Study Of Modeling In Myeloma Cell Culuture Processes

Mammalian cell culture is the process by which mammalian cells are grown under controlled conditions in vitro. Mammalian cell culture together with traditional microbial recombinant bacterial processes forms the core of fermentation-based manufacturing. Mammalian cell cultures represent the major source for a number of very high-value biopharmaceutical products, including monoclonal antibodies (MAbs), viral vaccines, and hormones. To present, mammalian cell culture has been an important part of study and application in biotechnology and medicine. It plays a key role in industrial practice. Therefore, more and more attentions have been paid to mammalian cell culture. In these years, biopharmaceutical industry based on large-scale mammalian cell culture developed very fast. Many products have been put into markets and well profit has been obtained.The use of mathematical relationships to characterise distinct parts of the physiological behaviour of mammalian cells and the systematic integration of this information into a coherent, predictive model, which can be used for simulation, optimisation, and control purposes would contribute to efforts to increase productivity and control product quality. Many factors influence mammalian cell culture. The mechanism to control the cell growth and intracellular metabolism is very complex. Due to the highly specialised culture conditions and the susceptibility to either reduced productivity or cell death as a result of slight deviations in the culture conditions. Products of mammalian cell culture are produced in relatively small quantities. By mathematic model, the cell culture process can be described quantificationally.The main efforts and contribution of this dissertation are as follows: 1, a macrokinetic and regulator model for myeloma cell culture based on metabolic balance of pathways was built. Myeloma cell line (X63-Ag8.653) was used to investigate the modeling of cell growth and metabolic behaviors. Firstly, glucose and glutamine were regarded as the main substrates in cell culture based on the metabolic flux analysis. The Stoichiometric balance model was built. And then, a regulator metabolic model of glucose was introduced to describe the glucose consuming. At last, the relationship between substrate feeding rates and the key state variables, such as cell density, substrates and metabolites concentrations, were established. 2, a cell cycle model driven by specific growth rate was established. The specific growth rate, which can drive the duration of each phase of cell cycle, was obtained from macrokinetic model. The following contents are included in this dissertation.(1) Metabolic analysis of mammalian cell lineMany nutrients are necessary in mammalian cell culture. The intercellular metabolic pathways are very complex. By analyzing the main metabolic pathways, including glycolysis, glutaminolysis, pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, coupling with the involved experimental data, the relationship between inputs and outputs is estimated. Glucose and glutamine are considered as the major carbon, nitrogen and energy sources in myeloma cell culture media. They provide carbon skeleton and energy for cell growth. Byproducts, such as lactate, ammonia and alanine, are produced during cell culture process. The cell cycle processing was also influenced by substrate metabolism. As the consumption of substrates, cells would accumulate in G0/G1. The rate of cells in S phase would decline. Cells in G2/M maintain constant.(2) Macrokinetic and regulator model for myeloma cell cultureA macrokinetic and regulator model for myeloma cell culture is proposed based on metabolic balance of pathways. The model is composed of substrate uptake model, stoichiometry balance model and bioreactor model. A regulator metabolic model coupling with a Monod model was introduced to describe the glucose uptake. The stoichiometry balance model is composed of two modes. First, a rapid-glycolysis mode describes the rapid glycolysis and glutaminolysis rates to produce lactate and ammonia. Second, in the low-glycolysis mode, the specific glucose consumption rate and the glycolysis rate are declined and the formation of lactate is terminated. The specific glycolysis rate is regarded as the key variable to induce the metabolic shift between these two modes. After coupling the stoichiometry balance model with a bioreactor model, the relationship between substrate feeding rates and the key state variables, such as cell density, substrates and metabolites concentrations, were established. Finally, the probability of expanding the model to secretable cell lines is investigated. Some relationships between protein secreting and specific growth rate are displayed according to literatures.(3) Cell cycle model for myeloma cell lineThe mechanism of cell cycle is analyzed. A myeloma cell cycle model was built according to the model of budding yeast. After coupling with the macrokinetic model, a completed macrokinetic-cell cycle model was established. The relationship between the cell specific growth rate and distribution of each phase in cell cycle was obtained.(4) Model validationA series of experiments were carried out for model validation. The testing results indicate that the cell growth and products concentration can be well described by the macrokinetic model proposed, and the proportion cells in each phase of cell cycle can be described by the cell cycle model with reasonable accuracy. Furthermore, the sensitivity analysis of model parameters is carried out. The number of batch dependent parameters is then reduced by fixing those insensitive parameters. Comparison between the models provided in the thesis and those models in literatures was done. The characteristic and the modification direction of the models were obtained. According to many unpredictable factors exist in cell culture process, rolling prediction approach was used to predict and simulate an experiment which it's feeding strategy caused shock in the culture environment.