Microfluidic Chip-Based <i>In Vitro</i> Screening System of Synthetic Signal Peptides for Enhanced Monoclonal Antibody Production in Chinese Hamster Ovary Cells

Establishing an <i>in vitro</i> screening system for synthetic signal peptides in Chinese hamster ovary (CHO) cells is crucial, as selecting an appropriate signal peptide ensures efficient secretion and enhances therapeutic protein production. However, expanding library sizes to increase peptide diversity and improving screening efficiency by minimizing false positives remain significant challenges. In this study, we optimized a synthetic-biology-driven <i>in vitro</i> screening system by incorporating the Beacon optofluidic system to minimize false positives and generating stable CHO cell pools in serum-free suspension culture to accommodate larger libraries. This platform enabled the high-throughput screening of diverse signal peptide variants at the single-cell level, exceeding the limits of conventional fluorescence-activated cell sorting (FACS)-based methods. Using this system, we successfully identified novel synthetic signal peptides for the heavy chain (HC) and light chain (LC) that enhanced the specific protein productivity (<i>q</i>_p) of the monoclonal antibody (mAb). The selected synthetic signal peptide combinations improved <i>q</i>_p in both transient and stable gene expression systems, with the best-performing combination increasing <i>q</i>_p by up to 2.23-fold compared with the native signal peptide. Additionally, substituting the native signal peptide with a screened synthetic signal peptide did not significantly affect the <i>N</i>-terminal cleavage, <i>N</i>-linked glycosylation, size heterogeneity, and biological activity of the mAb. Overall, the synthetic-biology-based <i>in vitro</i> screening system developed in this study enabled the discovery of novel synthetic signal peptides that significantly improved mAb productivity in CHO cells without compromising product quality and function.