Systematic evaluation of transposon vector elements to establish high-producing stable CHO cell pools achieving > 10 g/L monoclonal antibody titers

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. • Most current cell line development (CLD) platforms rely on transposon technology in combination with multi-gene vectors. This article is the first publication specifically describing the development of an entire high-performance multi-gene expression vector. Existing literature was assessed for vector elements, and suitable components were selected and screened alongside novel elements to generate an optimized multi-gene vector. • The resulting construct included Cricetulus griseus CHUB2, a novel fusion promoter (mCMV enhancer combined with the TATA box, TSS, and 5’UTR of the human hemoglobin gene), SV40 and hCMV enhancers, and WPRE. This enabled cell pools to exceed 10 g/L of trastuzumab, with clonal populations reaching up to 15 g/L with comparable product quality as a commercial reference. A difficult-to-express BEAT® format composed of three subunits was produced at 2.5 g/L in pools. • A PiggyBac variant with added nuclear localization signals and expressed under a strong promoter increased pool performance by 10–30% compared to wild-type PiggyBac.