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Genomic Analysis and Engineering of Chinese Hamster Ovary Cells for Improved Therapeutic Protein Production

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Genomic Analysis and Engineering of Chinese Hamster Ovary Cells for Improved Therapeutic Protein Production Genomic analysis and engineering of Chinese Hamster Ovary cells for improved therapeutic protein production A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Sofie Alice O’Brien IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor: Professor Wei-Shou Hu May 2020 Ó Sofie Alice O’Brien 2020 Acknowledgements First, I would like to express gratitude to my advisor, Professor Wei-Shou Hu, for his help and guidance throughout the years. I have learned so much from him and grown both as a scientist and as a person over my time in graduate school. I will always be thankful for the time and effort he put into my training, and I will carry his lessons with me for the rest of my career. Much of my work was the result of collaborations with many other talented scientists. I would like to thank Dr. Gary Dunny, Dr. Aaron Barnes, Dr. Rebecca Erickson, and the rest of the Dunny lab for their support at the beginning of my PhD. I thank Dr. Michael Smanski and Dr. Nik Somia for their help with vector design and the transgene swapping project. I am also grateful to Dr. Juhi OJha and Dr. Paul Wu for our collaboration on the development of an algorithm for integration site analysis. Thank you to Dr. Casim Sarkar, Dr. Samira Azarin, and Dr. Scott McIvor for serving on my thesis committee, and providing valuable feedback on my work. I am especially thankful for the wonderful members of the Hu lab who have supported me during graduate school. Thank you, Dr. Arpan Bandyopadhyay, for introducing me to the lab and helping me get started. To Dr. Christopher Stach, thank you so much for being a great teacher in the lab, I learned a lot about vector design and cell engineering from our many discussions. Thank you to all of the other members of the Hu group, current and former, that I was privileged to know: Meghan McCann, Dr. Conor O’Brien, Jen One, Dr. Kevin Ortiz-Rivera, Zion Lee, Thu Phan, Hansol Kim, Min Lu, Yen- An Lu, Janani Narayan, Dr. David Chau, Dr. Tung Le, Dr. Dong Seong Cho, Dr. Kyoungho Lee, Dr. Hsu-Yuan Fu, Dr. Ravali RaJu, Dr. Yonsil Park, and Dr. Liang Zhao. I am so grateful for the memories we made together at our group parties, going apple picking, and going to the lake. Thank you to the undergraduates I had the privilege of working with throughout graduate school: Alicia Zhang, Marina Raabe, Clarissa Kraft, and Daniela Batres. Training all of you and watching you progress as scientists has been one of the most rewarding experiences during my PhD. I was able to accomplish much more with your help. i Special thanks to Victoria Roberts for advice on figure design – my publications have really benefited from beautiful figures created with her help. I would also like to thank the many friends I made throughout my time at Minnesota. Thank you to Meghan McCann, Jen One, Jen Markou, Erica Connell, and Beth Harris for our girl’s board game group and long discussions about careers and life. To Meghan McCann, Andrew Allman, and Breezy Wentz, thank you for being great friends and neighbors. To Jacob and Tiffany Held, thank you for being our first friends in Minnesota, I will miss our regular dinners. To Jen One, thank you for helping me get through TAing and being my nerd buddy. To Matt Quan, thank you for your positivity, puns, and our numerous get-togethers. Finally, I would like to thank my family for their never-ending support during the past 5+ years. Thank you to my parents, Vadim and Anna Bluvstein, and my sisters Julia, Rachel, and Debbie for believing in me. To my husband Conor O’Brien, I cannot thank you enough for your love and support. Thank you for being there for me, both as a partner and as a lab mate. I would not have made it through this without you. ii Dedication To my parents, Vadim and Anna, and to my husband Conor. iii Abstract Protein biologics have transformed the field of medicine in recent years. These complex molecules are produced in living cells, primarily Chinese Hamster Ovary (CHO) cells. Due to the importance of these therapeutic proteins to disease treatment, it is essential to improve the efficiency of their production, both to promote the development of new therapies, and to bring down the cost of manufacture. One of the most important components of the production process is the development of a cell line for protein production. Many features of a cell line, such as cellular growth, metabolic behavior, and the integration site of the gene encoding the protein, influence the resulting productivity and quality of the protein produced. In this work, characteristics of desirable integration sites are investigated using genomics, transcriptomics, and epigenomics, and an exploration of cellular metabolic behavior was performed through kinetic modeling and cell engineering. In order to develop a cell line for therapeutic protein production, DNA encoding the product gene is integrated into the host cell genome. After selection, single cell cloning is performed to isolate clonal cell lines that originated from a single cell. For cell lines generated using random integration of the product gene, the integration site of the transgene becomes a unique signature for each cell clone and can be used for verification of clonality. An algorithm for the determination of integration sites from next generation sequencing data was developed and was able to distinguish between cell lines derived from different parent clones. In addition to clonality verification, knowledge of the integration site can be used for clone selection, as the integration site of a transgene can affect its expression as well as the stability of that expression over time. To study regions of the genome which may have inherent instability, a clonal cell line with high productivity of IgG was successively single cell cloned, and high and low producing clones were isolated. We found that a region of the genome containing an integration site repeatedly lost copies in low producing clones, suggesting that some genomic regions are liable to instability. To expand this work, whole genome sequencing from 23 cell lines was utilized to identify common regions of the genome prone to structural variability. The regions deleted iv in each cell line roughly clustered by host cell, showing that different cell lineages may have different vulnerable regions. Though overall a low percentage of the genome is vulnerable to deletions, certain regions were more prone to variability than others. The influence of the genomic landscape on transgene expression was further evaluated by identifying the integration sites of a randomly integrated transgene delivered via lentivirus to the CHO genome. A correlation was found between high expression of the transgene and high transcriptional activity and accessibility of the region surrounding the integration site. Using destabilized GFP as a reporter gene, high producing clones were isolated with this method. Furthermore, the transgene was replaced with a new product gene, highlighting a streamlined method for cell line development. Though the integration site can be a maJor contributor to cell line productivity, it is not sufficient to obtain a high producing cell line. Other traits, such as metabolic behavior, can have a large influence on the growth and protein production capabilities of the cell line. Through the use of a kinetic model and cell engineering of a protein producing CHO cell, we investigated both the robustness of cell metabolism during the growth stage, but also reduced waste metabolite production in late stage culture. This study highlights the difficulty of manipulating metabolism, but also the utility of model-based predictions towards cell line engineering. All together, these studies highlight some of the technological advances that are pushing improvements in cell line development. Through the increased use of omics technologies, a better understanding of a “good” integration site is being obtained. This together with the recent improvements in genome engineering techniques is allowing for targeted integration of transgenes or host cell engineering cassettes into pre-selected locations. These technologies will continue to drive the development of next generation cell lines with high, stable expression of transgenes and ideal behavior for high density culture. v Table of Contents Abstract .............................................................................................................................. iv List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii List of Abbreviations ........................................................................................................ xv 1 Introduction ................................................................................................................. 1 1.1 Thesis organization .............................................................................................. 1 2 Cell culture bioprocessing - the road taken and the path forward .............................. 3 2.1 Summary .............................................................................................................. 3 2.2 Introduction .......................................................................................................... 3 2.3 Cell Culture Process Technology ........................................................................
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