UNRAVELING CHO METABOLIC PROCESSES USING STABLE ISOTOPE LABELING TO ENHANCE BIOPROCESS PERFORMANCE
Embargo until
2026-12-01
Date
2022-09-14
Authors
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Publisher
Johns Hopkins University
Abstract
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.
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Keywords
bioprocessing, recombinant protein therapeutics, stable isotope labeling, 13C labeling, mammalian cells, CHO cells, metabolomics, metabolic flux analysis (MFA), kinetic modeling, glycosylation, product quality, amino acids, media formulations, cell line engineering