Transcriptome analysis in mammalian cell culture: Applications in process development and characterization.

University of Minnesota Ph.D. dissertation. August 2009. Major: Chemical Engineering. Advisor: Wei-Shou Hu. 1 computer file (PDF); xii, 211 pages, appendix p. 196-211.The advent of recombinant protein therapeutics more than two decades ago fundamentally transformed healthcare paradigms and has since improved the quality of life of millions of people. The production of these complex products is typically carried out in cultured mammalian cell lines, with a few cell lines accounting for the majority of production. Chief among these is the Chinese hamster ovary (CHO) cell line. Despite its importance, little genomic information is currently available in the public domain for this cell line. Consequently, our lab has devoted significant efforts to the development of genomic tools for CHO, including custom Affymetrix microarrays. These tools enable the global study of cellular gene expression. This thesis research has applied these transcriptome analysis tools to further understand the process of recombinant protein production. The course of bringing a recombinant protein product to production scale involves a series of complex and often lengthy steps. As demand for these products continues to increase, there is a need to streamline process development efforts. One approach to facilitate this process is to increase our fundamental understanding of cell culture processes. Microarrays are well-suited to this application, and in this work, transcriptome analysis has been used to characterize multiple facets of cell culture process development, including cell line development and modulation of process parameters in fed-batch cultures. We found significant variation amongst clonal gene expression profiles during cell line development, an observation which could be exploited to develop gene expression-based clone screening protocols. We also uncovered the widespread impact process parameters can have on cellular gene expression. In particular, we found that raw material source, namely different hydrolysate lots, has a profound effect on the transcriptional signatures of fed-batch cell culture processes. As next-generation sequencing technologies become increasingly mature and cost-effective, they are now being applied to the study of gene expression. We have used ultra high-throughput sequencing to investigate the deep transcriptome of CHO cells. We found that the technology correlated well with microarrays, and displayed a significantly broader detection range. Through this analysis, we also identified a number of transcriptionally-active regions in the CHO genome. The unprecedented depth achievable through next-generation sequencing now allows us to set genome sequencing firmly in our sights