This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports work to characterize cell lines producing recombinant monoclonal antibodies with an objective of identifying the characteristics of high productivity cell lines. Monoclonal antibodies represent a significant fraction of the recently approved and pipeline biopharmaceuticals, providing critical new therapies for diseases such as breast cancer and rheumatoid arthritis. However, cell-line development currently requires screening hundreds to thousands of clones to identify cell lines exhibiting high levels of specific productivity and rapid growth rates, limiting the rate at which compounds can be brought into clinical trials and ultimately, to market. The ability to improve productivity from a rational approach will improve the rates of bioprocess development, which will, in turn, improve human health by allowing novel biopharmaceutical products to reach the market more quickly and inexpensively.
The intellectual merit of this proposal is to analyze the epigenetic differences (heritable traits that do not involve changes in the DNA sequence) between highly productive cell lines and culture conditions and lower productivity systems to understand regulation of transcription. Specifically, we will compare (i) parental cell clones and their methotrexate-amplified progeny, to identify epigenetic changes that occur upon gene amplification; (ii) cell clones arising from independent transfection events, to identify the interactions between transgene localization and epigenetic modifications, and (iii) butyrate-treated and untreated cell clones, to identify the epigenetic changes that occur upon treatment with a histone-deacetylase inhibitor known to increase specific productivity.
The broader impacts of the proposed work are (i) to provide fundamental knowledge to the biotechnology and biopharmaceutical industry thus speeding bioprocess development, which will, in turn, have a significant impact on human health by allowing novel biopharmaceutical products to reach the market more quickly and inexpensively; (ii) to train graduate and undergraduate students, with a particular emphasis on undergraduates from historically minority institutions, in mammalian cell bioprocessing, meeting a critical need in the biotechnology/biopharmaceutical industry; (iii) to incorporate the results of the proposed work into instructional materials for undergraduate and graduate courses including a new graduate course at the Biomanufacturing Training and Education Center at NC State; (iv) to continue outreach activities, particularly focusing on recruiting young women into scientific careers.
Monoclonal antibodies represent breakthrough therapeutics, treating a wide range of conditions including cancer, infectious disease, and rheumatoid arthritis. Because these drugs are complicated proteins, they are generally produced in cultured animal cells. A critical, rate-limiting step in bringing these drugs to market is the time it takes to develop a cell line and a process for culturing those cells most effectively. This project has focused on developing a fundamental understanding of these processes, to expedite bringing drugs into clinical trials and ultimately to market. This project has focused on two scientific goals, first, understanding epigentic regulation of antibody production in Chinese hamster ovary (CHO) cells, and second, investigating cellular responses to hyperosmotic stress, also in CHO cells. The first aspect of the project attempted to understand the role of epigenetic modifications of DNA and transcription factors in controlling protein expression. Epigenetics broadly describes changes in DNA structure other than sequence and changes in interactions of proteins with DNA that affect the rate of transcription of a DNA sequence, and ultimately the yield of that protein. A critical aspect of epigenetic modifications is that they can be inherited from a parent cell to a daughter cell. When examining a collection of CHO cell clones, all producing the same antibody, we found that some clones made much more antibody than we expected based upon the amount of DNA they had for the antibody genes. To understand this phenomenon, we first examined the location of the genes in the chromosome (Figure 1), but found no differences. We then examined the interactions of transcription factors (proteins that lead to transcription of RNA from DNA and ultimately to protein protein synthesis) with the DNA (Figure 2) and found that there were differences in the interactions. We have since then examined modifications of the DNA and found that there are changes in the DNA structure between the clones. As part of this analysis of epigenetic changes, we received a small travel supplement to visit a laboratory in Hungary which has signficant expertise in epigenetic analysis. With this group, we developed some novel tools to analyze DNA-protein interactions and are currently evaluating their effectiveness. In the analysis of the effects of increasing osmolarity on CHO cell culture, we worked collaboratively with Pfizer to explore the effects of adding various salts and sugars into the culture medium. We found that the cellular responses to salts and sugars were different and we characterized the intracellular accumulation of protective compounds with the various increases in salts and sugars. This project has supported two graduate students (including one woman) and one postdoctoral fellow and has trained several undergraduate students and high school students including a number of women and underrepresented minorities. One of the graduate students went on to two postdoctoral positions and is currently applying for industrial research positions. The other graduate student will graduate in approximately six months. The postdoctoral fellow has recently accepted a position with Regeneron Pharmaceuticals.
