Background & Aim The field of cellular immunotherapy is growing rapidly. Natural killer (NK) cells are part of the innate immune system and have wide potential for therapeutic application. Although CAR-T cells are now commercially available, NK cells are still in pre-commercial development and testing. It is likely that much higher numbers of NK cells will be required to achieve therapeutic results compared to that of antigen-specific T cells. With the development of feeder cells expressing membrane-bound IL-21, it has become possible to achieve large-scale expansion of non-senescent NK cells. To date, most studies have expanded NK cells at clinical scale by sequential application of open systems (G-rex) or small-volume closed systems. Achieving large-scale expansion of GMP-grade NK cells requires multiple systems operating in parallel, which increase labor costs, material costs and potential for contamination. To develop a large-scale process for NK-cell expansion in compliance with regulatory requirements, we combined the CliniMACS Prodigy (Miltenyi Biotec) and Xuri W25 (GE Healthcare) into one closed-system process. Methods, Results & Conclusion In collaboration with Miltenyi, we optimized a custom program for the Prodigy to perform Ficoll density-gradient selection of mononuclear cells, CD3-based magnetic depletion of T cells, periodic addition of and forced interaction with feeder cells, and on-demand addition of media for the first phase of NK-cell culture, culminating in transfer of the cells to a secondary culture bag. We then applied a semi-continuous perfusion program on the Xuri for the second phase. We identified optimal glucose and pH levels to support log-phase expansion, and refined the Prodigy program to optimize CD3-depletion and allow efficient volume reduction steps for media exchanges. On the Xuri, pH and dissolved oxygen (DO) monitoring was utilized to identify optimal rocking rates, rocking angles and perfusion parameters to allow sufficient NK cell-feeder cell contact and achieve high cell densities. Finally, NK cells produced by this approach demonstrated very high cytotoxicity (mean 65.2% at 1.25:1 E:T ratio). With these optimized protocols, we achieved high-purity (≥99%, n=3) and high-density (∼107 cells/mL) conditions to yield 5 × 1010 high-activity NK cells in a 5L final culture volume from one donor buffy coat. We anticipate realizing both reduced cost and increased regulatory compliance of NK cell production by using the automated closed systems of Prodigy and Xuri in sequence.