Next‐Generation Glutamine Synthetase Expression Vector for Therapeutic Protein Manufacturing Using CHO Cells

The glutamine synthetase expression system is widely used for therapeutic protein biomanufacturing in Chinese hamster ovary (CHO) cells. Here, we present the design of an enhanced glutamine synthetase vector for generating CHOK1SV GS-KO stable pools that produce recombinant proteins at multigram-per-liter levels. We first developed a synthetic promoter, LHP-1, for high-level constitutive recombinant gene transcription, achieving up to 123% increase in titer across monoclonal and bispecific antibody formats compared to the viral CMV promoter. We then demonstrate, for the first time, that the bacterial origin of replication (Ori) can function as a core promoter to drive glutamine synthetase selection in CHO cells. In this novel glutamine synthetase vector, Ori and LHP-1 each play dual roles: Ori acts as both the plasmid replication origin and a minimal glutamine synthetase promoter, while LHP-1 drives product expression and optimal enhancement of glutamine synthetase activity without the use of methionine sulfoximine (MSX) supplementation. Together, these two elements further increased product titers by up to 73% compared to the vector harboring the SV40 promoter to drive glutamine synthetase gene expression with 50 µM MSX. Notably, the LHP-1 Ori-GS system effectively maintained high bulk pool titers of up to 5.7 g/L with no decline after 30 days-supporting consistent product yield and quality during culture scale-up for toxicology material supply. Further cell line development showed that clones derived from the LHP-1 Ori-GS pool achieved a 23% higher median titer while maintaining long-term expression stability compared to those generated with the SV40-GS vector, with the top-performing Ori-GS clone reaching 8.3 g/L. Overall, the MSX-free LHP-1 Ori-GS system offers a robust platform for both rapid, high-yield material generation from bulk pools and efficient progression to high-producing clonal cell lines.