Development and application of two-dimensional capillary electrophoresis for online mass spectrometry of CE(SDS)-separated proteins

Size-based protein separation by CE(SDS) is indispensable for process and quality control in the biopharmaceutical industry and is commonly applied for purity assessment on behalf of drug approval. To overcome the challenge of uncertain peak identification of CE(SDS)-separated fragments and impurities, here, the first online hyphenation with mass spectrometry is presented. This hyphenation is achieved by introducing a second electrophoretic, MS-compatible CZE dimension after the first generic CE(SDS) separation. Both dimensions are hyphenated via a 4-port nanoliter valve that is commercially available from VICI AG International. As soon as the peak of interest of the CE(SDS) separation reaches the sample loop, the separation is stopped. Due to the very stable SDS-protein complex that can not be separated electrophoretically in the CZE dimension alone, a decomplexation with a solvent and a cationic surfactant is required. Therefore, simultaneously to the separation in the 1D, methanol as presample and CTAB (solved in methanol : water (1:1)) as postsample zone are injected and positioned via a C4D detector in the second CZE dimension. The peak of interest is then transferred via a heart-cut approach between the decomplexation zones in the 2D. With this set-up, it was possible to demonstrate the proof of principle. Universal application of the mass spectrometric CE(SDS) protein characterization is presented by soybean protein analysis. By analyzing several mAb fragments, the suitability and power of the 2D system for pertinent biopharmaceutical questions are proven. The intact mass determination facilitate identification of these fragments directly from generic CE(SDS) separation for the first time. In addition, the analysis allows a deeper insight, revealing migration order shifts and detection of several fragments, migrating simultaneously in the CE(SDS) dimension. Detection of these proteoforms resulting from only one CE(SDS) peak demonstrates the necessity of a more detailed investigation that can only be achieved by mass spectrometry hyphenation. One major drawback of this 4-port nanoliter valve approach is the system and measurement instability due to frequently occurring current leakage. This current leakage is mainly related to the utilized 4-port nanoliter valves material and design, with distances of less than 1 mm between the separation dimensions. Additionally, the exact positioning of the CE(SDS) peak from the 1D in between the decomplexation zones in the 2D is crucial for successful decomplexation and directly related to MS signal intensity. For these main reasons, further development of the nanoliter valve and method adaptation was the primary aim of the second part of this work to improve system and measurement stability. Therefore, an 8-port nanoliter valve was developed and designed. To allow immediate and unambiguous identification and characterization of CE(SDS)- separated peaks, the optimized system was hyphenated with top-down mass spectrometry. After successful hyphenation of the 2D system with the Orbitrap MS via nanoflow sheath liquid interface, method compatibility of the developed top-down MS method with the decomplexation strategy was tested. It was possible to prove that concentration down to 0.03 mg/mL of NIST mAb LC can be analysed by top-down MS analysis. Furthermore, the presence of intra disulfide bonds was verified by intact mass determination and MS2 fragmentation. In the last part, the identification and characterization of stress-induced fragments of two different mAbs were performed. Both mAb fragments were identified as Fc fragments of the HC, and the cleavage mechanism could be suggested. This identification was only feasible by MS2 top-down measurements and not possible by intact mass analysis alone, representing the value of the third part of this work. The here presented work demonstrates the first online mass spectrometric characterization of generic CE(SDS)-separated proteins. It is shown that intact mass determination strongly supports the identification of these proteins and mAb fragments. The observation of unexpected migration order shift and several masses of proteoforms by analyzing only one CE(SDS) peak confirm the ambiguity of optical detection alone. Top-down experiments verify the strength of direct identification of mAb fragments and PTMs like the presence of disulfide bonds, which can not be distinguished by intact mass determination alone. This direct and unambiguous identification of CE(SDS)-separated proteins provides a detailed insight. It facilitates the understanding of protein stability and fragmentation, thereby supporting the production, safety and efficacy assurance of biopharmaceuticals

Sistemas pasivos de iluminación y ventilación natural que influyen en el diseño de centro comercial en el distrito de Trujillo, 2020

Los centros comerciales son parte fundamental en el desarrollo económico y social de una ciudad y/o urbanización para el desarrollo de microclimas confortables, es por ello, que tales equipamientos están proyectados a brindar al público una iluminación y ventilación natural adecuada o en óptimas condiciones, mejorando la calidad de vida de los usuarios que afrontan el problema de trabajar en entornos climáticos no confortables, a través de temperaturas óptimas, generando espacios agradables para su buen funcionamiento. La presente tesis de investigación tiene como objetivo determinar de qué manera los sistemas pasivos de iluminación y ventilación natural condicionan el diseño de un centro comercial en el distrito de Trujillo, basados en la búsqueda de crear entornos climáticos óptimos para la funcionalidad de las actividades comerciales, debido a que mientras mayor iluminación y ventilación natural ingrese al objeto arquitectónico este será más confortable y saludable para los usuarios Se empleó como metodología el análisis de casos arquitectónicos en donde se pueden apreciar el uso de estos sistemas mencionados y su relación con el diseño arquitectónico del edificio para el control solar, de manera que se obtuvieron diferentes lineamientos de diseño para el objeto arquitectónico en mención. Además, se analizaron diversas necesidades, características y problemas en el lugar, por lo tanto, se hizo investigaciones para que el proyecto funcione considerando el confort en espacios interiores y exteriores mediante elementos arquitectónicos, aprovechando las condiciones climáticas. Finalmente, se desarrolló el programa arquitectónico y se determinó el área de terreno requerido para emplazar el objeto arquitectónico de la presente tesis de investigación

Membrane curvature in cell biology: An integration of molecular mechanisms.

Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists

The use of ketogenic diets in cancer patients: a systematic review

Ketogenic diets are a widely known, yet controversial treatment for cancer patients. In this review, we summarize the clinical evidence for anti-tumor effects, as well as the effects on anthropometry, quality of life, adverse events and adherence in cancer patients. In April 2019, a systematic search was conducted searching five electronic databases (EMBASE, Cochrane, PsychInfo, CINAHL and Medline) to find studies analyzing the use, effectiveness and potential harm of a ketogenic diet in cancer patients of any age as sole or complementary therapy. From all 19.211 search results, 46 publications concerning 39 studies with 770 patients were included in this systematic review. The therapy concepts included all forms of diets with reduced carbohydrate intake, that aimed to achieve ketosis for patients with different types of cancer. Most studies had a low quality, high risk of bias and were highly heterogeneous. There was no conclusive evidence for anti-tumor effects or improved OS. The majority of patients had significant weight loss and mild to moderate side effects. Adherence to the diet was rather low in most studies. Due to the very heterogeneous results and methodological limitations of the included studies, clinical evidence for the effectiveness of ketogenic diets in cancer patients is still lacking