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While extensive research has focused on optimizing plasmid design, host cell characteristics, and selection strategies to enhance cell line development, this study specifically investigates strategic expression plasmid design to improve both productivity and product quality of therapeutic proteins in Chinese Hamster Ovary (CHO) cells using transposon technology. Our findings demonstrate that increasing light chain and heavy chain copy numbers while maintaining balanced expression significantly enhances productivity and mitigates purity risks. Furthermore, we reveal that consolidating all chains of asymmetric molecules into a single plasmid, rather than distributing them across multiple plasmids, substantially improves both productivity and purity. For molecules exhibiting poor purity due to imbalanced chain expression, we demonstrate that incorporating an additional copy of the under\u2010expressed chain can yield significant purity improvement. Based on these findings, we propose a customized plasmid design and pool development workflow to ensure high success rates for cell lines producing structurally complex molecules.<\/jats:p>","DOI":"10.1002\/biot.70104","type":"journal-article","created":{"date-parts":[[2025,8,13]],"date-time":"2025-08-13T13:05:33Z","timestamp":1755090333000},"source":"Crossref","is-referenced-by-count":2,"title":["Tailoring Plasmid Design Based on Chain Expression in Cell Line Development for Enhanced Monoclonal and Bispecific Antibody Production"],"prefix":"10.1002","volume":"20","author":[{"given":"Shenghai","family":"Liu","sequence":"first","affiliation":[{"name":"Biologics Technical Development Global Technical Operations &amp; Manufacturing, BeOne Medicines (Shanghai) Co., Ltd. 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