Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr^® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow^® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon^® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.

The demand for effective downstream processing of adeno-associated virus (AAV) is increasing as gene therapies advance toward broader clinical applications. Robust, efficient, and scalable ultrafiltration and diafiltration (UF|DF) operations are essential for generating high-quality AAV preparations, with tangential flow filtration (TFF) serving as a critical unit operation for vector concentration, impurity reduction, and buffer exchange while maintaining viral functionality. Development of TFF processes requires careful consideration of membrane characteristics—including chemistry, pore size or channel architecture—as these parameters directly influence vector retention, fouling behavior, and overall process efficiency. Equally important is the optimization of critical process parameters such as recirculation rate, transmembrane pressure (TMP), and total processing time, all of which govern hydrodynamic performance and product quality. This study assessed two Sartocon^® Hydrosart^® TFF cassette architectures—ECO-Screen and E-Screen—for the ultrafiltration and diafiltration of AAV8 clarified lysate. Through flux characterization and controlled small-scale evaluations, cassette-specific operating regions were defined. Both configurations supported high viral genome retention; however, the E-Screen geometry achieved faster processing and superior removal of host–cell protein and DNA contaminants, whereas the ECO-Screen format allowed for efficient operation under reduced pump rates and, therefore, lower shear conditions. Reproducibility assessments demonstrated minimal run-to-run variability, confirming the robustness of the optimized operating parameters. A 10-fold scale-up further validated the linearity and predictability of the UF|DF process, with consistent impurity-reduction profiles and only modest deviations in viral recovery. Collectively, these findings provide a quantitative basis for rational cassette selection in AAV purification workflows and establish a scalable, scientifically grounded UF|DF framework applicable across development and manufacturing scales.

Mitochondria are highly dynamic organelles that interchange their contents mediated by fission and fusion. However, it has previously been shown that the mitochondria of cultured human epithelial cells exhibit a gradient in the relative abundance of several proteins, with the perinuclear mitochondria generally exhibiting a higher protein abundance than the peripheral mitochondria. The molecular mechanisms that are required for the establishment and the maintenance of such inner-cellular mitochondrial protein abundance gradients are unknown. We verified the existence of inner-cellular gradients in the abundance of clusters of the mitochondrial outer membrane protein Tom20 in the mitochondria of kidney epithelial cells from an African green monkey (Vero cells) using STED nanoscopy and confocal microscopy. We found that the Tom20 gradients are established immediately after cell division and require the presence of microtubules. Furthermore, the gradients are abrogated in hyperfused mitochondrial networks. Our results suggest that inner-cellular protein abundance gradients from the perinuclear to the peripheral mitochondria are established by the trafficking of individual mitochondria to their respective cellular destination.