Probing CHO cells metabolism using metabolomics and fluxomics tools

Chinese hamster ovary (CHO) cells are preferred mammalian hosts for industrial production of therapeutic glycoproteins such as monoclonal antibodies (mAbs), used to treat cancer and immunological disorders. In these cells, the glutamine synthetase (GS) expression system has been widely used for efficient selection of high-yielding clones. However, fundamental knowledge on the metabolic behavior of GS-CHO cells in culture and its impact on product yields have not been yet systematized. The overall objective of this thesis was to comprehensively analyse the metabolome and fluxome of CHO cells exploring how the metabolism is affected by clonal variability and culture conditions. This information is then used to generate hypotheses on how metabolic wiring impacts mAb production.(...)PTDC/BBB-BSS/0518/201

A roadmap for interpreting 13C metabolite labeling patterns from cells

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. [superscript 13]C tracer analysis, although less information-rich than quantitative [superscript 13]C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrientcontributions. Here, we review selected key issues in interpreting [superscript 13]C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiment

Systems biotechnology of baculovirus-producing insect cells

Dissertation presented to obtain the Ph.D degree in Ciências da Engenharia e Tecnologia, especialidade Biotecnologiacritical step in the biopharmaceutical industry is the capacity to properly manufacture biological products in animal cell cultures. The ability to exploit the full potential of the underlying “cellular factories” is essential to reduce manufacturing costs and improve yield and product quality, thus freeing up resources to pursue other added value activities, such as clinical development and pharmacovigilance of new products. Bioprocess optimization efforts have mostly relied on empirical experience and rational, hypothesis-driven studies, rather than by fully leveraging the knowledge of how complex cells behave in culture. Despite the vast amount of information available on different cellular features, specific productivity improvements have been essentially incremental. The relatively recent developments in Systems Biology and computational methodologies present a paradigm-shifting opportunity to develop biotechnology processes with high efficiencies in a consistent, systematic way. (...

Deciphering Chronometabolic Dynamics Through Metabolomics, Stable Isotope Tracers, And Genome-Scale Reaction Modeling

Synchrony across environmental cues, endogenous genetic clocks, sleep/wake cycles, and metabolism evoke physiological harmony for organismal health. Perturbation of this synchrony has been recently correlated with a growing list of pathologies, which is alarming given the ubiquity of sleep deprivation, mistimed light exposure, and altered eating schedules in modern society. Deeper insights into clocks, sleep, and metabolism are necessary to understand these outcomes. In this work, extensive metabolic profiles of circadian systems were obtained from the development of new liquid chromatography mass spectrometry (LC-MS) metabolomics methods. These methods were applied to Drosophila melanogaster to discern relative influences of environmental and genetic drivers of metabolic cycles. Unique sets of metabolites oscillated with 24-hour circadian periods under light:dark (LD) and constant darkness (DD) conditions, and ultradian rhythms were noted for clock mutant flies under LD, suggesting clock-independent metabolic cycles driven by environmental inputs. However, this metabolomic analysis does not fully capture the inherently dynamic nature of circadian metabolism. These LC-MS methods were adapted to analyze isotope enrichments from a novel 13C6 glucose injection platform in Drosophila. Metabolic flux cycles were noted from glucose carbons into serine, glutamine and reduced glutathione biosynthesis, and altered under sleep deprivation, demonstrating unique energy and redox demands in perturbed sleep/wake cycles. Global isotopolome shifts were most notable in WT flies after lights-on, suggesting a catabolic rush from glucose oxidation early in the active phase. As the scope of these isotope tracer-based metabolomic analyses expand, attributing labeling patterns to specific reactions requires consideration of genome-scale metabolic networks. A new computational approach was developed, called the IsoPathFinder, which uncovered biosynthetic paths from glucose to serine, and extends to glycine and glutathione production. Carbon flux into glutamine was predicted to occur through the TCA cycle, supported by enzyme thermodynamics and circadian expression datasets. This tool is presented as a new mechanism to simulate additional isotope tracer experiments, with broad applicability beyond circadian research. Collectively, a new set of analytical and computational tools are developed to both produce dynamic metabolomic data and improve data interpretability, with applications to uncover new chronometabolic connections