Development of an Accelerated Platform for Vector Optimization using RMCE Facilitated Mini-pool Cloning


  • Rena So, Chemical Engineering, University of Delaware

Faculty Mentor(s)

  • Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware


Conventional means of optimizing biopharmaceutical production is a time and labor-intensive process due to the heterogeneity observed within bulk populations. To address this issue, we propose a novel approach that focuses on vector and cell line optimization to achieve greater homogeneity and consistency in biopharmaceutical expression. The goal of this project is to develop cell lines with integrated, stable landing pads, ensuring consistent and reliable expression of desired genes. Leveraging recombinase-mediated cassette exchange (RMCE) and the phiC31 enzyme, this platform offers a versatile and efficient solution for vector optimization. The landing pad design includes markers for positive and negative selection of integration and loss following recombinase-mediated cassette exchange (RMCE). This includes puromycin as a positive selection marker for enrichment of integration, internal eGFP and external mCherry for dual fluorescence selection of properly integrated landing pad, and a thymidine kinase gene for negative selection of the landing pad by conferring ganciclovir sensitivity To further scale up the process, mini-pool cloning is utilized, allowing for the simultaneous screening of multiple cell clones. Tracking mini-pool titers over time, such as stability, protein quality, and cell heterogeneity, can improve gene and vector performance. As an application of this workflow, we utilized Tocilizumab in addition to other monospecific and bispecific antibodies. However, the platform’s capabilities are not limited to these applications and can be a generalized platform for doing any vector optimization method.