Augmenting therapeutic protein production in CHO cells: A proline‐based selection strategy for enhanced productivity and product quality

Abstract Chinese hamster ovary (CHO) cells have emerged as the predominant mammalian host for the production of therapeutic recombinant proteins, including monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), and fusion proteins. To meet the growing demand for biologics and reduce manufacturing costs, the exploitation of efficient cell line development platforms is essential. Over the past decades, various selection markers, such as dihydrofolate reductase (DHFR), glutamine synthetase (GS), and antibiotic resistance genes, have been widely utilized in the development of production cell lines. In this study, we introduce the proline selection system, an alternative metabolic selection strategy, as an efficient approach to optimize our CHO cell line development platform. By employing yeast PRO1 and PRO2 genes as selection markers, proline selection effectively complements GS selection to establish high‐producing cell lines for both mAbs and bsAbs. In particular, the integration of PRO1 and PRO2 genes into a single plasmid, in conjunction with the GS gene, significantly enhances productivity for asymmetric molecules. Optimized chain configuration across proline and GS selection plasmids can further boost protein yield. Additionally, the overexpression of regulator proteins can be leveraged with proline selection to enhance antibody production or fine‐tune product quality. Taken together, the incorporation of proline selection into CHO cell line development, particularly when combined with GS selection, provides a consistent and streamlined strategy to meet the growing demand for high‐quality biologics in the pharmaceutical industry.