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.

The growing demand for large quantities of high‐purity lentiviral vectors (LVs) has driven the development of scalable, cost‐effective purification strategies. Membrane chromatography is particularly well‐suited for purifying large biological entities like LVs due to its low mass transfer resistance and minimal back pressure. Among these technologies, anion exchange (AEX) chromatography serves as a key unit operation during the intermediate purification stage of industrial‐scale bioprocesses. Sartobind Convec D, a weak AEX membrane with reduced ligand density, lack of hydrogel grafting, and adjusted pore size distribution, was specifically designed for LVs capture. While AEX is commonly employed immediately after clarification to capture LVs, incorporating a tangential flow filtration (TFF) step beforehand can offer several advantages, including product concentration, buffer exchange, and removal of low‐molecular‐weight impurities. Hydrosart High‐Performance TFF membranes, composed of regenerated cellulose, are characterized by high flux rates and are marketed as well‐suited for viral vector purification. This study investigated the integration of these two technologies into intermediate purification workflows for LVs. Specifically, the impact of introducing a TFF step prior to AEX chromatography (TFF‐AEX workflow) was compared to a process utilizing only AEX chromatography following harvest and clarification (AEX‐only workflow). In the AEX‐only workflow, Sartobind Convec D membranes were used directly after clarification to capture LVs. These membrane adsorbers can accommodate high flow rates, making them suitable for early downstream processing. In the TFF‐AEX workflow, clarified LVs harvests were first concentrated and diafiltrated using Hydrosart TFF membranes, allowing for product enrichment, buffer conductivity adjustment, and removal of smaller contaminants prior to AEX loading. As a result, the TFF‐AEX workflow demonstrated improved dynamic binding capacity and enhanced overall impurity removal compared to the AEX‐only approach.

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Plasmid DNA (pDNA) is a key raw material for advanced therapeutic modalities, including gene therapy, mRNA production, and DNA vaccination, requiring efficient and scalable downstream processing strategies. Increasing demand places stringent requirements on purification processes in terms of yield, impurity removal, preservation of plasmid topology, and robustness at scale. Membrane chromatography offers significant advantages for pDNA purification due to convective mass transfer, enabling high binding capacity at short residence times and straightforward linear scale-up. For the first time, a sequential two-step membrane chromatography platform was developed and optimized for the capture and polishing of pDNA. Capture was performed using Sartobind^® Q, a strong anion exchange membrane adsorber, allowing selective binding of negatively charged pDNA through electrostatic interactions with quaternary ammonium ligands under controlled buffer conditions. The capture achieved high plasmid recovery (97%) while enabling substantial removal of RNA and other process-related impurities. Process parameters were optimized to balance dynamic binding capacity, product recovery, and impurity clearance while maintaining plasmid integrity. Polishing was conducted using Sartobind^® Phenyl, operated under cosmotropic conditions, enabling further impurity removal and improved separation of plasmid isoforms. This step resulted in enrichment of the supercoiled (sc) pDNA fraction relative to open circular (oc) and linear forms, while maintaining high recovery (94%). The integrated process achieved an overall pDNA purity of 98%, meeting typical regulatory expectations. Process applicability was demonstrated using a second plasmid construct of larger size, highlighting the flexibility of the platform while indicating that optimization may be required for different plasmid formats. Overall, the combination of anion exchange capture and hydrophobic interaction polishing provides a robust, scale-up potential and high-throughput membrane chromatography-based platform to produce high-quality pDNA suitable for advanced therapeutic manufacturing.