Recombinant protein therapeutics have transformed healthcare by paving the way for the treatment of refractory illnesses like cancer and arthritis. Chinese hamster ovary (CHO) cells are the major workhorse for the production of these therapeutics. Striving for continual improvements in the productivity and quality of protein produced in CHO cells, many process enhancements have been successfully implemented. However, many processes are still empirical, and we have little understanding of the mechanisms for these methods. The availability of genomic resources for CHO cells has ushered in a `genomics' era in bioprocessing. Genomic resources can now be employed to understand and improve cell lines and processes to enhance the productivity and quality of protein therapeutics produced by CHO cells. Seeking the development of genomic resources for CHO cells, the Chinese hamster genome and transcriptome were sequenced, assembled and annotated. Such transcriptomic resources can be used to study the inherent transcriptomic variability in CHO cells. The genetic cues identified from the study of the variability in the glycosylation pathway genes opens up several opportunities to manipulate protein quality. The relative expression of isozymes in CHO cells affect metabolic characteristics, which in turn may potentially impact product quality or even process robustness. The comparative study of isozymes can give important clues for cell engineering and process development. The isozyme distribution in CHO cells indicates a very high overall glycolytic rate, insinuating to the possibility of manipulating glycolytic flux for improving processes. Engineering superior metabolism through cell engineering can be used to reduce glycolytic flux in the late stage of the fed batch culture to reduce lactate accumulation. A novel dynamic promoter was used to drive the expression of a fructose transporter selectively in the late stages of the culture. By maintaining adequately low fructose levels in the late stage, the glycolytic flux was reduced significantly to induce lactate consumption. Since lactate accumulation is well accepted to be detrimental to productivity, this phenotype is desired for bioprocessing. In addition to such high productivity processes, high producing cells are also desired. The lengthy process of cell line development transforms non-producing cells to high producers. The molecular changes in this transformation were elucidated by studying the transcriptome of CHO cells during cell line development. We hypothesize that methotrexate treatment not only increases the transgene copy number, but also enriches cells with superior growth, energy metabolism, and secretion capabilities. This leads to an enriched population of high producers. The sustenance of high productivity over several generations depends on the stability of the integration site of the transgene. Two methods for identifying the cell's transgene integration site were developed and optimized. These methods can be applied for high throughput investigation of stability of integration sites.The application of genomics in bioprocessing has sparked a systems approach to investigate genetic regulation. This knowledge paved the way for controlling cellular metabolism and achieve stable and high producing cell lines and processes. Such genome scale analyses have a great potential to advance the capacity of CHO cells for biopharmaceutical applications.
University of Minnesota Ph.D. dissertation. June 2014. Major: Chemical Engineering. Advisor: Professor Wei-Shou Hu. 1 computer file (PDF); xi, 192 pages.
Genomic and transcriptomic approaches for the advancement of CHO cell bioprocessing.
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