This study describes the development and experimental verification of a modified harvest system to enhance Factor VIII (FVIII) yield in an alternating tangential flow (ATF) perfusion culture. The main innovation of the modified harvest system is the use of check and pinch valves, eliminating the need of a peristaltic pump for harvest. The system was applied to perfusion cultures of Chinese hamster ovary cells, which co-express both recombinant human FVIII (rhFVIII) and von Willebrand factor (vWF). The modified harvest system showed comparable cell growth with the conventional harvest system using a peristaltic pump. The perfusion rate was successfully controlled using the system. In addition, the modified harvest system achieved an approximately 13.6-fold increase in the final concentration yield of FVIII activity and a 1.47-fold increase in the production yield of FVIII activity compared with a peristaltic pump. Enhancement of the yield of FVIII activity resulted from the reduction of FVIII antigen (FVIII:Ag) retention. As a result of transmembrane pressure (TMP) measurement, the reduction of the retained FVIII:Ag was due to the increased TMP, which was caused by the characteristic function of a check valve, compared with a peristaltic harvest system. The modified harvest system developed in this study could be useful to enhance the production yield of other recombinant proteins in ATF perfusion culture. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.