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

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

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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

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

Extracellular Enzyme Kinetics Scale With Resource Availability

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales