Abstract

Constructing expression cell lines using site-specific integration (SSI) technology has become a prominent focus in cell line development (CLD) because of its potential to minimize clonal variation. However, this technology has not yet been widely adopted in commercial biopharmaceutical manufacturing. A major reason is the low yield of SSI-derived cell lines. To improve yield, we designed landing pad locus-specific synthetic promoters by screening for potent transcription factor regulatory elements (TFREs) within an SSI platform. We then combined these TFREs to create novel, strong promoters. All synthetic promoters exhibited reporter expression comparable to that of the CMV promoter, with some up to 20% stronger, despite being only one-third the length of the CMV promoter. When expressing a monospecific antibody, fed-batch titers from these synthetic promoters were up to 45% higher than those from the CMV promoter. We also used synthetic promoters to express a bispecific antibody via SSI. The titer from a pure synthetic promoter combination was ~2.7 g/L, lower than that from an all-CMV promoter combination (~3.8 g/L). These findings demonstrate that established SSI platforms can be used to design efficient context-specific promoters, leading to significant improvements in antibody expression and greatly enhancing the value of SSI applications in CLD.