 Hello, this is Shulita Deshary from Androzea, this research group in chemical engineering at Penn State. It's a great opportunity to showcase our work on virus filtration that has recently been published in biotechnology and bioengineering. The article is entitled, Probing Effects of Pressure Release on Virus Capture During Virus Filtration Using Conformal Microscopy. I hope you will find it interesting and informative. The consequence of viral contamination in bioprocessing are enormous. Viral contamination can compromise patient's safety, lead to product free calls and shortage of life-saving therapeutics. Therefore, viral clearance is an integral part of the purification process for all therapeutic proteins. Virus filtration membranes can provide a robust size-based mechanism for virus removal. It is a highly sized selective process which allows transmission of therapeutic products such as proteins while retaining virus particles. Most virus filters used today have a pore size around 20 nanometer, providing high removal of even the smallest viruses. Although commercial virus filtration membranes provide high initial removal of small viruses, in many cases the extent of virus retention declines significantly during the virus filtration. This decline in retention can be very dramatic. In addition, virus retention can decrease after a process disruption that might occur when using multiple feed tanks or due to operator changeover. However, the mechanisms leading to this loss in virus retention are still poorly understood. In this work, we applied fluorescence confocal microscopy to directly visualize virus capture through the depth of the virus filter. Experiments were performed with a model bacteriophage, a bacterial virus covalently labeled with a fluorescent dye, for example, fluorescence. A suspension of labeled bacteriophage were used to challenge the virus filter at a safe transmembrane pressure. At the completion of the experiment, a small piece of the membrane was placed under confocal microscope. Here you can see the Z-stacked image of the Altypo DV-20 virus filter after it was challenged with a bacteriophage labeled with the green dye fluorescent. The fars are clearly visible in a single zone near the entrance of the DV-20 filter. The virus capture profiles are very different for a membrane used in an experiment with a process disruption. In this case, the labeled fars were filtered through the membrane at a constant pressure. The pressure was then released for several minutes and the system was repressurized and the filtration continued. The confocal image obtained after the filtration showed two separate green bands near the entrance of the DV-20 filter. However, the origin of these multiple bands is unclear, since it is impossible to determine which virus entered the filter before and after the pressure release. In the current work, we developed a novel approach to study virus capture after a process disruption. We labeled two batches of fudge, one with Psi-5, which is a red fluorescent dye and one with Cyber Gold, which is a green fluorescent dye. The virus filtration experiment was performed by initially challenging the filter with red labeled fudge. The pressure was released and then the filter was challenged with the green labeled fudge. Confocal images were then obtained using different lasers to selectively excite the red and green virus. The beauty of this work is that by using viruses with different floors and dyes, we were able to differentiate between virus that were captured during the initial virus challenge and those that were captured after the pressure disruption. This slide shows typical confocal images for the DV-20 and virus-all-pro filters. The red virus in the DV-20 filter are clearly seen in two bands demonstrating that the virus that were captured before the pressure disruption migrate deeper into the filter. Our hypothesis is that the previously captured virus are able to diffuse out of the pores during the pressure release before migrating deeper into the filter. This is consistent with the observed loss of virus retention after the pressure release. The virus capture profiles in the virus-all-pro filter were very different. Multiple bands are seen but each band shows both red and green virus. The green virus in the band near the filter exit are actually located deeper in the filter. This very different profile is consistent with the observation that virus retention by the virus-all-pro filter was unaffected by the process disruption. These results clearly demonstrate that the material properties and pore morphology of the virus filter have a large effect on the details of virus capture and retention. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure. To know more about this work, please check our article on BNB's current issue. Thank you for taking the time to watch this presentation.