The Innovative Multi-Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression in CHO Cells

The Innovative Multi-Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression in CHO Cells | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 2 May 2025 V1 Latest version Share on The Innovative Multi-Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression in CHO Cells Authors : Lei Cao , Daoyuan Na , Jun Cheng , Liang Zhao 0000-0001-8077-3617 , Qian Ye 0000-0002-1101-2088 [email protected] , and Wen-Song Tan 0000-0001-9857-4798 Authors Info & Affiliations https://doi.org/10.22541/au.174620524.44890569/v1 Published Biotechnology and Bioengineering Version of record Peer review timeline 360 views 184 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cell line development of Chinese hamster ovary (CHO) faces persistent challenges in selecting high-producing clones, particularly for complex therapeutic proteins requiring multigene co-expression. To address this, a novel tyrosine-based selection system incorporating three selection markers was developed based on three crucial enzymes in tyrosine biosynthesis: pterin-4α carbinolamine dehydratase 1 (PCBD1), phenylalanine hydroxylase (PAH), and quinoid dihydropteridine reductase (QDPR). Following systematic evaluation of sgRNA editing efficiency, a two-step knockout strategy was implemented: initial PCBD1 ablation followed by concurrent PAH/QDPR disruption, generating a triple-knockout CHO-Tyr-ko cell line. This engineered chassis exhibited strict tyrosine auxotrophy that was specifically rescued by pcbd1 / pah / qdpr co-expression. The coupling of triple-fluorescent reporters ( mCherry / copGFP / mTagBFP2 ) with three selection markers ( pcbd1 / pah / qdpr ) via expression cassettes in tyrosine-based selection system demonstrated enhanced reporter expression levels, improved population homogeneity, and achieved 97.49% enrichment of triple-positive cells under tyrosine deprivation. Moreover, combined with optimized tyrosine supplement strategies, recombinant CHO-Tyr-ko-H/L/L cells selected in tyrosine-free medium attained a monoclonal antibody (mAb) titer of 0.35 g/l in fed-batch culture and 1.60 g/l in perfusion culture. In conclusion, the novel tyrosine-based selection system, featuring three selection markers under unified selection pressure, provides an alternative for recombinant protein expression and paves a new avenue for multigene co-regulation engineering in CHO cells. Supplementary Material File (bb-the innovative multi-marker selection system based on tyrosine synthesis pathway for monoclonal antibody expression in cho cells.docx) Download 132.62 KB File (figure legends.docx) Download 3.18 MB File (table.docx) Download 33.77 KB Information & Authors Information Version history V1 Version 1 02 May 2025 Peer review timeline Published Biotechnology and Bioengineering Version of Record 9 Mar 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords chinese hamster ovary cells fed-batch；perfusion monoclonal antibody selection markers tyrosine-based selection system Authors Affiliations Lei Cao East China University of Science and Technology State Key Laboratory of Bioreactor Engineering View all articles by this author Daoyuan Na East China University of Science and Technology State Key Laboratory of Bioreactor Engineering View all articles by this author Jun Cheng East China University of Science and Technology State Key Laboratory of Bioreactor Engineering View all articles by this author Liang Zhao 0000-0001-8077-3617 East China University of Science and Technology State Key Laboratory of Bioreactor Engineering View all articles by this author Qian Ye 0000-0002-1101-2088 [email protected] East China University of Science and Technology State Key Laboratory of Bioreactor Engineering View all articles by this author Wen-Song Tan 0000-0001-9857-4798 East China University of Science and Technology State Key Laboratory of Bioreactor Engineering View all articles by this author Metrics & Citations Metrics Article Usage 360 views 184 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Lei Cao, Daoyuan Na, Jun Cheng, et al. The Innovative Multi-Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression in CHO Cells. Authorea . 02 May 2025. DOI: https://doi.org/10.22541/au.174620524.44890569/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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