(141a) Exploring Virus Retention during Virus Filtration Using Confocal Microscopy with Fluorescently-Labeled Bacteriophage

Although virus filtration is a well-established method for combating the inherent risk of viral contamination in the production of therapeutic proteins, there are still significant uncertainties regarding the underlying mechanisms controlling virus retention.  Previous studies have reported a significant decline in virus retention during constant pressure virus filtration with certain filters (but not others).  Several studies showed that a temporary release in the transmembrane pressure can cause a significant transient increase in virus transmission.  However, there is currently no detailed understanding of the factors controlling these phenomena or the underlying physical basis for the changes in virus retention.  The objective of this work was to employ confocal microscopy with fluorescently labeled bacteriophage to directly probe virus capture in several different virus filters (Viresolve Pro, Viresolve NFP, and DV20) having different pore morphology.  A model bacteriophage (phix-174) was labeled with either SYBR Gold (green fluorescence) or Cy5 (red).  The use of dyes with different excitation and emission wavelengths makes it possible to distinguish between phage that were captured during the initial challenge and after the pressure release.  Bacteriophage retention during the normal flow filtration was quantified using a plaque forming assay.  Images obtained with the DV20 filter showed two distinct bands of phage at the end of a pressure release experiment, with the band that penetrated deeper into the filter consisting primarily of phage that were captured before the pressure release.  The behavior of the Viresolve membranes was very different, with the phage captured in a more diffuse region deep within the filter upstream of the retentive skin layer of the membrane.   The confocal images and retention data were analyzed using an internal polarization model that accounts for the accumulation of phage within a reservoir zone within the filter.  The results provide important insights into the mechanisms controlling virus retention as well as the effects of membrane morphology on virus capture during virus filtration.