Background <p>Fed-batch culture is a well-established and widely adopted platform for industrial antibody production using Chinese Hamster Ovary (CHO) cells. However, conventional fixed feeding strategies often fall short in meeting the dynamically changing nutrient demands of cells, leading to metabolic imbalance and suboptimal productivity. Oxygen Uptake Rate (OUR), as a real-time indicator of cellular respiratory activity, is tightly coupled to nutrient metabolism and holds strong potential for guiding adaptive, demand-driven feeding strategies.</p> Methods <p>To understand the decline in specific productivity (Q_P) during the late stationary phase (LSP) under a conventional reference feeding (RF) strategy, we examined whether it stemmed from cell-intrinsic metabolic changes or from environmental stressors such as nutrient imbalance, by-product accumulation, and osmotic pressure. Based on these insights, we developed an OUR-based continuous feeding (OBCF) strategy and benchmarked its performance against the RF strategy. Mechanistic understanding was elucidated through metabolic flux and transcriptional analyses.</p> Results <p>The RF strategy resulted in a mismatch between nutrient supply and cellular demand during LSP, triggering osmotic stress and limiting antibody expression. In contrast, the OBCF strategy dynamically aligned nutrient delivery with cellular respiration, thereby mitigating osmotic stress and reshaping intracellular metabolism. Notably, OBCF enhanced pyruvate utilization and TCA cycle activity, promoted amino acid catabolism, and suppressed by-product accumulation. These metabolic improvements led to a 52% increase in specific productivity (Q_P) and a 32% increase in total antibody yield during LSP, along with reduced batch-to-batch variability.</p> Graphical abstract <p></p>