Diffusion integrity testing of small‐area sterilizing‐grade membrane filters in single‐use assemblies

Sterile filtration represents the final critical step in biomanufacturing, where a sterilizing-grade membrane filter removes microbial contamination from the drug product - making filter integrity verification essential. While bubble point and gas-liquid diffusion tests are established non-destructive methods for hydrophilic membranes, diffusion testing offers high sensitivity to small defects and operates at lower test pressures. However, applying diffusion testing to small-area filters integrated into closed single-use assemblies remains challenging because pressure changes measured by automated integrity testers can arise not only from membrane diffusion, but also from assembly-related effects such as gas-permeable tubing, upstream volume and thermal equilibration. For low-area devices, these background contributions can become comparable to or greater than the membrane signal, reducing test sensitivity and complicating interpretation. This work presents a practical method for implementing diffusion-based integrity testing of small-format sterilizing-grade filters in final sealed single-use assemblies. The approach quantifies assembly-specific background contribution and incorporates it into an adjusted diffusion specification, allowing membrane-specific diffusion to be resolved without altering the assembled system. By accounting for system-derived measurement noise, the method enables reliable end-user integrity assessment of closed assemblies that cannot be reconfigured to reduce upstream volume or replace high-permeability tubing materials.