Systematic Investigation of Impact of Antifoam and Extracellular Vesicles on Fouling of Hollow Fiber Filters in Intensified Perfusion Processes Highlights the Key Impact of Antifoam

ABSTRACT Ultra‐high cell density perfusion cultures are becoming increasingly attractive for biologics manufacturing due to their ability to boost productivity and offer a flexible manufacturing footprint. However, these intensified perfusion processes pose challenges, particularly concerning the performance of hollow fiber filters used as cell retention devices. To facilitate their implementation in biologics manufacturing, it is crucial to understand the factors driving filter fouling and develop innovative strategies to mitigate this issue. In this study, we developed a small‐scale model to investigate various factors in cell culture that affect filter fouling. We systematically examined individual components, such as antifoam and extracellular vesicles, to assess their impact on filter performance. Our data suggest that the extent and mechanism of fouling differ among these components, likely due to variations in particle size distribution and properties. Additionally, our results indicate that simethicone‐based antifoam accumulates over time in perfusion cultures, significantly impacting fouling. We observed better cell health in perfusion runs with minimal antifoam addition. Pellet fractions isolated by ultracentrifugation at 10,000 g and 100,000 g from “No antifoam” perfusion culture exhibited markedly improved filter performance in the offline model, highlighting the negative impact of antifoam in perfusion cultures. Conversely, an alternative antifoam variant that does not rely on simethicone showed better filter performance in the offline model, emphasizing the role of both antifoam and membrane composition in fouling tendencies. This study is the first to systematically examine the impact of individual components in perfusion cultures on filter fouling. Further investigations will be essential to develop the next generation of robust perfusion processes.