Synonymous codon variants identified by in vitro screening enhance etanercept production in CHO cells

Codon optimization has become a common strategy for enhancing recombinant protein expression in mammalian systems; however, improvements in codon usage-based computational metrics do not always translate into enhanced production performance. Herein, we established a Chinese hamster ovary (CHO) cell-based in vitro screening framework to identify synonymous coding sequences that enhance therapeutic protein production in CHO cells. Focusing on etanercept (ETN), a therapeutic Fc-fusion protein, we introduced a 5′ coding region-focused synonymous codon library into Flp-In™ CHO cells using Flp/FRT-mediated site-specific integration at a defined genomic locus. High-producing variants were enriched using a fluorescence-activated cell sorting-based antibody detection assay and evaluated under stable gene expression conditions. Three variants (ETN-Syn-4, ETN-Syn-14, and ETN-Syn-25) yielded 2.08-, 2.75-, and 2.08-fold higher specific protein productivity (qp), respectively, compared with the native sequence. Notably, the mRNA abundance of the three top-producing variants showed only modest changes compared to the native sequence. Moreover, the selected variants also showed differences in sequence characteristics. Importantly, the N-terminal processing, N-linked glycosylation, size heterogeneity, and binding activity of synonymous codon variant-derived ETN were comparable to native ETN. Collectively, this screening platform enables the empirical identification of synonymous codon variants that enhance ETN production in CHO cells. Key points Cell-based screening system identified novel synthetic codon variants in CHO cells.Selected synonymous codon variants showed distinct codon-usage characteristics.Selected codon variants increased ETN production with comparable quality.