Abstract
Chinese hamster ovary (CHO) cells constitute the industry-standard platform for the production of complex therapeutic proteins, yet genomic heterogeneity arising from random integration leads to clonal variability and unstable expression, necessitating a robust cell line development process to efficiently isolate stable, high-expressing clones. By employing an optimized integration strategy, recombinant cell line performance can be enhanced through improved genomic stability and increased productivity. In this study, GFP reporter analysis demonstrated that the PiggyBac system significantly boosts the yield of both stable and high-expression clones. Subsequently, transposase modified with a nuclear localization signal (NLS) exhibited superior stability in recombinant cell lines and polyclonal pools. The nucleoplasmin NLS resulted in transgene integration into genomic loci that promote enhanced and more stable expression, thereby improving clonal distribution. Furthermore, this strategy also increased recombinant mAb expression by 97% in pools while enhancing average and specific productivity in derived cell lines. Additionally, recombinant suspension cells generated with the optimized system exhibited comparable performance, with over 50% of minipools showing robust growth. Overall, these findings highlight the promising utility of the NLS-optimized PiggyBac system for improving transgene stability and expression while streamlining the cell line screening process.