Optimization of amino acid composition in CHO cell perfusion medium using definitive screening design and 1 H NMR ‐based consumption profiling

Abstract Perfusion culture is acknowledged as a promising platform for sustained high‐density cell production, while concurrently necessitating stringent control over medium nutrient composition. A multi‐component medium optimization strategy has been developed in this study, integrating the targeted feeding approach (TAFE), 1 H nuclear magnetic resonance ( 1 H NMR) analysis, and definitive screening design (DSD). Nine pivotal amino acids were selected through quantitative profiling of cellular uptake kinetics and literature evidence. Their concentrations were optimized using a DSD within only 24 experimental runs. The optimized formulation was demonstrated to maintain stable cell density, high viability (>97%), and excellent monoclonal antibody production in both shake flask semi‐perfusion (38.63 pg/cell/day) and 3L bioreactor systems (45–61.5 pg/cell/day), while significantly reducing the accumulation of lactate and ammonium. These results suggest that the proposed strategy can effectively enhance both productivity and metabolic stability, offering excellent scalability and engineering applicability. This work provides a novel and efficient pathway for the development of perfusion culture media in biopharmaceutical manufacturing.