Combination of small-molecule additives enhances recombinant protein expression in CHO cells via NF-κB pathway.

Chinese hamster ovary (CHO) cells are a key platform for the production of recombinant therapeutic proteins. Supplementing serum-free medium (SFM) with small-molecule additives is an effective strategy for enhancing recombinant protein production. This study screened three small-molecule additives (baicalein, procyanidin and soy isoflavone) from eight candidates for their positive effects on recombinant protein expression. The optimal concentrations of these three additives were determined using the Design of Experiment (DOE) approach. The results showed that the expression of secreted alkaline phosphatase (SEAP) and adalimumab increased by 3.14- and 3.43-fold at optimal concentrations of 172.7 μM baicalein, 800 μM procyanidin and 1.00 mM soy isoflavone. Flow cytometry analysis demonstrated that the three additives combination effectively arrested cell cycle at G0/G1 phase and inhibited apoptosis. Transcriptome sequencing revealed that differentially expressed genes (DEGs) exhibited significant enrichment in the NF-κB signaling pathway. Combination of baicalein, procyanidin and soy isoflavone significantly increased NF-κB phosphorylation and the expression of p21, cyclin D1, Bcl-2 and Bcl-xL, whereas it significantly decreased the expression of Bax and caspase-3. Our findings indicated that baicalein, procyanidin and soy isoflavone synergistically enhanced recombinant protein expression by activating the NF-κB signaling pathway, offering a potentially effective approach for improved recombinant protein production.