Downstream Workflows for Intermediate Purification of Lentiviral Vectors Using Tangential Flow Filtration and AEX Membrane Chromatography.

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.