Asymmetrical flow field-flow-fractionation in pharmaceutical analytics

Due to enormous progress in recombinant DNA techniques and methodology, a multitude of biosynthetic, pharmaceutically relevant polypeptides and proteins became available in the past decade and have been employed in numerous pharmaceutical products. Concomitantly, substantial progress was made in pharmaceutical formulation development of peptides and proteins, inasmuch as many challenges in formulating these compounds in products with optimal therapeutic effects and shelf life were successfully approached. Additionally, new drug delivery systems – e.g., based on polymeric materials – will most likely enlarge the spectrum of future proteinic dosage forms, where today solutions and lyophilized products take center stage. Yet, due to the proneness of proteins to degradation - what can affect pharmaceutical relevant features such as biological activity and immunogenicity -, scrutinizing the homogeneity of protein formulations is of utmost importance. Hence, the development and implementation of new analytical techniques in order to keep pace is highly desired. It was the aim of this thesis to evaluate the applicability of asymmetrical flow field flow fractionation (AF4) in pharmaceutical protein analytics, to compare AF4 performance with established state-of-the-art methods and to reveal the effectivity of inherent AF4 characteristics in demanding analytical tasks. The Theoretical Section encompasses Chapter 2, wherein the family of field-flow fractionation techniques is introduced, as well as Chapter 3 (attending to protein aggregation) and Chapter 4, which provides an insight into multi-angle light scattering. The Theoretical Section is summarized in Chapter 5. Chapter 6 attends to the general applicability of (semi-)chromatographic AF4 in protein analysis. The correlation of cross flow progression with increased resolution was exemplified by the separation of human serum albumin (HSA), thereby rendering the (base-line) separation of HSA specimen into monomer, dimer, trimer and tetramer possible. Due to the AF4 feature to discretionarily alter the resolution power within one separation run, the fractionation of higherorder aggregates and insoluble, precipitated protein was successfully performed. System variables and parameters of fractionation were investigated, revealing that sample loads differing more than two orders of magnitude did not negatively affect data reproducibility. Whereas up to now cross flow intensity was deemed to predominantly account for contingent sample loss during AF4 experiments, analysis of proteins with varying hydrophilicity proved the preceding focusing step to contribute notably for that phenomenon. How to overcome potential drawbacks such as sample-membrane interactions by adequate choice of the ultrafiltration membrane as well as carrier liquid composition was illustrated. Chapter 11. Summary, conclusions and prospective 177 Given the background that effective AF4 fractionations are due to differences in analyte size – i.e., in diffusion coefficients -, the separation of equal-sized proteins is prima facie considered to be impractical. Yet, the retaining impact of sample-membrane interaction was evidenced to decrease the effective diffusion coefficient, resulting in successful fractionations of proteins differing ~1% in size (i.e., G-CSF, 19.6 kDa versus IFN-α2a, 19.4 kDa). In this realm, the normal mode elution order of smaller analytes eluting prior to larger ones was shown to be invertible, exemplified by the elution of a 40 kDa analyte prior to IFN-α2a. AF4 potency in analysis of insoluble high molecular weight (hmw) aggregates was compared with data derived by established methods such as light obscuration and Coulter technique, verifying the competitiveness of AF4. A comparative study of AF4 with size exclusion chromatography (SE-HPLC) unveiled SE-HPLC to inhere higher recovery rates and AF4 to exhibit greater resolution. Coupling both techniques with multi-angle light scattering (MALS) detection systems disclosed SE-HPLC to induce artifacts concerning hmw aggregate quantification. Moreover, in contrast to SE-HPLC, AF4 was capable of seizing undissolved and precipitated protein specimen, thus making AF4 a promising alternative in the analysis of protein pharmaceuticals. AF4´s ability to separate undissolved sample components proved to be an indispensable feature in the analysis of a pharmaceutical protein formulated within siliconized disposable syringes, which was attended to in Chapter 7. During long-term storage, visible particulate matter developed sporadically within the syringe volumes, raising the question of the particles´ origin. Since protein instabilities were not to be accounted for being the particle source – verified by several analytical methods -, silicone oil detachment and subsequent coalescence came into question, as the barrel siliconization process was lacking a final heat curing step. Thus, an AF4 application was developed, intending to separate μm sized silicone oil droplets. The task was approached by analysis of silicone oil emulsions, followed by the fractionation of ultrasoundstressed syringe volumes containing detached and coalesced silicone oil after stress exertion. Unambiguously, detached silicone oil was evidenced by AF4 to account for visible particulate matter in the syringe volumes, corroborated by MALS and refractive index detection as well as light microscopy and syringe frictional drag analysis. Subsequently to artificially induced protein aggregation of particle-containing syringe volumes, AF4 was able to separate silicone oil droplets, protein monomer and aggregates as individual fractions within one single run. Finally, AF4 enabled access to data on protein drug stability and insights into protein adsorption tendencies on coalesced silicone oil specimen – thereby providing valuable data which otherwise would have required a variety of various analytical techniques. Chapter 11. Summary, conclusions and prospective 178 In Chapter 8 the suitability of AF4 in overall-characterization of gelatin nanoparticles was explored. The efficacy of providing hmw gelatin bulk material by various desolvation steps was evaluated by SE-HPLC and AF4. Due to the absence of shear degradation phenomena, AF4 was demonstrated to enable more moderate separation conditions than SE-HPLC, verified by on-line determination of analyte molecular weight via MALS. Gelatin nanoparticles were characterized by means of AF4/MALS with respect to size and size distributions and the data were compared to results of photon correlation spectroscopy (PCS) and scanning electron microscopy (SEM). Because of the precedent separation step via AF4, data derived by MALS revealed a greater veracity than PCS results, where the size assessment of nanoparticles relied on batch experiments. Whereas PCS attributed unloaded and DNA plasmid loaded nanoparticles virtually unimodal size distributions, both AF4/MALS and SEM demonstrated the nanoparticles to span a broad size range. Furthermore, loaded and unloaded nanoparticles were unveiled to exhibit only minimal differences in size, thus providing information on the interplay of nanoparticles and plasmid strands. For the first time, the separation of nanocolloidal drug carrier and designated pharmaceutical payload was established. Additionally to drug carrier characteristics, data on the loading efficacy could be yielded. Furthermore, nanoparticle shelf life stability and extent of potential drug decomplexation could be determined. Bearing in mind colloidal, polymer-based drug delivery carriers gaining increasing importance, that very AF4 application is expected to accommodate the demand for accurate analytics, as the pharmaceutical product can be characterized in both qualitative and quantitative terms. In Chapter 9 a case study of particulate matter analysis of a pharmaceutical antibody solution is presented, wherein individual vials of one production lot developed visible components at random during long-term storage. In order to (a) provide evidence on the presence of the contamination, (b) to attempt particulate entitiy quantification and (c) to elucidate particles´ nature, a multiplicity of analytical techniques were applied, encompassing particle counting (optical inspection, light obscuration, light microscopy), protein characterization techniques (SE-HPLC, polyacrylamide gel electrophoresis, AF4, microcalorimetry) and particle separation techniques (sterile filtration, AF4). Attempts to isolate the particulate components by AF4 or filtration techniques provided no further indications of the particle´s origin. Virtually no alterations in protein characteristics were monitored between contaminated and particle-free vial volumes, respectively. Solely, microcalorimetric data of contaminated vial volumes resembled those of immunoglobulin solutions exposed to heat stress prior to analytics. Consequently, protein instabilities were assumed not to cause the visible contamination. Chapter 11. Summary, conclusions and prospective 179 The topic of liquid protein parenterals, protein instability and particulate matter was completed by presenting a formulation process of an immunoglobulin into a liquid formulation in Chapter 10. Prevalent strategies and mainstream trends of liquid protein formulation were introduced by reviewing latest publications on the issue. Parameters revealing decisive influence on the protein´s long-term stability such as solution pH as well as type and concentration of excipients were evaluated by means of accelerated stability studies at various storage temperatures. Additionally, processing parameters, e.g., freeze/thawing, were assessed evaluating criteria in terms of surfactant and buffer choice. The addition of NaCl was shown to detract from protein stability and to facilitate the formation of particulate matter. Non-deleterious alternatives of salt additives were discovered. On the other hand, the addition of polyols such as mannitol and sorbitol was demonstrated to notably contribute to the immunoglobulin stability. Preferential accumulation at the native state protein was thought to be the mechanism for reducing aggregation phenomena of the protein. Besides, the extent of fragmentation was reduced by polyols, indicating a second pathway of stabilization, which was hypothesized to be hampering of oxidation processes. Due to detailed investigations, a proposal pertaining an optimal formulation could be made in the course of that case study. This thesis has shown that asymmetrical flow field-flow fractionation (AF) can effectively be used to monitor protein stability in a broad variety of pharmaceutical formulations. Especially in the characterization of the most common outcome of physical instability – i.e., protein aggregation – the potential of AF4 has comprehensively been demonstrated. Moreover, AF4 applications and separation tasks within pharmaceutical analytics considered hitherto impractical or at least highly challenging were successfully performed. Facing increasingly complex liquid- or colloidal-based formulations, with this knowledge practice and research in pharmaceutical analytics can take a notable step forward

Los jóvenes y su sentido de la vida

Los jóvenes buscan un sentido, pero desde sí mismos. Han trasladado los muebles de la vieja casa tradicional y, por el momento, los tienen almacenados en el pasillo de su nuevo espacio mental sin saber cómo colocarlos en los distintos compartimentos de su nuevo hogar íntimo. Un conjunto de valores ampliamente compartidos sustentan una ética mínima, que permite articular la diversidad de sentidos

Preventing Distributed Denial-of-Service Attacks on the IMS Emergency Services Support through Adaptive Firewall Pinholing

Emergency services are vital services that Next Generation Networks (NGNs) have to provide. As the IP Multimedia Subsystem (IMS) is in the heart of NGNs, 3GPP has carried the burden of specifying a standardized IMS-based emergency services framework. Unfortunately, like any other IP-based standards, the IMS-based emergency service framework is prone to Distributed Denial of Service (DDoS) attacks. We propose in this work, a simple but efficient solution that can prevent certain types of such attacks by creating firewall pinholes that regular clients will surely be able to pass in contrast to the attackers clients. Our solution was implemented, tested in an appropriate testbed, and its efficiency was proven.Comment: 17 Pages, IJNGN Journa

Applying Human-Centered Design Methods in Industry – a Field Report

Human-centered design methods are nowadays widely used in the design of consumer products. These methods aim at designing products with a high usability and a positive user experience (UX). However, in the domain of the design of industrial machines, design is often driven by functional requirements mostly neglecting the usability and user experience of products. Together with a medium-sized manufacturer of industrial laundry machines we applied the human-centered design process in an industrial context. In this field report, we describe the human-centered design methods applied in the project, the adaptations we had to make in the process and the challenges and opportunities for applying human-centered design in an industrial environment in general

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In this paper, we review different approaches on proximity tracing apps which are supposed to automate the current labor-intensive contact tracing approach conducted by national health officials. The purpose of these apps is to automatically notify people who are at risk of being infected with SARS-CoV-2 to interrupt infection chains as early as possible. However, a privacy-preserving and yet functional and scalable design of such apps is not trivial and in some parts leads to counter-intuitive properties. This paper reviews the most prominent European approaches, DP-3T, the German variant NTK \u27 of PEPP-PT, and its closely related concept ROBERT. We discuss their design decisions from a privacy perspective and point out the fundamentally different adversary models assumed by the approaches. In addition, we touch on practical aspects such as scalability and ease of implementation

Kiri Friedrich Georg Wilhelm Struve'le

Fraunhofer, Joseph, 1787-1826, saksa optikStruve, Friedrich Georg Wilhelm, 1793-1864, astronoom, Tartu Ülikooli õppejõud, Peterburi TA liigeTeatab refraktori väljasaatmisest 21. aug. 22 kasti

INFACT technology watch report

This research has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement nº 776487. Furthermore, some of the authors (B.J. and V.H.-S.) were supported by the Spanish Ministry of Science Innovation and Universities under the framework of the R&D project RTI2018-098966-B-I00.Summary: This report presents a bibliometric study on patents and scientific publications related to the following technologies involved in INFACT: airborne electromagnetic methods, airborne gravity gradiometry, airborne magnetometry and drone-borne hyperspectral imaging. A statistical analysis of the documents reveals the main players, technology trends and collaboration patterns via bibliometric techniques

Adhesion and Cohesion

The phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry. This review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials. The forces involved in surface tension, surface wetting, chemical adhesion, dispersive adhesion, diffusive adhesion, and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given. Substrate surface chemistry and its influence on adhesion, together with the properties of adhesive materials, are evaluated. The underlying mechanisms involved in adhesion failure are covered. The relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed

Magnetorheological landing gear: 2. Validation using experimental data

Aircraft landing gears are subjected to a wide range of excitation conditions with conflicting damping requirements. A novel solution to this problem is to implement semi-active damping using magnetorheological (MR) fluids. In part 1 of this contribution, a methodology was developed that enables the geometry of a flow mode MR valve to be optimized within the constraints of an existing passive landing gear. The device was designed to be optimal in terms of its impact performance, which was demonstrated using numerical simulations of the complete landing gear system. To perform the simulations, assumptions were made regarding some of the parameters used in the MR shock strut model. In particular, the MR fluid's yield stress, viscosity, and bulk modulus properties were not known accurately. Therefore, the present contribution aims to validate these parameters experimentally, via the manufacture and testing of an MR shock strut. The gas exponent, which is used to model the shock strut's nonlinear stiffness, is also investigated. In general, it is shown that MR fluid property data at high shear rates are required in order to accurately predict performance prior to device manufacture. Furthermore, the study illustrates how fluid compressibility can have a significant influence on the device time constant, and hence on potential control strategies

SPH analysis of inkjet droplet impact dynamics

This paper presents a novel Smoothed Particle Hydrodynamics (SPH) framework for analysis of droplet impact dynamics in a 3D inkjet printing process. Results obtained are validated against experimentally derived high-speed imaging data. The numerical framework is based on the Smoothed Particle Hydrodynamics approach of Monaghan et al [1] which has been proven to be efficient and effective for analysis of dynamic fluid flow problems involving free surface interfaces. The SPH approach has been augmented through addition of the kernel gradient correction scheme proposed by Belytschko et al [2] and stabilization terms of Marrone et el [3]. This correction provides a more accurate approximation of the boundary forces including surface tension which dominate at typical inkjet droplet lengthscales (<100 µm). Analysis is expedited through adoption of the OpenACC programming paradigm to enable GPU based computation. Numerical analyses have been validated against analytical solutions, reference macroscale problems and through comparison with experimental high speed imaging data of the inkjet printing process. The experimental setup consisted of a Fuji Dimatix SL-128 inkjet printhead jetting an acrylate based 3D printing build material onto a glass substrate. Images of a single inkjet droplet impacting onto the glass slide were captured at a rate of 100,000 frames per second, with droplet diameter assessed using a weight test approach. Qualitative comparison of the numerical and experimental results showed a good agreement, indicating that the implemented framework is effective for analysis of the fluidic aspects of the printing process. The model is able to assist in tackling manufacturing issues that can detrimentally influence the quality of manufactured parts through provision of insight into the process