Development of a simplified intensified fed-batch CHO cell culture processbased on a commercial single-feed strategy

To reduce the operational complexity of conventional dual-feed fed-batch processes while maintaining high productivity, an intensified cell culture strategy integrating a commercial single-feed medium with N-1 perfusion-based high-density inoculation was developed using a CHO-K1 cell line expressing a bispecific antibody. Six commercially available single-feed media were first evaluated in shake-flask cultures, and the selected medium achieved cell growth, metabolic performance, antibody titer, and product quality comparable to those of a conventional dual-feed process. To further improve productivity, N-1 perfusion intensification enabled inoculation at 10 × 10⁶ cells/mL in 2 L bioreactors. High-density cultures entered the stationary phase earlier while maintaining elevated viable cell densities throughout production. When combined with the single-feed strategy, The IFB-Single process achieved a final antibody titer of 9.4 g/L, approximately 24% higher than that of the conventional FB process (7.6 g/L). Although its titer was lower than that of IFB-Dual (10.2 g/L), IFB-Single exhibited lower culture osmolality, suggesting an opportunity for further feed optimization. Product quality attributes, including monomer content, charge variants, and N-glycan profiles, remained comparable across all conditions. These results demonstrate that combining single-feed supplementation with high-density inoculation provides a practical and scalable strategy for process intensification, simplifying operation while enhancing manufacturing performance without compromising product quality.