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Transgenic plants have the potential to produce recombinant proteins on an agricultural scale, with yields of several tons per year. The cost-effectiveness of transgenic plants increases if simple cultivation facilities such as greenhouses can be used for production. In such a setting, we expressed a novel affinity ligand based on the fluorescent protein DsRed, which we used as a carrier for the linear epitope ELDKWA from the HIV-neutralizing antibody 2F5. The DsRed-2F5-epitope (DFE) fusion protein was produced in 12 consecutive batches of transgenic tobacco (Nicotiana tabacum) plants over the course of 2 years and was purified using a combination of blanching and immobilized metal-ion affinity chromatography (IMAC). The average purity after IMAC was 57 ± 26% (n = 24) in terms of total soluble protein, but the average yield of pure DFE (12 mg kg−1) showed substantial variation (± 97 mg kg−1, n = 24) which correlated with seasonal changes. Specifically, we found that temperature peaks (>28°C) and intense illuminance (>45 klx h−1) were associated with lower DFE yields after purification, reflecting the loss of the epitope-containing C-terminus in up to 90% of the product. Whereas the weather factors were of limited use to predict product yields of individual harvests conducted for each batch (spaced by 1 week), the average batch yields were well approximated by simple linear regression models using two independent variables for prediction (illuminance and plant age). Interestingly, accumulation levels determined by fluorescence analysis were not affected by weather conditions but positively correlated with plant age, suggesting that the product was still expressed at high levels, but the extreme conditions affected its stability, albeit still preserving the fluorophore function. The efficient production of intact recombinant proteins in plants may therefore require adequate climate control and shading in greenhouses or even cultivation in fully controlled indoor farms.
Chromatography is the workhorse of biopharmaceutical downstream processing because it can selectively enrich a target product while removing impurities from complex feed streams. This is achieved by exploiting differences in molecular properties, such as size, charge and hydrophobicity (alone or in different combinations). Accordingly, many parameters must be tested during process development in order to maximize product purity and recovery, including resin and ligand types, conductivity, pH, gradient profiles, and the sequence of separation operations. The number of possible experimental conditions quickly becomes unmanageable. Although the range of suitable conditions can be narrowed based on experience, the time and cost of the work remain high even when using high-throughput laboratory automation. In contrast, chromatography modeling using inexpensive, parallelized computer hardware can provide expert knowledge, predicting conditions that achieve high purity and efficient recovery. The prediction of suitable conditions in silico reduces the number of empirical tests required and provides in-depth process understanding, which is recommended by regulatory authorities. In this article, we discuss the benefits and specific challenges of chromatography modeling. We describe the experimental characterization of chromatography devices and settings prior to modeling, such as the determination of column porosity. We also consider the challenges that must be overcome when models are set up and calibrated, including the cross-validation and verification of data-driven and hybrid (combined data-driven and mechanistic) models. This review will therefore support researchers intending to establish a chromatography modeling workflow in their laboratory.
Proteins are important ingredients in food and feed, they are the active components of many pharmaceutical products, and they are necessary, in the form of enzymes, for the success of many technical processes. However, production can be challenging, especially when using heterologous host cells such as bacteria to express and assemble recombinant mammalian proteins. The manufacturability of proteins can be hindered by low solubility, a tendency to aggregate, or inefficient purification. Tools such as in silico protein engineering and models that predict separation criteria can overcome these issues but usually require the complex shape and surface properties of proteins to be represented by a small number of quantitative numeric values known as descriptors, as similarly used to capture the features of small molecules. Here, we review the current status of protein descriptors, especially for application in quantitative structure activity relationship (QSAR) models. First, we describe the complexity of proteins and the properties that descriptors must accommodate. Then we introduce descriptors of shape and surface properties that quantify the global and local features of proteins. Finally, we highlight the current limitations of protein descriptors and propose strategies for the derivation of novel protein descriptors that are more informative.
The book covers various numerical field simulation methods, nonlinear circuit technology and its MF-S- and X-parameters, as well as state-of-the-art power amplifier techniques. It also describes newly presented oscillators and the emerging field of GHz plasma technology. Furthermore, it addresses aspects such as waveguides, mixers, phase-locked loops, antennas, and propagation effects, in combination with the bachelor's book 'High-Frequency Engineering,' encompassing all aspects related to the current state of GHz technology.
Self metathesis of oleochemicals offers a variety of bifunctional compounds, that can be used as monomer for polymer production. Many precursors are in huge scales available, like oleic acid ester (biodiesel), oleyl alcohol (tensides), oleyl amines (tensides, lubricants). We show several ways to produce and separate and purify C18-α,ω-bifunctional compounds, using Grubbs 2nd Generation catalysts, starting from technical grade educts.
The research group focuses on the characteristics in the land-and cityscapes of the Drielanden-zone, which contribute to generate common identities, as well as on those features that trigger differences and specificities of the adjacent countries that enrich the perception of the zone. In this research, the instruments of cartography and land survey system serve to detect and localize the fragmented appearance of relevant historic elements. These analytic procedures help to develop strategies for infrastructures and processes that gradually initiate local forms of cross-border tourism. The architectural research displays how top-down and bottom-up interventions can be combined in order to guarantee a sustainable use and development of the considered area.
In many instances, freight vehicles exchange load or information with plants that are or will soon be Industry4.0 plants. The Wagon4.0 concept, as developed in close cooperation with e.g. port or mine operations, offers a maximum in railway operational efficiency while providing strong business cases already in the respective plant interaction. The Wagon4.0 consists of main components, a power supply, data network, sensors, actuators and an operating system, the so called WagonOS. The Wagon OS is implemented in a granular, self-sufficient manner, to allow basic features such as WiFi-Mesh and train christening in remote areas without network connection. Furthermore, the granularity of the operating system allows to extend the familiar app concept to freight rail rolling stock, making it possible to use specialised actuators for certain applications, e.g. an electrical parking brake or an auxiliary drive. In order to facilitate migration to the Wagon4.0 for existing fleets, a migration concept featuring five levels of technical adaptation was developed. The present paper investigates the benefits of Wagon4.0-implementations for the particular challenges of heavy haul operations by focusing on train christening, ep-assisted braking, autonomous last mile and traction boost operation as well as improved maintenance schedules