Figure 8 - available via license: CC BY
Content may be subject to copyright.
Glycolysis and Glutaminolysis. 5, 50 Molecules colored in green are generated, while molecules in red are used in the transformations. High concentration of ATP stops conversion of pyruvate into acetyl CoA, alpha-ketoglutarate (oxaloacetate) into citrate and oxoglutarate into succinyl CoA.
Source publication
Chinese hamster ovary (CHO) epithelial cells are one of the most used therapeutic medical lines for the production of different biopharmaceutical drugs. They have a high consumption rate with a fast duplication cycle that makes them an ideal biological clone. The higher accumulated amounts of toxic intracellular intermediates may lead to lower orga...
Context in source publication
Context 1
... is metabolized into nucle- otide monosaccharides that are transported into ER and GA. The pathway of glycolysis is highly regulated to sus- tain sufficient concentration of ATP (Figure 8). In rapidly dividing cells glucose and glutamine consumptions are major steps for energy supply. ...
Citations
... The switch in lactate metabolism from its production as part of an inefficient overflow metabolism to its consumption in a more efficient metabolic phase, for example, is a well-known and desired phenomenon that is not yet fully understood mechanistically [18]. Several hypotheses on the cause of this phenomenon exist (see e.g., [18][19][20]) and were included in some models with different strategies based on the introduction of a redox variable [10] or switching function [21], the usage of two different kinetics for the lactate dehydrogenase [7], or simply making the reaction reversible [22]. ...
Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism.
... Several hypotheses on the cause of this phenomenon exist (see e.g. [16][17][18]) and were included in some models with different strategies based on the introduction of a redox variable [10] or switching function [19], the usage of two different kinetics for the lactate dehydrogenase [7] or simply making the reaction reversible [20]. ...
Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high seeding density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells. Using a metabolic network and flux balance analysis, we compared standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched in amino acids. We observed high glycolytic fluxes and depletion of asparagine simultaneously with the lactate shift building kinetic models around this and other observations. An ensemble of kinetic models assessed the impact of a feeding medium enriched with additional asparagine, revealing a missing lactate shift without enhancing mAb productivity in the experiments. This research provides valuable model-based insights into cellular metabolism during HSD processes, laying the foundation for refined CHO cell-based mAb production. The proposed hypothesis on the regulations guides future experimental validation and bioprocess optimization for enhanced mAb production efficiency.
Optimizing mammalian cell growth and bioproduction is a tedious task. However, due to the inherent complexity of eukaryotic cells, heuristic experimental approaches such as, metabolic engineering and bioprocess design, are frequently integrated with mathematical models of cell culture to improve biological process efficiency and find paths for improvement. Constraint-based metabolic models have evolved over the last two decades to be used for dynamic modelling in addition to providing a linear description of steady-state metabolic systems. Formulation and implementation of the underlying optimization problems require special attention to the model's performance and feasibility, lack of defects in the definition of system components, and consideration of optimal alternate solutions, in addition to processing power limitations. Here, the time-resolved dynamics of a genome-scale metabolic network of Chinese hamster ovary (CHO) cell metabolism are shown using a genome-scale dynamic constraint-based modelling framework (gDCBM). The metabolic network was adapted from a reference model of CHO genome-scale metabolic model (GSMM), iCHO_DG44_v1, and dynamic restrictions were imposed to its exchange fluxes based on experimental results. We used this framework for predicting physiological changes in CHO clonal variants. Because of the methodical creation of the components for the flux balance analysis optimization problem and the integration of a switch time, this model can generate sequential predictions of intracellular fluxes during growth and non-growth phases (per hour of culture time) and transparently reveal the shortcomings in such practice. As a result of the differences exploited by various clones, we can understand the relevance of changes in intracellular flux distribution and exometabolomics. The integration of various omics data into the given gDCBM framework, as well as the reductionist analysis of the model, can further help bioprocess optimization.
The development and optimization of cell culture media for biotech applications is a fundamental step of process development. The composition of cell culture media requires an ideal blend of amino acids, vitamins, nucleosides, lipids, carbohydrates, trace elements and other components. The ability to monitor these constituents is required to ensure that cells receive sufficient nutrients to facilitate growth, viability and productivity. Analysis of cell culture media is challenging due to the range and diversity of compounds contained in this matrix and normally requires time consuming methods. A rapid, simple and sensitive microfluidic chip CE-MS method is described to monitor amino acids in chemically defined cell culture media from a Chinese hamster ovary cell line cultured over a period of 10 days. The described platform enabled the separation of 16 amino acids in less than 2 minutes and without the requirement for extensive sample preparation. The analytical parameters evaluated were precision, linearity, limit of detection and limit of quantification. The majority of essential amino acids were present in cell culture growth in high concentrations compared to non-essential amino acids. Over the course of the 10 days cell culture the concentration of certain amino acids declined by up to 39%. Microfluidic chip based CE-MS methods can be used effectively to obtain the consumption rates of amino acids in cell culture media during cell growth and to perform at-line monitoring and screening of cell culture status.
