Virus filtration in biopharmaceutical downstream process ensures viral safety with removal efficiency greater than 99.99%. Despite its robust performance, it has been reported that viruses can escape through the membrane under certain conditions. This study aimed to investigate virus breakthrough using Viresolve® Pro membrane with PP7 bacteriophage under high bacteriophage titer, high protein concentration, flow interruption, and low-flux operation. The results show high virus removal performance (LRV >6.8) up to 109 PFU/mL for 200 L/m2, with the first phage breakthrough observation over 1012 PFU/m2 of phage challenge in the membrane. As the protein concentration increased in the coexistence condition, a greater number of phages passed through the membrane. During the post-buffer flush and the operation at low filtrate flux, diffusion plays an important role in the escape of phages, which were detected as high as 1012 PFU/m2, but as low as 109 PFU/m2. These results suggest that the virus-retentive membrane may become susceptible to undesired virus breakthrough when exposed to phage challenges of > 1012 PFU/mL, providing important key factors to ensure viral safety during the downstream process of biopharmaceutical production.