467766 Use of Fluorescent Nanospheres to Study Capture Behavior in Virus Filtration Membranes

Tuesday, November 15, 2016: 9:45 AM
Mission I (Parc 55 San Francisco)
Fatemeh Fallahianbijan, Chemical Engineering, Pennsylvania State University, state college, PA, Sal Giglia, Millipore, Bedford, MA, Christina Carbrello, EMD Millipore, Bedford, MA and Andrew Zydney, Chemical Engineering, The Pennsylvania State University, University Park, PA

Virus clearance is an essential part of the downstream process to remove the viral contamination from biotherapeutics. Several recent studies have demonstrated the complex behavior of commercial virus filters, including the observed decline in virus retention with increasing volumetric throughput and the transient loss in virus retention in response to a process disruption observed with some (but not all) virus filters. Although fluorescently labeled viruses can be used to directly visualize virus capture in different virus filters, the preparation of these fluorescently-labeled viruses is challenging and the results can be influenced by the presence of both proteinaceous and cellular debris. The objective of this work was to use fluorescently labeled nanospheres with confocal microscopy to probe the capture behavior of several commercial virus filters.

Carboxylate-modified fluorescently labeled polystyrene nanospheres with 20, 40, and 100 nm size were obtained from ThermoFisher. Filtration experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes. The membranes were examined by laser scanning confocal microscopy after challenge with one or more of the nanospheres. Images obtained after filtration of the 20 nm nanospheres show very similar capture profiles to those obtained with fluorescently labeled ϕX174 bacteriophage (25 nm in size), demonstrating the validity of using the nanospheres as models for small viruses. Results obtained with mixtures of the 20, 40, and 100 nm nanospheres show that the different size nanoparticles are captured at slightly different depths within the Viresolve® Pro filter, consistent with the graded pore size within this highly asymmetric membrane. Experiments performed in which the virus filtration was “disrupted” part way through the experiment show multiple capture zones within the Ultipor® DV20 membrane, consistent with the migration of previously captured particles within the filter when the pressure is released. This type of migration was not seen with the Viresolve® Pro, which also provided highly robust virus retention even in response to a pressure disruption. The results from this study clearly demonstrate the potential of using small fluorescently labeled nanospheres as a powerful tool to study the capture behavior in virus filtration membranes.


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