Improved Lentiviral Vector Biomanufacturing for Cell and Gene Therapy Applications

The aim of this NIIMBL-funded project is to provide the industry with a lentivirus production process that yields large quantities of highly potent vector with fast facility turnaround times.

Industry Need

Current production processes, in which host mammalian cells are used to generate

lentivirus, are inefficient, resulting in low yields, lack of scalability, and high biomanufacturing costs. With clinical trials and FDA approval of T-cell immunotherapy, efficient lentiviral production processes appropriate for large scale manufacturing are needed.

Approach

The aim of the NIIMBL-funded project was to provide the industry with a lentivirus production process that yields large quantities of highly potent vector with fast facility turnaround times.

Impacts

Development of a platform process to mitigate lentiviral vector supply shortages for cell and gene therapy

Improved viral vector production process yield, processing times, and measurement accuracy

Value Statement/Outcomes

By implementing high-throughput LVV process development—including microscale plasmid transfection screening, scalable HEK293 suspension culture, and integrated upstream/downstream workflows—an organization will reduce development costs by minimizing experimental burden and optimizing DNA and plasmid ratios. This approach will accelerate timelines for CAR-T and gene therapy vector production through predictive scale-up and rapid purification strategies and will ensure consistent product quality and regulatory compliance via robust analytical methods and optimized chromatography processes—all while enabling scalability from microscale to bioreactor production.

Outputs/Deliverables

Upstream Outcomes/Highlights • Developed a screening platform using 96-deep well plates for high density HEK293 suspension cell growth of up to 4 x 107 cells/mL. • Identified optimal DNA content, DNA:PEI ratio, cell density and four plasmid ratio for lentiviral production. • Showed that the results obtained on the microscale platform were scalable to shake flasks and bioreactors. • Performed screening studies to test the impact of nutrient supplements on LVV production in a fed-batch process. • Established processes for high cell density perfusion culture of HEK293 cells using a novel perfusion device

Downstream Outcomes/Highlights • Developed a high throughput screening method to evaluate the stability of lentiviral vectors in mobile phase conditions across a broad range of pH and salt concentration to inform selection of purification process conditions. • Evaluated chromatographic media including resins, membranes and monoliths for lentiviral vector purification potential. • Performed bench scale process development and developed a two-column purification process for lentiviral vectors. • Developed a novel two stage flow through chromatography process that can be utilized to purify lentiviral vectors • A comprehensive suite of analytical methodologies were developed

Developed a novel two stage flow through chromatography process that can be utilized to purify lentiviral vectors

Publications

Ghosh, R., Koley, S., Gopal, S., Rodrigues, A. L., Dordick, J. S., & Cramer, S. M. (2022). Evaluation of lentiviral vector stability and development of ion exchange purification processes. Biotechnology Progress, 38(6). https://doi.org/10.1002/btpr.3286

Gopal, S., Osborne, A. E., Hock, L., Zemianek, J., Fang, K., Gee, G., Ghosh, R., McNally, D., Cramer, S. M., & Dordick, J. S. (2021). Advancing a rapid, high throughput screening platform for optimization of lentivirus production. Biotechnology Journal, 16(10), 2000621. https://doi.org/10.1002/biot.202000621

Valkama, A., Oruetxebarria, I., Lipponen, E., Leinonen. H., Kayhu, P., Hynynen, H., Turkki, V., Malinen, J., Miinalainen, T., Heijura, T, Parker, N., Yla-Herttuala, S, & Lesch, H. (2020). Development of Large-Scale Downstream Processing for Lentiviral Vectors. Molecular Therapy, doi: 10.1016/j.omtm.2020.03.025.

Posters

Cattaneo, M. & Spanjaard, R., Process Intensification using the VHU(TM) Perfusion System for increased Lentiviral Vector Yield, NIIMBL National Meeting, Washington, DC, June 27, 2019.

Hock, L., Zemianek, J., McNally, D., & Ganesa, C., Development of a Perfusion Bioreactor Platform for High-Density Suspension Hek293 Cells, ASGCT 2020, Virtual, May 12, 2020.

Or, E., DiSpirito, C., Ganesa, C., Fang, K., Gee, G., & McNally, D., Development of a Scalable Downstream Process for Lentiviral Vector Production, ASGCT 2020, Virtual, May 12, 2020.

Presentations

Cattaneo, M., A Viral Harvest Unit (VHU™) for the Continuous Harvesting of Lentivirus from Suspension Cell Cultures, Integrated Continuous Biomanufacturing IV, Cape Cod, MA, October 6, 2019.

Cattaneo, M., Continuous Perfusion for Scaling Up Lentiviral Vector Production using Transient Transfection and Stable Producer HEK293 Cell Lines, Advanced Therapeutic Medicinal Products BioMAN-MIT Summit, Cambridge, MA, December 11, 2019.

Ghosh, R., Koley, S., Gopal, S., Dordick, J. S., & Cramer, S., Development of Downstream Purification Process for Lentiviral Gene Therapy Vector, Purification of Gene Therapy Products Symposium, Boston, MA, October 2, 2019.

Ghosh, R., Koley, S., Gopal, S., Dordick, J.S., Cramer, S.M., Presenter, Development of Downstream Purification Process for Lentiviral Vector. March 20, 2019.

Gopal, S., Ghosh, R., Osborne, A.E., Hock, L., Ramchetty, A., Poirier, M., Uddin, T., Zemianek, J., Fang, K., Gee, G., Ozturk, S., Ganesa, C., McNally, D., Cramer, S.M., Dordick, J.S., Scaled Down Optimization of Transfection for Lentivirus Production for Use as Gene Therapy Vectors, ASGCT Conference, Virtual, May 12, 2020.

Gopal,S., Ghosh, R., Osbourne, A.E., Hock,L., Ramchetty, A. Poirier,M., Uddin, T., Zemianek, J., Fang, K., Gee, G, Ozturk, S., Ganesa, C., McNally, D., Cramer, S.M., Dorick, J.S., Scaled Down Optimization of Transfection for Lentivirus Production for Use as Gene Therapy Vectors, ACS Conference Virtual, August 16, 2020.

Improved Lentiviral Vector Biomanufacturing for Cell and Gene Therapy Applications (PC1.0-012), NIIMBL Member Forum, Virtual, March 25, 2021.