Systematic evaluation of transposon vector elements to establish high-producing stable CHO cell pools achieving...

Rapid generation of cell populations capable of producing industrially relevant levels of complex biologics remains challenging. Using wild-type PiggyBac transposase and a CHO-K1 glutamine synthetase (GS) knockout cell line, we screened known and novel vector elements in a multi-gene vector (containing two expression cassettes and one resistance marker) for their impact on accumulated titer in stable fed-batch pools. The optimal combination included the Cricetulus griseus polyubiquitin gene UbC (CHUB2) enhancer, a fusion promoter combining the murine cytomegalovirus (CMV) enhancer with the TATA box, transcription start site (TSS), and 5' UTR of the human β-hemoglobin gene, human SV40 and CMV enhancers placed between cassettes, and the WPRE element (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) in the 3' UTR. Expression remained stable over 30 population doubling levels (PDLs). This enabled the generation of pools producing > 10 g/L of trastuzumab in a standardized fed-batch process within 5 weeks post-transfection, with product quality comparable to the commercial reference. Clonal populations reached up to 15 g/L with low variability in expression between clones. To further increase titers, we engineered a PiggyBac variant with additional nuclear localization signals and expressed it under a strong promoter. The improved transposase increased pool performance by 10-30%. Suitability of the vector for complex molecules was demonstrated by achieving up to 2.5 g/L in pools expressing a difficult-to-express multispecific antibody format.