摘要： Virus filtration is a highly effective method in the downstream processing of biotherapeutic products to provide effective removal of potential infectious agents based on a size exclusion mechanism. The direct visualization of viruses retained inside the filter membrane represents a valuable tool to get a deeper understanding of the filtration process and to explain observations of virus breakthrough under particular operating conditions. Parvoviruses, which are used as worst-case models in validation studies, were purified and labeled with fluorescent dyes to detect their retention pattern inside the filter membrane using laser scanning microscopy. Critical factors influencing the reproducibility and accuracy of the approach were identified and optimized. The retention profiles revealed detectable differences between viruses, suggesting that the use of bacteriophages or nanoparticles as surrogates is limited in their applicability to accurately predict the behavior of parvoviruses in filter membranes. The established method enables a direct and quantitative analysis of the virus retention profile, adding a valuable tool to the conventional measurement of the viral load reduction to better understand the mechanism underlying the removal of viruses during nanofiltration of biotherapeutic products.