(466a) Use of Site Specific Recombinases to Engineer New Cell Lines for Therapeutic Protein Biomanufacturing

Authors: 
O'Brien, S. A., University of Minnesota, Twin Cities
Stach, C., University of Minnesota, Twin Cities
Lee, K., University of Minnesota, Twin Cities
McCann, M. G., University of Minnesota, Twin Cities
Somia, N., University of Minnesota, Twin Cities
Smanski, M. J., University of Minnesota, Twin Cities
Hu, W. S., University of Minnesota, Twin Cities
Traditional cell line development for therapeutic protein biomanufacturing involves random integration and amplification of a product gene to endow cells with high transcript level of the product gene and high production capabilities. Subsequently, single cell cloning is performed and a clone with high productivity that retains its productivity over a long cultivation period is selected as the production cell line. This process is time and resource intensive, and could be shortened by improving the stability and homogeneity of cells for production. We have established a streamlined cell line development process by generating a template cell line with a “landing pad” that contains an antibody gene surrounded by recombination sites. Through recombinase mediated cassette exchange (RMCE), a new product gene can be quickly swapped in to take its place in the landing pad and generate a new producing cell line. The template cell line was created using single copy integration of lentivirus and screened for high productivity. Additionally, the cell line has a high transcript level of the product gene and has been shown to be stable in its productivity over long term culture, demonstrating the feasibility of single copy integration for protein production.

By RMCE, new cell lines producing TNFR:Fc fusion protein or Erythropoietin were generated from the template cell line. First, we optimized the use of RMCE by performing recombination transiently between plasmids to determine the optimal donor DNA to recombinase ratio. Then, using this information, we generated pools of cells with new product genes. The pools were then subcloned to characterize the variation between cells that have had targeted integration of a new product gene. We compared the productivity, transcript levels, and growth of different clones to identify the potential for RMCE to replace traditional cell line development. If a high producing cell line can be changed to produce a new product gene and maintain its productivity, we could vastly improve the cell line development process by shortening the time to obtain a high producing, stable cell line.