Increasing capacity utilization and lowering manufacturing costs are critical for pharmaceutical companies to improve their competitiveness in a challenging environment. Development of next generation cell lines, improved media formulations, application of mature technologies and innovative operational strategies have been deployed to improve yields and capacity utilization. This article describes a large-scale perfusion strategy for the N-1 seed train bioreactor that was successfully applied to achieve higher inoculation cell densities in the production culture. The N-1 perfusion at 3,000-L scale, utilizing a inclined settler, achieved cell densities of up to 158 x 105 cell mL1 at perfusion rates of 2950 L day1 and a retention efficiency of >85%. This approach increased inoculation cell densities and decreased cultivation times by approximate to 20% in a CHO-based, fed-batch antibody manufacturing process while providing comparable culture performance, productivity, and product quality. The strategy therefore yielded significant increase in capacity utilization and concomitant cost improvement in a large scale cGMP facility. Details of the strategy, the cell retention device, and the cell culture performance are described in this article. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 29: 222-229, 2013.