Single particle tracking reveals through-membrane diffusion of bacteriophage during process disruption of virus filtration

In the biopharmaceutical industry, virus filters are crucial for ensuring the removal of endogenous and adventitious viruses as part of the viral clearance strategy. Although traditionally described as a size-exclusion mechanism, virus retention has a process-dependent nature where challenging conditions, such as process disruptions, may compromise membrane retention and significantly increase virus filtrate concentrations. The detailed mechanisms underlying this loss of retention are challenging to determine using traditional breakthrough experiments. In this work, single particle tracking and kinetic simulations were employed to connect individual particle behavior to the observed macroscopic losses in virus retention. Our experiments, using fluorescently labeled ΦX174 bacteriophage as a model parvovirus, replicated conditions representative of process disruptions within the Pegasus SV4, a homogeneous polymeric virus filtration membrane. During flow, phage particles retained were trapped within relatively large cavity spaces that had downstream constrictions aligned with the flow direction; the trapped particles were dynamic and exhibited significant intra-cavity motion. Upon flow stoppage, particles escaped from these retention locations rapidly, with approximately 90 % of previously trapped particles being remobilized for process disruption times ranging from 2 to 10 min, suggesting that local cavity escape had reached saturation at these timescales. Diffusion experiments within the membrane revealed isotropic and Fickian motion, hindered by more than an order of magnitude compared to diffusion in unconfined liquid. Despite the reduced mobility within the membrane, the substantial diffusion coefficient of 4.19 ± 0.06 μm2/s indicated that virus particles could travel tortuous but non-retentive pathways through the membrane on length scales equal to or greater than the membrane thickness during a disruption event. A 1D kinetic Monte-Carlo simulation successfully connected single-particle behavior to macroscopically observed virus release, indicating that significant diffusive release into the filtrate can occur even without the resumption of flow. This work provides crucial insights into the retention behavior of homogeneous membranes during periods of disruption, enabling the design of more robust mitigation strategies and filter designs. [Display omitted] •Single particle tracking related phage transport to ensemble observations.•Phage particles were d