Conversion of an intensified fed-batch to an integrated continuous bioprocess

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Cattle grazing for invasive Phragmites australis (common reed) management in Northern Utah wetlands

This fact sheet provides suggestions for how cattle grazing can be integrated into Phragmites management programs in Northern Utah

Metabolic flux analysis and population heterogeneity in mammalian cell culture

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2000.Includes bibliographical references (p. 189-206).Metabolic flux and population heterogeneity analysis were used to develop relations between mammalian cell physiology and specific culture environments and to formulate strategies for increasing cell culture performance. Mitochondrial characteristics associated with respiration, membrane potential, and apoptosis along with physiological state multiplicity involving both metabolism and apoptotic death played a key role in this research. Research involving the accurate calculation of metabolic flux and the analysis of cellular behavior occurring in continuous cultures set the stage for subsequent research on physiological state multiplicity. This phenomena was observed in continuous cultures when at the same dilution rate, two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations. Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the tri-carboxylic (TCA) cycle for energy generation. This metabolic adaptation was unlikely due to favorable genetic mutations and was implemented in subsequent research aimed at improving cell culture performance. A hypothesis stating that mitochondrial physiology and cellular physiology are correlated was tested and confirmed. A mammalian cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123. The MMP sorted subpopulations were subjected to apoptosis inducers, and the apoptotic death was characterized both morphologically through the determination of apoptosis related chromatin condensation and also biochemically through the measurement of caspase-3 enzymatic activity. The results showed dramatic differences in apoptotic death kinetics with the higher MMP subpopulations demonstrating a higher resistance to apoptotic death. These results were applied in the development of novel fed-batch feeding and operating strategies. The first strategy showed that overfeeding cells later in culture leads to an increase in culture viable cell concentration, viability, and productivity. The second strategy showed that cell populations with a higher mean MMP are able to resist apoptosis during fed-batch culture. These results indicate that mammalian cell populations have considerable flexibility in their ability to redistribute metabolic flux in central carbon metabolism. Furthermore, these cell populations contain subpopulations that vary in their resistance to apoptotic death. The analysis of mitochondrial physiology and metabolic flux led to these discoveries, and these areas will play a key role in future mammalian cell culture research.by Brian D. Follstad.Ph.D

A CDMO perspective toward the implementation of continuous bioprocessing stand- alone and integrated offerings

The challenge involved in integrating unit operations for continuous bioprocessing is a significant impediment to implementation of the technology in the industry. The benefit of continuous bioprocessing can be better understood when the components of the technology are analyzed under multiple factors including modalities, protein quality attributes and stability, specific productivity and overall cost-benefit of implementation and operation of the technology. Contract Development and Manufacturing Organizations (CDMO) need to provide a portfolio of offerings that cover the needs of diverse groups and process needs. For example, processes with lower productivity and unstable molecules can benefit from a perfusion system while more stable molecules with high productivity may need to focus on the benefits of a continuous capture to address a potential bottleneck on the downstream. Please click Additional Files below to see the full abstract

A procedure for the estimation over time of metabolic fluxes in scenarios where measurements are uncertain and/or insufficient

<p>Abstract</p> <p>Background</p> <p>An indirect approach is usually used to estimate the metabolic fluxes of an organism: couple the available measurements with known biological constraints (e.g. stoichiometry). Typically this estimation is done under a static point of view. Therefore, the fluxes so obtained are only valid while the environmental conditions and the cell state remain stable. However, estimating the evolution over time of the metabolic fluxes is valuable to investigate the dynamic behaviour of an organism and also to monitor industrial processes. Although Metabolic Flux Analysis can be successively applied with this aim, this approach has two drawbacks: i) sometimes it cannot be used because there is a lack of measurable fluxes, and ii) the uncertainty of experimental measurements cannot be considered. The Flux Balance Analysis could be used instead, but the assumption of optimal behaviour of the organism brings other difficulties.</p> <p>Results</p> <p>We propose a procedure to estimate the evolution of the metabolic fluxes that is structured as follows: 1) measure the concentrations of extracellular species and biomass, 2) convert this data to measured fluxes and 3) estimate the non-measured fluxes using the Flux Spectrum Approach, a variant of Metabolic Flux Analysis that overcomes the difficulties mentioned above without assuming optimal behaviour. We apply the procedure to a real problem taken from the literature: estimate the metabolic fluxes during a cultivation of CHO cells in batch mode. We show that it provides a reliable and rich estimation of the non-measured fluxes, thanks to considering measurements uncertainty and reversibility constraints. We also demonstrate that this procedure can estimate the non-measured fluxes even when there is a lack of measurable species. In addition, it offers a new method to deal with inconsistency.</p> <p>Conclusion</p> <p>This work introduces a procedure to estimate time-varying metabolic fluxes that copes with the insufficiency of measured species and with its intrinsic uncertainty. The procedure can be used as an off-line analysis of previously collected data, providing an insight into the dynamic behaviour of the organism. It can be also profitable to the on-line monitoring of a running process, mitigating the traditional lack of reliable on-line sensors in industrial environments.</p

Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.COMET center acib: Next Generation Bioproduction, which is funded by BMK, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria and Vienna Business Agency in the framework of COMET - Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG; D.S., J.S., M.W., M.H., D. E.R. This work has also been supported by the PhD program BioToP of the Austrian Science Fund (FWF Project W1224)info:eu-repo/semantics/publishedVersio

Global Patterns and Controls of Nutrient Immobilization On Decomposing Cellulose In Riverine Ecosystems

Microbes play a critical role in plant litter decomposition and influence the fate of carbon in rivers and riparian zones. When decomposing low-nutrient plant litter, microbes acquire nitrogen (N) and phosphorus (P) from the environment (i.e., nutrient immobilization), and this process is potentially sensitive to nutrient loading and changing climate. Nonetheless, environmental controls on immobilization are poorly understood because rates are also influenced by plant litter chemistry, which is coupled to the same environmental factors. Here we used a standardized, low-nutrient organic matter substrate (cotton strips) to quantify nutrient immobilization at 100 paired stream and riparian sites representing 11 biomes worldwide. Immobilization rates varied by three orders of magnitude, were greater in rivers than riparian zones, and were strongly correlated to decomposition rates. In rivers, P immobilization rates were controlled by surface water phosphate concentrations, but N immobilization rates were not related to inorganic N. The N:P of immobilized nutrients was tightly constrained to a molar ratio of 10:1 despite wide variation in surface water N:P. Immobilization rates were temperature-dependent in riparian zones but not related to temperature in rivers. However, in rivers nutrient supply ultimately controlled whether microbes could achieve the maximum expected decomposition rate at a given temperature

Fastsettelse av pensjonsforutsetninger blant norske børsnoterte selskaper

Temaet i denne masterutredningen er fastsettelsen av pensjonsforutsetninger blant norske børsnoterte selskaper. Formålet er å undersøke om de de fastsatte forutsetningene har en sammenheng med selskapers karakteristika. Reviderte IAS19 som hadde virkning fra 01.01.2013 fjernet muligheten for å utsette regnskapsføringen av estimatavvik som oppstår som følger av endringer i pensjonsforutsetningene. Dette estimatavviket gir informasjon om hvordan selskaper har truffet med sine forutsetninger. Det blir gjennomført statistisk testing for å undersøke om det foreligger sammenhenger mellom fastsatte pensjonsforutsetninger og tre karakteristika ved selskapet. De tre karakteristikaene er egenkapitalfinansiering, synlighet og lønnsomhet. Testingen blir gjort ved bruk av regresjonsmodeller. Utredningen finner statistisk signifikant sammenheng mellom selskapenes lønnsomhet og fastsatte pensjonsforutsetninger. Sammenhengen tilsier at selskaper med lav lønnsomhet setter forutsetninger som medfører en lavere pensjonsforpliktelse enn selskaper med høyere lønnsomhet. Det blir ikke funnet statistisk signifikante sammenhenger mellom selskapers egenkapitalfinansiering og fastsatte pensjonsforutsetninger. Heller ikke selskapers synlighet og fastsatte pensjonsforutsetninger viser statistisk signifikant samvariasjon. Det blir funnet statistiske forskjeller mellom årene 2012 og 2013. Dette skyldes blant annet innføring av ny dødelighetstabell i 2013 som gir negative estimatavvik. I denne utredningen konkluderes det kun på at det foreligger en statistisk signifikant sammenheng mellom selskapers lønnsomhet og fastsatte pensjonsforutsetninger. Årsaken til denne samvariasjonen forsøker oppgaven ikke å konkludere på

Mass Loss: A Quantitative Synthesis of Leaf Decomposition in Streams and Rivers

Freshwater ecosystems play a significant role in the global carbon (C) cycle, emitting roughly 1.2 Pg C y/-1 to the atmosphere. Results from recent studies in freshwaters show that ecosystem respiration increases with elevated temperature faster than primary production. Hence, CO2 evasion is predicted to increase as global temperature rises. The majority of the CO2 degassed from heterotrophic streams and rivers comes from the decomposition of allochthonous leaf litter inputs. However, temperature is one of myriad intrinsic and extrinsic factors controlling decomposition. Through the Meta-Analysis and SynthesiS of Leaf decOmposition in StreamS (MASS LOSS) project, we have compiled over 1200 records of leaf litter decomposition from control treatments in streams and rivers located on 5 continents. With the goal of improved prediction of decomposition rate response to global change in mind, we are using this database to answer the following questions: What is the apparent activation energy of decomposition? Does it vary based on leaf bag mesh size or taxonomy? How much variation in decomposition is explained by temperature vs. other intrinsic (e.g., leaf chemistry) and extrinsic (e.g., stream order, macroinvertebrate density) factors? Do interactions exist? Are these patterns similar to those observed for terrestrial ecosystems