RESUMO
The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.In contrast to single recombinant proteins, the glycan profiles of viral vaccines are far more complex and difficult to predict. The example of influenza A virus shows that hemagglutinin, the major antigenic determinant, has three to nine N-glycans, which may influence the antigenicity and efficacy of the vaccine. Glycosylation of the influenza A virus has been largely unmonitored in the past as production has been from eggs, where glycan profiles of antigens are difficult if not impossible to control. Over the past decade, however, there have been various commercial influenza vaccines made available from cell technology using animal host cells. Analysis of glycosylation control shows that the type of host cell has the greatest influence on the final analyzed glycan profile. Other factors such as the virus strain, the cultivation system, or various process parameters have been shown to have only a minor effect on the glycosylation pattern. We predict that the analysis of glycan profiles in viral vaccines will become increasingly important in the development and consistent manufacturing of safe and potent vaccines. Graphical Abstract.
Assuntos
Vacinas contra Influenza , Animais , Anticorpos Monoclonais/metabolismo , Glicosilação , Polissacarídeos , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: In biogas plants, complex microbial communities produce methane and carbon dioxide by anaerobic digestion of biomass. For the characterization of the microbial functional networks, samples of 11 reactors were analyzed using a high-resolution metaproteomics pipeline. RESULTS: Examined methanogenesis archaeal communities were either mixotrophic or strictly hydrogenotrophic in syntrophy with bacterial acetate oxidizers. Mapping of identified metaproteins with process steps described by the Anaerobic Digestion Model 1 confirmed its main assumptions and also proposed some extensions such as syntrophic acetate oxidation or fermentation of alcohols. Results indicate that the microbial communities were shaped by syntrophy as well as competition and phage-host interactions causing cell lysis. For the families Bacillaceae, Enterobacteriaceae, and Clostridiaceae, the number of phages exceeded up to 20-fold the number of host cells. CONCLUSION: Phage-induced cell lysis might slow down the conversion of substrates to biogas, though, it could support the growth of auxotrophic microbes by cycling of nutrients.
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Bacteriófagos/genética , Biocombustíveis/microbiologia , Consórcios Microbianos , Interações Microbianas , Proteoma , Anaerobiose , Bactérias/genética , Bactérias/virologia , Bacteriófagos/metabolismo , Reatores Biológicos/microbiologia , Fermentação , Metano/metabolismo , RNA Ribossômico 16SRESUMO
Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis. Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used. Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase. SIGNIFICANCE: Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases.
Assuntos
Colite Ulcerativa , Doença de Crohn , Fezes/microbiologia , Microbioma Gastrointestinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Colite Ulcerativa/metabolismo , Colite Ulcerativa/microbiologia , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Complex microbial communities are the functional core of anaerobic digestion processes taking place in biogas plants (BGP). So far, however, a comprehensive characterization of the microbiomes involved in methane formation is technically challenging. As an alternative, enriched communities from laboratory-scale experiments can be investigated that have a reduced number of organisms and are easier to characterize by state of the art mass spectrometric-based (MS) metaproteomic workflows. Six parallel laboratory digesters were inoculated with sludge from a full-scale BGP to study the development of enriched microbial communities under defined conditions. During the first three month of cultivation, all reactors (R1-R6) were functionally comparable regarding biogas productions (375-625 NL Lreactor volume-1 d-1), methane yields (50-60%), pH values (7.1-7.3), and volatile fatty acids (VFA, <5 mM). Nevertheless, a clear impact of the temperature (R3, R4) and ammonia (R5, R6) shifts was observed for the respective reactors. In both reactors operated under thermophilic regime, acetic and propionic acid (10-20 mM) began to accumulate. While R4 recovered quickly from acidification, the levels of VFA remained to be high in R3 resulting in low pH values of 6.5-6.9. The digesters R5 and R6 operated under the high ammonia regime (>1 gNH3 L-1) showed an increase to pH 7.5-8.0, accumulation of acetate (>10 mM), and decreasing biogas production (<125 NL Lreactor volume-1 d-1). Tandem MS (MS/MS)-based proteotyping allowed the identification of taxonomic abundances and biological processes. Although all reactors showed similar performances, proteotyping and terminal restriction fragment length polymorphisms (T-RFLP) fingerprinting revealed significant differences in the composition of individual microbial communities, indicating multiple steady-states. Furthermore, cellulolytic enzymes and cellulosomal proteins of Clostridium thermocellum were identified to be specific markers for the thermophilic reactors (R3, R4). Metaproteins found in R3 indicated hydrogenothrophic methanogenesis, whereas metaproteins of acetoclastic methanogenesis were identified in R4. This suggests not only an individual evolution of microbial communities even for the case that BGPs are started at the same initial conditions under well controlled environmental conditions, but also a high compositional variance of microbiomes under extreme conditions.
Assuntos
Biocombustíveis , Microbiota , Plantas/metabolismo , Plantas/microbiologia , Proteômica , Amônia/análise , Anaerobiose , Biocombustíveis/análise , Reatores Biológicos , Análise por Conglomerados , Ácidos Graxos Voláteis/análise , Metagenômica/métodos , Metano/biossíntese , Proteômica/métodos , Espectrometria de Massas em Tandem , TemperaturaRESUMO
BACKGROUND: Methane yield and biogas productivity of biogas plants (BGPs) depend on microbial community structure and function, substrate supply, and general biogas process parameters. So far, however, relatively little is known about correlations between microbial community function and process parameters. To close this knowledge gap, microbial communities of 40 samples from 35 different industrial biogas plants were evaluated by a metaproteomics approach in this study. RESULTS: Liquid chromatography coupled to tandem mass spectrometry (Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer) of all 40 samples as triplicate enabled the identification of 3138 different metaproteins belonging to 162 biological processes and 75 different taxonomic orders. The respective database searches were performed against UniProtKB/Swiss-Prot and seven metagenome databases. Subsequent clustering and principal component analysis of these data allowed for the identification of four main clusters associated with mesophile and thermophile process conditions, the use of upflow anaerobic sludge blanket reactors and BGP feeding with sewage sludge. Observations confirm a previous phylogenetic study of the same BGP samples that was based on 16S rRNA gene sequencing by De Vrieze et al. (Water Res 75:312-323, 2015). In particular, we identified similar microbial key players of biogas processes, namely Bacillales, Enterobacteriales, Bacteriodales, Clostridiales, Rhizobiales and Thermoanaerobacteriales as well as Methanobacteriales, Methanosarcinales and Methanococcales. For the elucidation of the main biomass degradation pathways, the most abundant 1 % of metaproteins was assigned to the KEGG map 1200 representing the central carbon metabolism. Additionally, the effect of the process parameters (i) temperature, (ii) organic loading rate (OLR), (iii) total ammonia nitrogen (TAN), and (iv) sludge retention time (SRT) on these pathways was investigated. For example, high TAN correlated with hydrogenotrophic methanogens and bacterial one-carbon metabolism, indicating syntrophic acetate oxidation. CONCLUSIONS: This is the first large-scale metaproteome study of BGPs. Proteotyping of BGPs reveals general correlations between the microbial community structure and its function with process parameters. The monitoring of changes on the level of microbial key functions or even of the microbial community represents a well-directed tool for the identification of process problems and disturbances.Graphical abstractCorrelation between the different orders and process parameter, as well as principle component analysis of all investigated biogas plants based on the identified metaproteins.
RESUMO
Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.
Assuntos
Cromatografia de Afinidade/métodos , Eritropoetina/química , Eritropoetina/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Serotonina/metabolismo , Configuração de Carboidratos , Eritropoetina/metabolismo , Humanos , Ácido N-Acetilneuramínico/química , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Serotonina/químicaRESUMO
Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution.
Assuntos
Proteínas Arqueais/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Metano/biossíntese , Methanomicrobiales/metabolismo , Methanosarcinales/metabolismo , Proteoma/análise , Biocombustíveis , Reatores Biológicos , Eletroforese em Gel de Poliacrilamida/métodos , Focalização Isoelétrica/métodos , Metagenoma , Methanomicrobiales/química , Methanomicrobiales/genética , Methanosarcinales/química , Methanosarcinales/genética , Consórcios Microbianos/fisiologia , Plantas/metabolismo , Proteólise , Espectrometria de Massas em Tandem , Temperatura , ResíduosRESUMO
In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin-Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.
Assuntos
Meios de Cultura/química , Citosol/química , Células Epiteliais/metabolismo , Animais , Cães , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Células Madin Darby de Rim Canino , Redes e Vias Metabólicas , MetabolomaRESUMO
Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10â»5, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log10 HAU/100 µL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log10 HAU/100 µL and TCID50 of 1 × 109 and 2.37 × 109 infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.
Assuntos
Apoptose , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Células Madin Darby de Rim Canino/fisiologia , Células Madin Darby de Rim Canino/virologia , Carga Viral , Animais , Adesão Celular , Sobrevivência Celular , Células Imobilizadas , Cães , Cultura de Vírus/métodosRESUMO
Continuous size exclusion chromatography for the separation of cell culture-derived influenza virus from contaminating proteins was established successfully. Therefore, an open loop simulated moving bed (SMB) setup with one column per zone was applied. Several operating conditions were tested and overall trends were found to be in agreement with expectations derived from theory. Furthermore, the separation performance was compared to an optimized conventional batch chromatography. The yield of influenza virus in the product fraction, based on a hemagglutination assay, was 70% (SMB) and 80% (batch), respectively. The amount of contaminating protein per product was 0.61µgkHAU(-1) (SMB) compared to 0.29µgkHAU(-1) (batch). This corresponds to a reduction of the respective amount in the feed solution by 60% and 80%, respectively. For both processes, the estimated amount of total protein per vaccine dose would meet the level required for manufacturing of human influenza vaccines prepared in cell cultures. Depending on the strategy chosen for sanitization and equilibration of columns the calculated overall productivity for the SMB process was up to 3.8 times higher compared to the batch mode. SMB, therefore, has the potential to replace single column discontinuous chromatography in order to design more efficient purification trains for production of cell culture-derived influenza vaccines.
Assuntos
Cromatografia em Gel/métodos , Orthomyxoviridae/isolamento & purificação , Animais , Reatores Biológicos , Cães , Testes de Hemaglutinação , Vacinas contra Influenza/síntese química , Vacinas contra Influenza/química , Células Madin Darby de Rim Canino , Orthomyxoviridae/química , Orthomyxoviridae/imunologia , Orthomyxoviridae/metabolismoRESUMO
In cell culture process development, monitoring and analyzing metabolic key parameters is routinely applied to demonstrate specific advantages of one experimental setup over another. It is of great importance that the observed differences and expected improvements are practically relevant and statistically significant. However, a systematic assessment whether observed differences in metabolic rates are statistically significant or not is often missing. This can lead to time-consuming and costly changes of an established biotechnological process due to false positive results. In the present work we demonstrate how well-established statistical tools can be employed to analyze systematically different sources of variations in metabolic rate determinations and to assess, in an unbiased way, their implications on the significance of the observed differences. As a case study, we evaluate differing growth characteristics and metabolic rates of the avian designer cell line AGE1.CR.pIX cultivated in a stirred tank reactor and in a wave bioreactor. Although large differences in metabolic rates and cell growth were expected (due to different aeration, agitation, pH-control, etc.) and partially observed (up to 79%), our results show that the inter-experimental variance between experiments performed under identical conditions but with different pre-cultures is a major contributor to the overall variance of metabolic rates. The lower bounds of the overall relative standard deviations for specific metabolic rates were between 4% and 73%. The application of available statistical methods revealed that the observed differences were statistically not significant and consequently insufficient to confirm relevant differences between both cultivation systems. Our study provides a general guideline for statistical analyses in comparative cultivation studies and emphasizes the necessity to account for the inter-experimental variance (mainly caused by biological variation) to avoid false-positive results.
Assuntos
Técnicas de Cultura de Células/métodos , Metabolismo/fisiologia , Modelos Biológicos , Modelos Estatísticos , Animais , Reatores Biológicos , Linhagem Celular , Meios de Cultura/metabolismo , Patos , Espaço Extracelular/metabolismo , Biologia de SistemasRESUMO
In biogas plants agricultural waste and energy crops are converted by complex microbial communities to methane for the production of renewable energy. In Germany, this process is widely applied namely in context of agricultural production systems. However, process disturbances, are one of the major causes for economic losses. In addition, the conversion of biomass, in particular of cellulose, is in most cases incomplete and, hence, insufficient. Besides technical aspects, a more profound characterization concerning the functionality of the microbial communities involved would strongly support the improvement of yield and stability in biogas production. To monitor these communities on the functional level, metaproteome analysis was applied in this study to full-scale agricultural biogas plants. Proteins were extracted directly from sludge for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent identification with mass spectrometry. Protein profiles obtained with SDS-PAGE were specific for different biogas plants and often stable for several months. Differences of protein profiles were visualized by clustering, which allowed not only the discrimination between mesophilic and thermophilic operated biogas plants but also the detection of process disturbances such as acidification. In particular, acidification of a biogas plant was detected in advance by disappearance of major bands in SDS-PAGE. Identification of proteins from SDS-PAGE gels revealed that methyl CoM reductase, which is responsible for the release of methane during methanogenesis, from the order Methanosarcinales was significantly decreased. Hence, it is assumed that this enzyme might be a promising candidate to serve as a predictive biomarker for acidification.
Assuntos
Biocombustíveis , Biomassa , Methanosarcinales/metabolismo , Proteoma/biossíntese , Proteômica , Proteínas de Bactérias , Methanosarcinales/genética , Proteoma/genéticaRESUMO
In biotechnology, mathematical models often consider changes in cell numbers as well as in metabolite conversion to describe different cell growth phases. It has been frequently observed that the cell number is only a delayed indicator of cell growth compared to the biomass, which challenges the principle structure of corresponding models. Here, we evaluate adherent cell growth phases in terms of cell number and biomass increase on the basis of detailed experimental data of three independent cultivations for Madin Darby canine kidney cells. We develop a model linking cell numbers and mean cell diameters to estimate cell volume changes during growth without the need for diameter distribution measurements. It simultaneously describes the delay between cell number and cell volume increase, cell-specific volume changes and the transition from growth to maintenance metabolism while taking different pre-culture conditions, which affect the cell diameter, into account. In addition, inspection of metabolite uptake and release rates reveals that glucose is mainly used for generation of cellular energy and glutamine is not required for cellular maintenance. Finally, we conclude that changes in cell number, cell diameter and metabolite uptake during cultivation contribute to the understanding of the time course of intracellular metabolites during the cultivation process.
Assuntos
Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Modelos Biológicos , Animais , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Processos de Crescimento Celular/fisiologia , Tamanho Celular , Simulação por Computador , Cães , Feminino , Glucose/metabolismo , Glutamina/metabolismo , Células Madin Darby de Rim Canino , Reprodutibilidade dos TestesRESUMO
A model has been developed for the determination of the correct absolute size of nanoparticles using a disc centrifuge; the method does not require externally measured or literature derived particle densities. The principle of this method is the altered settling velocity of particles in fluids with different viscosities and/or densities, with the use of a linear regression analysis for the calculation of particle densities. This allows a fast particle density determination with at least two measurements using a disc centrifuge and a small subset of easily measurable parameters. Furthermore, correctness of the model is evaluated using viruses and nonbiological particles of known densities.
Assuntos
Centrifugação com Gradiente de Concentração/métodos , Nanopartículas/química , Centrifugação com Gradiente de Concentração/instrumentação , Tamanho da Partícula , Propriedades de Superfície , ViscosidadeRESUMO
To mitigate the diesel particle pollution problem, diesel vehicles are fitted with modern exhaust after-treatment systems (ATS), which efficiently remove engine-generated primary particles (soot and ash) and gaseous hydrocarbons. Unfortunately, ATS can promote formation of low-vapor-pressure gases, which may undergo nucleation and condensation leading to formation of nucleation particles (NUP). The chemical nature and formation mechanism of these particles are only poorly explored. Using a novel mass spectrometric method, online measurements of low-vapor-pressure gases were performed for exhaust of a modern heavy-duty diesel engine operated with modern ATS and combusting low and ultralow sulfur fuels and also biofuel. It was observed that the gaseous sulfuric acid (GSA) concentration varied strongly, although engine operation was stable. However, the exhaust GSA was observed to be affected by fuel sulfur level, exhaust after-treatment, and driving conditions. Significant GSA concentrations were measured also when biofuel was used, indicating that GSA can be originated also from lubricant oil sulfur. Furthermore, accompanying NUP measurements and NUP model simulations were performed. We found that the exhaust GSA promotes NUP formation, but also organic (acidic) precursor gases can have a role. The model results indicate that that the measured GSA concentration alone is not high enough to grow the particles to the detected sizes.
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Poluentes Atmosféricos/análise , Monitoramento Ambiental/métodos , Espectrometria de Massas/métodos , Nanopartículas/análise , Ácidos Sulfúricos/análise , Emissões de Veículos/análise , Monitoramento Ambiental/instrumentação , Tamanho da PartículaRESUMO
In cell culture-based influenza vaccine production significant efforts are directed towards virus seed optimization for maximum yields. Typically, high growth reassortants (HGR) containing backbones of six gene segments of e.g. influenza A/PR/8, are generated from wild type strains. Often, however, HA and TCID50 titres obtained do not meet expectations and further optimization measures are required. Flow cytometry is an invaluable tool to improve our understanding of mechanism related to progress of infection, virus-induced apoptosis, and cell-specific productivity. In this study, we performed infections with two influenza A/PR/8 variants (from NIBSC and RKI) and two A/PR/8-based HGRs (Wisconsin-like and Uruguay-like) to investigate virus replication, apoptosis and virus titres at different multiplicities of infection (MOI 0.0001, 0.1, 3). Flow cytometric analyses showed similar dynamics in the time course of infected and apoptotic cell populations for all four tested strains at MOI 0.0001. Interestingly, higher MOI resulted in an earlier increase of the populations of infected and apoptotic cells and showed strain-specific differences. Infections with A/PR/8 NIBSC resulted in an earlier increase in both cell populations compared to A/PR/8 RKI. The Uruguay-like reassortant showed the earliest increase in the concentration of infected cells and a late induction of apoptosis at all tested MOIs. In contrast, the Wisconsin-like reassortant showed strong apoptosis induction at high MOIs resulting in reduced titres compared to lower MOI. Maximum HA titres were unaffected by changes in the MOI for the two A/PR/8 and the Uruguay-like reassortant. Maximum TCID50 titres, however, decreased with increasing MOI for all strains. Overall, infections at very low MOI (0.0001) resulted not only in similar dynamics concerning progress of infection and induction of apoptosis but also in maximum virus yields. Highest HA titres were obtained for virus seed strains combining a fast progress in infection with a late onset of apoptosis. Therefore, both factors should be considered for the establishment of robust influenza vaccine production processes.
Assuntos
Apoptose , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/isolamento & purificação , Vírus Reordenados/patogenicidade , Cultura de Vírus/métodos , Animais , Reatores Biológicos/virologia , Técnicas de Cultura de Células , Cães , Citometria de Fluxo , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Vírus Reordenados/crescimento & desenvolvimento , Vírus Reordenados/fisiologia , Replicação ViralRESUMO
Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.
Assuntos
Enzimas/metabolismo , Células Epiteliais/fisiologia , Estresse Fisiológico , Adaptação Fisiológica , Animais , Técnicas de Cultura de Células , Linhagem Celular , Cães , Células Epiteliais/metabolismo , Rim/citologia , Rim/metabolismo , Redes e Vias MetabólicasRESUMO
Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 µL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.