Alternative splicing (AS) is a critical regulatory mechanism in mammalian cells, enabling functional diversity in mRNA transcripts. Yet, its role in optimizing Chinese Hamster Ovary (CHO) Cells for recombinant monoclonal antibody (mAb) production remains under-researched. In this comprehensive study, we characterized the AS landscape of industrial CHO producer cell lines, to successfully expand its engineerable design space. We conducted a large-scale RNA-Seq analysis on a cohort of 152 samples, comparing four industrial CHO cell lines (IgG1,2 and 4) across growth and stationary cell culture phases. To achieve this, we integrated differential gene expression (DGE) and differential transcript usage (DTU) analyses, employing rigorous statistical frameworks alongside novel metrics like the Differential Isoform Fraction (DIF) and Intron Exclusion Rate (IER) to quantify splicing activity and isoform switches. Our results highlight distinct, product-specific splicing profiles across the cell lines, with minimal overlap in DGE, DTU, and AS, indicating tailored cellular responses to the unique demands of each recombinant mAb. Data mining revealed a curated set of 12/179 alternatively spliced isoforms as potential engineering targets across producers, despite high product-specificity. A key finding was the identification of mAb light chain (LC) splicing efficiency as a potential critical bottleneck, strongly correlated with productivity in the stationary phase, where inefficient LC processing leads to a significant accumulation of unspliced mRNA. Additionally, host genome splicing activity (IER) declines over time, suggesting a redirection of splicing resources toward mAb transcripts, which impacts overall cellular homeostasis. Although the causal relationship between splicing efficiency and productivity remains under investigation, these findings underscore AS as a novel target for CHO cell optimization. We propose future research directions, including advanced sequencing technologies and targeted splicing interventions, to enhance mAb yields and advance bioprocessing strategies, laying a foundation for transformative applications in industrial biotechnology.
