ein Ziel dieser Arbeit. Dieses konnte durch Derivatisierung mittels 6-Aminochinolyl-N-hydroxysuccinimidylcarbamat (AQC) und anschließender Analyse durch enantioselektiver flüssigchromatographischer Ionenmobilitäts-Massenspektrometrie (LC-IM-MS) erreicht werden. Eine sehr schnelle dreiminütige Analysenmethode konnte entwickelt und zur Strukturaufklärung von therapeutischen Peptiden und eines natürlichen Lipopeptides eingesetzt werden. Die absolute Konfiguration eines Tetrapeptides als Bestandteil des natürlichen, antimikrobiellen Peptidpolyens‘ Epifadin konnte mittels chiraler LC-MS bestimmt werden, was wiederum entscheidend für die Strukturaufklärung war. In dieser Arbeit konnten alle acht Enantiomerenpaare erfolgreich getrennt werden und die Diastereomerentrennung wurde optimiert.Biopharmaceuticals have become an essential class of therapeutics and are used for different medical indications such as diabetes, cancer, inflammatory diseases, and infectious diseases. Monoclonal antibodies (mAbs) have the biggest share within the biopharmaceuticals regarding the drug approval numbers. However, the benefits in terms of high specificity and efficacy come with the drawback of higher cost and higher complexity. This complexity arises from the high molecular weight on the one hand and high structural heterogeneity on the other hand, making the analytical characterization and quality control of mAbs and other biopharmaceuticals a significant challenge. In addition to these protein-based biopharmaceuticals, the elucidation of the absolute configuration of therapeutic peptides and natural (lipo)peptides is also of particular interest for drug discovery. To address these challenges, different liquid chromatography (LC) and mass spectrometric (MS) methods were used for the more comprehensive analysis in the presented work. The first publication of this dissertation was dedicated to the analysis of charge variants of mAbs, which is an important quality attribute that might affect safety and efficacy of the drug product. To characterize the charge variants, the mAbs were analysed at the intact protein level and the subunit level after limited digestion and disulphide reduction using strong cation-exchange chromatography (SCX). The SCX method was systematically optimized to enable the separation of the maximum number of charge variants using a design of experiments (DoE) approach. The optimized SCX mobile phase, however, contains high concentrations of non-volatile salt in the mobile phase, which is incompatible with MS detection. On the other hand, MS analysis is essential for the identification of the charge variants. To overcome this limitation, an online two-dimensional liquid chromatographic (2D-LC) method was successfully developed, which uses SCX in the first separation dimension and reversed-phase (RP) LC in the second separation dimension, which can be used for de-salting prior MS analysis. An ultra-short analysis time (≤ 1 min) of the second dimension RP method was essential to establish a full comprehensive 2D-LC analysis. For this purpose, a column comparison study was performed using a set of monolithic and superficially porous particle (SPP) columns, and the separation efficiency and analysis speed were investigated. An even more comprehensive column comparison study focusing on the kinetic performance was done for the second work presented in this dissertation. A set of 13 RP protein separation columns including monolithic, SPP, and fully porous particle (FPP) columns was investigated regarding their capability to separate peaks in the shortest possible time. It could be demonstrated that SPP columns with a pore size of 400 Å and a thin, porous shell provided the best performance especially for large proteins such as mAbs. Proteins themselves can also be the potential targets of drug products such as the tumour suppressor protein p53 studied in publication III. Intact protein LC-MS was successfully used to investigate the binding efficiency and specificity of covalent inhibitors. Amino acids are the building blocks of proteins and peptides and most of these amino acids are chiral. As the biological activity is usually dependent on the absolute configuration of the amino acids, the enantioselective analysis is of utmost importance for structural elucidation and quality control. Therefore, one goal of the presented work was to develop a fast and comprehensive method to separate amino acids, their enantiomers, diastereomers, and constitutional isomers. This was achieved by derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and subsequent analysis by enantioselective liquid chromatography ion mobility-mass spectrometry (LC-IM-MS). A very fast three minutes short analysis method could be developed and was applied for the successful structure elucidation of a therapeutic peptide and a natural lipopeptide. The absolute configuration of a tetrapeptide originating from the natural antimicrobial peptide-polyene epifadin could be determined using chiral LC-MS, which was crucial for the structure elucidation. In this work, all eight enantiomer peak pairs could be successfully separated and the separation of the diastereomers was optimized

    Target Selection and Enhancement During Attentional Tracking

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    At any waking moment, we are bombarded with more sensory information than we can fully process. Attention is necessary to deal with the dynamic world we live in. One fundamental function of vision and attention is to keep track of moving objects, but what are the targets of attention during tracking? One of the first theories of attentional tracking predicted that targets would be selected at early processing stages. By employing the double-drift illusion, which dissociates physical and perceived positions of moving objects, we investigated which of these positions is selected for tracking. Contrary to earlier theories and in line with newer findings, targets were selected rather late in visual processing, at least after the construction of illusory percepts, for both covert (Chapter One) and overt tracking (Chapter Two). Furthermore, capacity and speed limits to attentional tracking are hemifield specific. Brain activity in many areas is known to covary with the number of tracked targets, but it was previously unknown whether this effect would also show hemifield bias. Only targets presented in the contralateral hemifield influenced activity in earlier visual areas, while both contralateral and ipsilateral targets affected activity in parietal and frontal areas associated with attention (Chapter Three). Due to the hemifield specific nature of the capacity limit, we conjecture that it should emerge where load dependent activity is strongly contralateral. Overall, the studies presented in this dissertation illuminate two different aspects of attentional tracking. While selection happens late in the visual hierarchy, capacity and speed limits appear to emerge early in visual processing

    LIPIcs, Volume 251, ITCS 2023, Complete Volume

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    LIPIcs, Volume 251, ITCS 2023, Complete Volum

    Advanced Filter Solutions for High-performance Millimetre and Submillimetre-wave Systems

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    This thesis is devoted to the investigation of advanced filter design solutions for high-performance millimetre and submillimetre-wave systems. Each of the proposed design solutions are enabled using waveguide-based technologies with the aim of advancing future generations of satellite communications, radar, and remote sensing. As trends for frequency allocations move to higher and higher frequency bands, engineers are faced with increasingly complex challenges such as the degradation of component performance, the inability to correctively tune the performance, or scenarios that all together make circuits infeasible. In light of these challenges, this work seeks to advance the current literature on filter design and proposes many unique design solutions for overcoming manufacturing and accuracy limitations, reducing the transmission losses, and reducing the overall design complexity. Each of the proposed filter solutions that are presented in this thesis are based on either a novel structural design or a novel technology. Each of the proposed designs are presented with functional prototypes as a means of verifying the theory. In the majority of cases, prototypes have been manufactured using high-precision computer numerical control (CNC) milling, and in several articles, exploratory activities with the use of alternative technologies such as stereolithography (SLA) 3D-printing and deep-reactive ion etching (DRIE) are presented. Prior to the presentation of the filter designs, an overview on the design and synthesis of millimetre-wave filters and diplexers is provided and serves as a foundation for the coupling matrix descriptions of symmetric and asymmetric resonator designs throughout this work

    Sensing Collectives: Aesthetic and Political Practices Intertwined

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    Are aesthetics and politics really two different things? The book takes a new look at how they intertwine, by turning from theory to practice. Case studies trace how sensory experiences are created and how collective interests are shaped. They investigate how aesthetics and politics are entangled, both in building and disrupting collective orders, in governance and innovation. This ranges from populist rallies and artistic activism over alternative lifestyles and consumer culture to corporate PR and governmental policies. Authors are academics and artists. The result is a new mapping of the intermingling and co-constitution of aesthetics and politics in engagements with collective orders

    Analysis of three body decays in quasi-real photoproduction

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    This thesis presents preliminary measurements of the moments of angular distribution for the K∗K + mesonic final state using a quasi-real, linearly polarised photon. Two of the main points of focus for this analysis were the development of the formalism for photoproduced vector-pseudoscalar decay and the tools by which the analysis was carried out. The formalism described how the moments of angular distribution could be extracted from the angular decay variables, and how they related to resonance decays in terms of partial waves. In extracting the moments, it was found that adapted Markov Chain Monte Carlo (MCMC) methods proved to be more effective in terms of extracting results from the data compared to the sole use of gradient descent based fitting algorithms. These measurements are complementary to other decay channels and production mechanisms, with the global aim of probing the existence of exotic mesons. Data from this analysis was taken by the CLAS collaboration at the Thomas Jefferson National Accelerator Facility (JLab) by scattering from a liquid hydrogen target using a 10.6 GeV electron. This work was done within the MesonEx program at CLAS12 that aims to map the spectrum of mesons, and in doing so, gain a greater understanding of Quantum ChromoDynamics (QCD) and the strong force

    Electrochemical Hydrogen Gas Production and Carbon Dioxide Sequestration via \u3ci\u3eIn Situ\u3c/i\u3e Formation of Nickel (II) Tris-(Pyridinethiolate) Derivatives

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    In the research presented, we use electrochemical techniques, such as cyclic voltammetry and controlled potential electrolysis, to study in situ formation of hydrogen evolution catalysts which also reduce carbon dioxide. Using pyridinethiol-based ligands and a nickel (II) precursor, both homo- and heteroleptic complexes were investigated as catalysts to produce alternative fuels while mitigating greenhouse gas emissions. Existing synthetic procedures to obtain these Ni(II) catalysts lead to low yields of the complexes and difficulties in crystallizing samples for further analysis, limiting sample size and restricting the number of studies. While current research is heavily focused on photochemically-driven experiments, given that solar radiation is highly inconsistent across the world, a major shift must be made to understand electrocatalysts and how they yield pertinent information about solar fuel production. Cyclic voltammetry was used to investigate production of hydrogen gas from the in situ formation of nickel(II) tris-(pyridinethiolate), 3P+Ni. Comparing a sample of the isolated versus the in situ catalyst, the voltammograms suggested that the complex not only self-assembled in solution but also performed proton reduction. Four separate homoleptic catalysts with electron donating and electron withdrawing groups were employed to explore how ligand modifications could influence the reduction potential needed to produce hydrogen gas. Although 3P+Ni did not have ligand substituents, it afforded the most positive reduction potential (-1.41 V v. SCE) and concurrently a low rate-constant (376 mM-1·s-1) for hydrogen production. 3(3-F)+Ni, in situ nickel(II) tris (3 (trifluoromethyl)pyridine-2-thiolate), exhibited the most negative reduction potential (-1.68 V v. SCE) and the highest rate constant (951 mM-1·s-1) for proton reduction. Using these findings, heteroleptic catalysts were proposed to develop catalysts with tunable reduction potentials. By using a 1:2:1 stoichiometric ratio (metal precursor, major ligand, and minor ligand), heteroleptic catalytic solutions were developed in situ and tested for hydrogen production. Reduction potentials for all the heteroleptic pairs were found to be more positive than their ligand modified homoleptic parent complex, i.e., heteroleptic 2P+1(3 F)+Ni (-1.53 V v. SCE) and 2(3-F)+1P+Ni (-1.50 V v. SCE) have lower overpotentials when compared to 3(3-F)+Ni (-1.68 V v. SCE). When comparing voltammograms for the heteroleptic pairs, similarities in the traces suggested that one hydrogen evolution electrocatalyst is formed for both ratio pairs. Despite the differences in the stochiometric ligand ratios, the electrochemical studies and reduction potentials are consistent with this theory. With respect to the rate constants, most of the catalytic solutions were also found to work better than the ligand modified parent analogues; therefore, the studies imply that in situ heteroleptic catalysts offer improvements without rigorous synthetic techniques. Expanding on the versatility of these in situ nickel(II) catalysts, preliminary experiments using controlled potential electrolysis and quantitative gas analysis of the headspace tested carbon dioxide reduction. Exploring homoleptic 3P+Ni and 3(3-F)+Ni, both electrocatalysts were found to produce a maximum partial pressure change of ~14-15% CH4, ~15-16% HCOOH, and ~21% CO. However, they were found to produce the highest pressure changes from H2 evolution (~49%) which identified 3P+Ni and 3(3-F)+Ni as non-selective for CO2 reduction over proton reduction catalysts. Three heteroleptic catalysts, namely 2P+1(3-F)+Ni, 2(3 M)+1P+Ni, and 2P+1(3-M)+Ni, were investigated similarly and where found to show a significant improvement in the pressure changes related to HCOOH (+ ~4-5%). Although these in situ heteroleptic catalysts were also deemed better proton reduction catalysts, they afforded selectivity for HCOOH formation that was not expected. Through this research, the benefits of in situ studies in yielding measurable hydrogen gas production through electrochemical studies is shown, as well as interesting results that showcase the further development of these Ni(II) catalysts for carbon dioxide reduction and formic acid formation

    Selected problems of materials science. Vol. 2. Nano-dielectrics metals in electronics. Mеtamaterials. Multiferroics. Nano-magnetics

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    The textbook examines physical foundations and practical application of current electronics materials. Modern theories are presented, more important experimental data and specifications of basic materials necessary for practical application are given. Contemporary research in the field of microelectronics and nanophysics is taken into account, while special attention is paid to the influence of the internal structure on the physical properties of materials and the prospects for their use. English-language lectures and other classes on the subject of the book are held at Igor Sikorsky Kyiv Polytechnic Institute at the departments of “Applied Physics” and “Microelectronics” on the subject of materials science, which is necessary for students of higher educational institutions when performing scientific works. For master’s degree applicants in specialty 105 “Applied physics and nanomaterials”.Розглянуто фізичні основи та практичне застосування актуальних матеріалів електроніки. Подано сучасні теорії, наведено найважливіші експериментальні дані та специфікації основних матеріалів, які потрібні для практичного застосування. Враховано сучасні дослідження у галузі мікроелектроніки та нанофізики, при цьому особливу увагу приділено впливу внутрішньої структури на фізичні властивості матеріалів і на перспективи їх використання. Англомовні лекції та інші види занять за тематикою книги проводяться в КПІ ім. Ігоря Сікорського на кафедрах «Прикладна фізика» та «Мікро-електроніка» за напрямом матеріалознавство, що необхідно студентам вищих навчальних закладів при виконанні наукових робіт. Для здобувачів магістратури за спеціальністю 105 «Прикладна фізика та наноматеріали»

    An efficient quantum memory in 167Er3+:Y2SiO5

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    This thesis investigates whether a quantum memory suitable for quantum communication applications can be developed using an erbium doped crystal. To assess the potential of the storage material, 167Er3+:Y2SiO5, the performance of two quantum memory protocols are characterised, the Atomic Frequency Comb (AFC) and Rephased Amplified Spontaneous Emission (RASE). As such, this work is a spiritual successor to two previous PhD projects, Kate Ferguson's non-classical demonstration of the RASE protocol using praseodymium, and Milos Rancic's high resolution spectroscopy and demonstration of long hyperfine coherence times in erbium. A telecom compatible quantum memory is vital for the DLCZ quantum repeater protocol, a critical device for quantum communications networks. A quantum memory designed for communications networks will need to meet several requirements: operate in the fibre optic telecommunications band, high recall efficiency, long storage time, and high bandwidth. Erbium is of interest as it has an optical transition within the telecommunications C-band (1530-1565 nm) and Rancic's thesis demonstrated the hyperfine coherence time needed for long storage times, 1.3 s. However, efficient quantum memories using erbium have not been demonstrated to date. This thesis will present an efficient quantum memory using erbium and discuss a pathway to demonstrate all the above criteria simultaneously. Techniques that were developed in Rancic's thesis are expanded in this thesis to create a new memory preparation process. The preparation process uses the long hyperfine lifetimes and large hyperfine splittings found in 167Er3+:Y2SiO5. Using this preparation, two quantum memory protocols were demonstrated, the Atomic Frequency Comb (AFC) and Rephased Amplified Spontaneous Emission (RASE), from Ferguson's thesis. In the AFC experiments, non-classical storage was demonstrated with a delay time of 0.66 us, an efficiency of 22%, and a bandwidth of 6 MHz. In the RASE experiments, an efficiency of 47% was demonstrated with a spin-state storage time of 27 us, and the potential to store 40 temporal modes. The initial results have shown orders of magnitude increases in storage times and efficiency over previous erbium memories. However, the efficiencies shown are not high enough for a quantum repeater demonstration. Cavity-enhancement offers a way to increase the efficiencies of both the AFC and RASE demonstrations. In the AFC chapter, cavity enhancement was discussed as a way to increase the efficiency, theoretically, to 96.6% with a 100 MHz bandwidth. These predicted efficiencies and bandwidths, using erbium, would meet three of the requirements needed for applications in a communications network, while Rancic has already demonstrated the remaining requirement in the same material. The next step for this work will be to realise the predicted efficiency and bandwidth, and then implement hyperfine rephasing for long storage times. In summary, this thesis expands on the works of Ferguson and Rancic to demonstrate quantum memories based in erbium. The demonstrations are promising so far, and proposed improvements to the experiment suggest that a quantum memory fit for quantum networks applications is possible. Furthermore, a pathway to an improved quantum memory is presented. Such a memory could be used in an initial quantum repeater demonstration
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