Elucidating dipeptide utilization and metabolism by CHO cells for improved cell culture performance

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Amino acid analysis to support a genome-scale nutrient minimization forecast algorithm for controlling essential amion acid levels in CHO cell cultures

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Tracking amino acid metabolism in CHO cell cultures using stable isotope labeling assisted metabolomics

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Elucidating amino acid metabolism in CHO cells

CHO cells require complex media for cell growth and protein production. The major components of industrial media are amino acids, however, relatively little is known about the metabolism of amino acids in CHO cell cultures. Here, we applied advanced 13C-flux analysis tools to elucidate the metabolic flow of the amino acids in a fed-batch CHO culture that overproduced IgG. Carbon flows were tracked throughout the growth phase and changes in metabolism were quantified when cells transitioned from growth phase to stationary phase. In addition, we quantified how changes in amino acids profiles in the medium translated to changes in cell growth, protein production and product quality attributes. To trace each amino acid individually, custom media formulations were used, where each medium formulation was depleted of a specific amino acid. A labeled 13C variant of the depleted amino acid was then added to the medium at the desired concentration. CHO cells were then grown in fed-batch culture. As the cells metabolized the labeled amino acids, this resulted in a redistribution of 13C-atoms which we quantified using GC-MS for both extracellular metabolites (including lactate, amino acids and the IgG product) and intracellular metabolites (including free intracellular metabolites, cell proteins, lipids and carbohydrates). We then estimated metabolic fluxes using state-of-the-art 13C-metabolic flux analysis. This allowed us to calculate the fraction of each amino acid that was used for cell growth, protein production, lactate formation and energy generation. We also investigated the effects of labeling in both the batch and fed-batch stationary phase. Finally, we investigated the effects of varying amino acid concentrations. Each 13C-labeled amino acid was added to the medium at a lower or higher concentration compared to the base medium. 13C-metabolic flux analysis was again performed and changes in fluxes were compared in order to determine the precise impacts of amino acid concentration changes on the flux profiles. Taking all of this data together, we are now building a predictive kinetic model that relates how the metabolism of CHO cells can be predicted from amino acid profiles. In future work, model predictions will be experimentally validated as a means of optimizing the amino acid composition of industrial culture media

Metabolic Engineering and Process Development Approaches to Enhance Biotherapeutics Production from Mammalian Cells

Mammalian cells especially Chinese Hamster Ovary (CHO) cells have been the dominating production platforms for monoclonal antibodies (mAbs) and other glycoproteins. Improving their productivity and product quality through cell line engineering and media optimization is the major focus of the biopharmaceutical industry. Basal media formulations differ across manufactures including Millipore Sigma, Lonza and Thermofisher Scientific. There is no standard basal media available that can be used as a baseline for experimentation. Supported by AMBIC (Advanced Mammalian Biomanufacturing Innovation Center) this study aims at developing a reference media formulation called AMBIC 1.0. Using this media platform that has performance characteristics similar to industrial standards will provide a better community-wide understanding of media components and result in a common, shared platform of knowledge about CHO cells. Cell growth and IgG productivity of two CHO cell lines was tested in different blends of legacy media provided by AMBIC member companies. From the media blend studies, the optimal media blend was formulated as AMBIC reference 1.0 medium. Further, the two cell lines were successfully adapted to grow in the new AMBIC 1.0 reference medium. While variability among cell culture media is a major concern, the cost of producing effective media formulations is also very high. Material costs of media can be reduced if mammalian cells are engineered to synthesize their own biomolecules and do not need to rely on the media for these nutrients. Amino acids are one of the major components of cell culture media. In this study, amino acid biosynthesis pathways including three essential branched chain amino acids – threonine, isoleucine and valine from bacteria and fungi were integrated in mammalian cells. Isoleucine and valine genes (ILV2, ILV3, ILV5, ILV6) were successfully transfected in CHO-K1 cells and their expression was confirmed using Western Blot analysis. Threonine producing U-2 OS osteosarcoma cell line was tested for threonine production using 13-C labeled aspartic acid and GC-MS analysis. GC-MS analysis confirmed that labeled threonine was produced from the labeled aspartic acid. This opens up avenues in cell line engineering to make mammalian cells self-sufficient

UNRAVELING CHO METABOLIC PROCESSES USING STABLE ISOTOPE LABELING TO ENHANCE BIOPROCESS PERFORMANCE

Chinese Hamster Ovary (CHO) cells are the preferred host systems for recombinant protein therapeutics production including monoclonal antibodies and fusion proteins. CHO cells have the ability to grow in high densities in suspension and produce glycosylation patterns that are compatible with humans. Improving CHO cell productivity and product quality by altering cellular metabolism and media formulations is the major focus of the biopharmaceutical industry. Amino acids (AAs) are the major components of cell culture media. However, they pose some major challenges in cell culture processes. Firstly, relatively little is known about the metabolism of AAs in cell culture. Secondly, while AAs are primarily used for biomass production and protein synthesis, some of them may also be diverted towards metabolic pathways generating by-products that are toxic to cell culture performance. Thirdly, supplying AAs to CHO cell cultures can be challenging especially glutamine, cysteine, and tyrosine due to their low solubility and poor stability. This can raise the risk of media precipitation leading to insufficient nutrient levels to meet cellular demands and thus, contribute to suboptimal culture performance. A possible alternative to these AAs is dipeptides since they are more soluble and stable. In this thesis, we applied stable isotope (13C) labeling assisted metabolomics, metabolite profiling, metabolic flux analysis, and kinetic modeling to address and mitigate the above-mentioned challenges. First, we applied advanced 13C labeling and GC-MS based analytics to elucidate the metabolic flow of AAs in batch and fed-batch CHO cell cultures that produced IgG. This allowed us to analyze relative pathway activities, assess nutrient contributions to metabolite production, and determine changes in AAs metabolism as cells transitioned from growth phase to protein production phase. Second, we employed stable isotope (13C) labeling assisted metabolomics to identify and characterize by-products of AA catabolism accumulating in CHO cell cultures. Third, we investigated the use of small molecule inhibitors of the hexokinase-2 (HK-2) enzyme to control glycolysis flux and reduce lactate accumulation in CHO cell cultures. Out of the several molecules screened, 2-deoxyglucose and 5-thioglucose were determined to significantly reduce lactate accumulation without negatively impacting cell growth and protein production. 13C metabolic flux analysis revealed a potential rewiring of CHO cell metabolism by these molecules including increases in TCA and oxidative phosphorylation fluxes. Lastly, we comprehensively elucidated the kinetics of dipeptide uptake and metabolism by applying and integrating data from 13C labeling experiments into a kinetic model. We demonstrated that dipeptides are initially cleaved inside the cells and later in the extracellular environment as well. The concentration of dipeptides as well as order and type of AAs in the dipeptides impacts dipeptide utilization rate. Using this information and aiming for controlled release of cysteine in cell culture, we evaluated the impact of replacing cysteine and cystine with Ala-Cys-Cys-Ala (ACCA) in cell culture media and feed. ACCA was found to support cell growth and protein production in the absence of cysteine and cystine in feed medium. Overall, the results from these studies provide valuable information to enhance CHO cell culture performance that could lead to improved raw materials, enhanced media and feed formulations, and more efficiently engineered CHO cell lines for therapeutics protein production

Towards a universal CHO reference platform with epigenome characterization for the biotechnology community

Chinese Hamster ovary (CHO) cells are widely used both by academic researchers and in the biotechnology industry. However, comparing studies across a wide spectrum of labs in the CHO community has been challenging due to the different variants of host cell line, culture media and proteins of interest used in the individual laboratories. Unfortunately, unlike other communities, there is no standard CHO platform that can be used as a baseline for experimentation and evaluation, leading to a limited understanding of how a result or innovation from one group may be applied to another group. This limits the pace at which innovations in cell line development are achieved by the community. As a result, there is a growing need to create and establish a common platform with the goal of comparability and compatibility across the CHO bioprocessing community. The Advanced Mammalian Biomanufacturing Innovation Center (AMBIC) is a US based academic-industrial-government collaborative initiative dedicated to developing improved upstream biomanufacturing methods. Together, AMBIC’s five academic and sixteen industrial members are working together to implement a newly developed CHO based reference platform that has performance characteristics similar to what is used in the industry. Our initial goals have been to identify reference production and host cell lines together with a common platform medium used in the production of model recombinant protein targets, including antibodies and other targets. These cell lines are also being used to develop standardized processes that can be comparable across AMBIC sites and within the CHO community. In concert, these reference platforms are being applied to evaluate and understand CHO cell line capabilities and processing parameters for improving the production platform. Our progress in establishing a reference cell host and the partner media and processing parameters will be described, and the role of such a reference standard in helping to define the scope of other AMBIC research endeavors will also be delineated. We believe such a reference CHO platform will facilitate a robust and dynamic CHO research and development environment and hasten progress in cell culture engineering in coming decades