 Hi, I'm Mara Innes. I'm here in Pfizer's new building in Kendall Square in the Biotech Hub in Cambridge, Mass, where researchers commit to discovering, investigating, and developing therapies for a variety of diseases. Pfizer's portfolio is one of the most robust in the industry and spans multiple indications, such as inflammation, immunology, oncology, neuroscience, cardiovascular, metabolic disease, and vaccines. I'm a postdoctoral fellow in cell line development, which is part of Biotherapeutics Pharmaceutical Science. We're responsible for making cell lines, both mammalian and microbial, which will produce biotherapeutics suitable for clinical and commercial development. For example, our group uses Chinese hapsir ovary cells, or cho-cells, to produce molecules such as monoclonal antibodies. Because we're relying on a biological system, there's a lot of variability in product quality and productivity of the cell lines. Therefore, we're always looking for ways to improve our processes and improve efficiency. The current process for making a stable recombinant cell line relies on random integration of an expression construct into the cho genome. This means that intensive cell line screening effort will be required in order to find a cell line with the appropriate productivity and product quality characteristics for clinical development. To address this challenge, scientists, including ourselves, have developed site-specific integration technology using recombinase-mediated cassette exchange, or RMCE. In this system, the host cell line contains a DNA construct with recombinase target sites at a location that is well characterized and highly transcriptionally active. By putting matching recombinase target sites on the targeting expression vector, we can ensure that our biotherapeutic expression cassette goes to the same genomic location every time. This means that most of the cell lines will have very similar performance characteristics, and cell line screening efforts can be significantly reduced. While most RMCE systems currently in use are based on flip or pre-recombinase, in this study we identified a novel RMCE system based on the phage-integrated BXV1. This enzyme is reported to be highly efficient in mammalian cells, and we hypothesized that this would translate to a highly accurate and efficient RMCE system. We first created host cell lines harboring either the BXV1 or flip RMCE system. We used CRISPR-Cas9 technology to integrate a large genomic construct, which we call a landing pad, into a well-characterized site in the cho genome. The only difference between these two constructs are the recombinase target sites for either BXV1 or flip. This allowed us to compare the two systems in the same genomic context. Once we establish the landing pad cell lines, we next delivered targeting vectors encoding monoclonal antibody to compare the efficiency and accuracy of these two systems. As with the landing pads, the only difference between the two targeting vectors are the recombinase target sites for either BXV1 or flip recombinase. In order to thoroughly evaluate both systems, we developed a handful of clonal cell lines. We showed that a higher percentage of cell lines derived from the BXV1 system expressed monoclonal antibody, and most importantly, 100% of clones expressing monoclonal antibody had the expression cassette integrated in the correct site. On the other hand, only 78% of clones expressing antibody derived from the flip system had their expression construct in the correct location in the genome. The superior precision of site-specific integration of the BXV1 recombinase means that cell line screening efforts can be significantly reduced. The impact of the BXV1 RMC system on cell line development will be twofold. Firstly, it will shorten the timeline from start of development to the clinic, and secondly, it will allow us to execute multiple projects simultaneously with the same given number of resources, allowing us to bring more candidates forward for development. If you want to learn more about our research, please look for our paper in biotechnology and bioengineering.