European Society for Animal Cell Technology (ESACT)
Join us in-person at the Lisbon Congress Center in Portugal for the European Society for Animal Cell Technology where Just-Evotec will be showcasing our science.
Who’s Attending?
- Sherman Ku, Principal Science at Just-Evotec Biologics
Poster
Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads
Background and novelty: Current methods for stably expressing recombinant protein therapeutics in CHO cells often rely on random genomic integration events which result in a widely heterogeneous cell population. Consequently, a significant portion of cell line development efforts involves extensive pool and clone screening to identify clones with high expression, growth, and product quality. Additionally, these integration methods preclude experiments such as variant library screening that involve stably overexpressing pools or libraries of molecules in a single cell culture. In these cases, it is difficult to determine whether differences in library member behavior (yield, degree of library enrichment, etc.) are due to true differences inherent to each variant or merely to due to variable genomic integration site(s).
Experimental approach: We developed two targeted integration systems that express high levels of recombinant protein in CHO cells. We first generated two clonal cell lines stably expressing enhanced green fluorescent protein (eGFP) reporter landing pads in genomic hot spots. Subsequently, three therapeutic protein molecules were used to test targeted integration, which were assayed for yield and productivity as well as characterized for landing pad copy number and integration fidelity by targeted locus amplification (TLA) and PCR. We additionally test for enrichment of cell subpopulations with fully saturated landing pads with ganciclovir (GCV) counterselection. Finally, we developed a small antibody library of ~100 variants through random pairing of 10 unique light chains and 10 unique heavy chains. We transfected this library into a cell line containing a single copy landing pad wherein each cell line would express a single variant. We performed puromycin selection for cells that had successfully taken up one of the variants and cell-sorted for variants that successfully paired and expressed. We determined the identity of successful chain pairs with next generation sequencing.
Results and discussion: We demonstrate functional integration of several donor vectors encoding both monoclonal antibodies and a Fc‐fusion molecule using Cre or PhiC31 recombinase mediated integration. TLA and PCR characterization of integrated cell lines showed correct targeting of landing pads. Post‐integration enrichment for fully saturated landing pads using GCV counterselection increased recombinant protein titer by 2–2.5‐fold and specific productivity by ∼3.4‐fold with observed potential off‐target random integration detected only following GCV enrichment. Finally, we demonstrate that library screening or CHO display is feasible in our study with the 100-member variant library.
Presented by Sherman Ku, Principal Scientist at Just-Evotec Biologics