Optimized Split Zeocin Selection System Enhances Monoclonal Antibody Productivity and Reduces High Mannose Levels in CHO Cells.

The limited variety of selection markers presents a challenge in the development and production of complex biologic therapeutics. Here, we developed and optimized a split zeocin selection system using a split bleomycin binding protein (BBP) to address these limitations. Two split bleomycin binding protein strategies (the N-terminal fragment paired with the heavy chain + the C-terminal fragment paired with the light chain (LC), or the reciprocal arrangement) were evaluated in multiple monoclonal antibodies. Our results demonstrate that the split zeocin system achieves comparable or higher titers than the conventional biantibiotic resistance system (blasticidin and zeocin) while significantly reducing high mannose levels. Notably, the use of separate open reading fragments driven by the SV40 promoter resulted in more efficient recovery of functional split BBP compared with internal ribosome entry site-driven systems. The increased LC gene copy number observed in the split system likely promotes excess light-chain expression, facilitating mAb assembly and improving glycan maturation. Further evaluation across key stages of cell line development─pool selection, single-cell cloning, 60-population doubling level stability studies, and bioreactor scale-up─validated the system's robustness, reproducibility, and scalability. These findings highlight the split zeocin system as a cost-effective and innovative approach for improving both the productivity and product quality in large-scale biopharmaceutical production.