Performance characteristics of virus filtration membranes: Protein fouling and virus retention

Preventing viral contamination is critical in the manufacturing of safe and effective biotherapeutic proteins. Virus filtration is an integral part of the overall strategy for viral clearance of cell culture-based proteins. Virus filtration membranes provide a robust size-based removal of viruses through a multi-layer structure that is uniquely designed to provide high levels of viral clearance and significant recovery of the protein products. The objective of this thesis is to develop a more complete understanding of the fundamental mechanisms governing protein transport and fouling, as well as virus retention, during virus filtration. This includes the role of solution conditions, protein concentration, and membrane structure on protein transport, membrane fouling, and the observed decline in virus retention during virus filtration.;Experimental studies were performed using three very different virus filters, the Pall UltiporRTM DV20, the Millipore ViresolveRTM Pro, and the Millipore ViresolveRTM 180 membranes operated in both the standard and reverse flow orientations. The ViresolveRTM membranes have an asymmetric structure with a retentive skin layer on top of a more open substructure. In contrast, the UltiporRTM DV20 membrane has a fairly homogenous (isotropic) pore structure. The decline in filtrate flux through the Viresolve membrane was primarily due to concentration polarization, with the extent of polarization dependent upon solution pH due to the pH dependence of the protein mass transfer coefficient. In contrast, the dominant mechanism of flux decline with the DV20 membrane was protein fouling due to the constriction of the membrane pores.;Several novel approaches were developed to probe the pore morphology and retention characteristics of the virus filtration membranes. First, a dextran sieving test was developed that was appropriate for the large pores in virus filters. The dextran sieving profiles depend on both the pore size and the asymmetric structure of the virus filter. For example, the dextran sieving profiles for the UltiporRTM DV20 membrane were slightly different in the two orientations, demonstrating that the DV20 membrane had a slightly non-homogeneous pore structure, in contrast to the highly asymmetric structure of the ViresolveRTM membranes. Second, a confocal scanning laser microscopy method was developed to directly visualize the capture of fluorescently labeled bacteriophage within the pore structure of the virus filters. Third, transmission electron microscopy was used to visualize the capture of nanometer-sized gold particles.;Several different hypotheses were examined to describe the reduction in virus retention including: small pore plugging, virus polarization, virus breakthrough, and virus internal polarization. Experimental data for retention of PP7 bacteriophage through the DV20 membrane were consistent with predictions of the internal virus polarization model, in which the accumulation of viruses within the membrane matrix (but in the liquid phase within the porous structure) leads to the observed reduction in virus retention. These results provide important insights into the key factors controlling the performance of virus filtration membranes for bioprocessing applications.