Establishing A Versatile Platform For Intensified Lentiviral Vector Manufacturing

By Chris Brown, Yeonji Kim, Charles Hill, Keen Chung, Jing Zhu, and Xiaojun Liu

Lentiviral vectors (LVVs) have become important tools in cell and gene therapy, valued for their ability to efficiently modify both dividing and non-dividing cell types. This, combined with their ability to integrate into the host genome for long-term expression, have made LVVs increasingly attractive for treating a wide range of diseases. However, current LVV production methods face significant challenges, including low titers, limited scalability, and high manufacturing costs – leading to a high cost of goods (COGs) and limiting adoption of these vectors. Through this work, we aim to develop a flexible, intensified LVV manufacturing process by systematically evaluating key process parameters and implementing effective process intensification strategies such as TFDF perfusion to improve LVV production performance.

In this work, we first evaluated LVV production via transient transfection in suspension at a small scale to develop a baseline production platform. This entailed the screening of several commercial LVV production systems, and the selection of the system which performed best on the basis of LVV functional titer and quality. We further refined the packaging plasmid system used, and transfection conditions were optimized to increase LVV productivity, resulting in a 10-fold increase in functional titer. This optimized process was then successfully scaled to 2L benchtop bioreactors, maintaining comparably high productivity to the small-scale runs, with titers exceeding 2.0x108 transduction units per milliliter (TU/mL).

Further, process intensification was achieved through the implementation of Repligen’s Tangential Flow Depth Filtration (TFDF) technology. TFDF combines both tangential flow and depth filtration principles through the use of a tubular depth filter with a 2-5 µm pore size operated in tangential flow mode. Use of this system resulted in a greater than 3-fold increase in cell density at time of transfection, increasing from 4.0x106 cells/mL in our batch process to over 13.0x106 cells/mL in perfusion while maintaining high cell viability. TFDF operation also enabled the continuous harvest of LVV for the duration of the study while retaining cells and cell debris within the bioreactor volume. Overall, this intensified process resulted in a significant increase in yield compared to our non-intensified process, with total titers harvested from the 2L perfusion bioreactor exceeding 1.9x1012 TU. Space yield in the bioreactor increased more than 4-fold from 2.0x108 TU/mLBR in batch operation to nearly 9.0x108 TU/mLBR in TFDF-mode operation, indicating an increase in cell-specific LVV production. This intensified process demonstrates significant potential to improve access to high-quality lentiviral vectors, allowing for equivalent LVV production at smaller scales and potentially obviating the need to scale beyond 200L, thereby reducing overall manufacturing costs and time. Future work will focus on further optimization of the TFDF perfusion process and its integration with downstream purification to develop a fully continuous LVV manufacturing platform.