INTRODUCTION
Lentiviral vectors are commonly used in the production of ex vivo cell therapies to transduce various cell types. Lentiviral vectors are sensitive to the shear forces of traditional continuous centrifugation systems. Additionally, significant yield losses due to adsorption effects can be seen during the primary clarification of lentiviral vectors with single-use filtration methods such as depth filtration. In this case study, we present data demonstrating the performance of the UniFuge® cell harvesting system for the clarification of lentiviral vectors. Key performance indicators including viral titer recovery and turbidity are presented for a range of conditions including batch size, cell viability, and feed rate.
THE CHALLENGE
Among the key challenges of supernatant clarification for downstream processing of lentiviral vectors are low titer recovery and loss of the vector’s activity. HEK293 cells are fragile and are easily damaged due to bioprocessing conditions such as shear stress. These damaged cells may release intracellular debris that could lead to inefficiencies and fouling in downstream processing. While some mild centrifugation techniques such as fluidized bed centrifugation reduce shear stress, they have slow processing times. The long processing times associated with fluidized bed centrifugation increase the risk of titer loss due to re-infection of the cells.
Filtration methods are also susceptible to titer loss as membranes may become easily fouled and lead to vector adsorption, elevated impurity, and slow processing time. As demand for cell therapies expands, there is a growing need for a mild centrifugation technique that maintains high titer recovery, minimizes turbidity, lower the filtration footprint, and streamlines processing time.
THE SOLUTION
The UniFuge Pilot offers a single-use, tubular bowl centrifugation solution that is ideal for shear-sensitive products such as HEK293 cells used to manufacture lentiviral vectors. The gentle operation recovers high viral titers. It also increases the economy of downstream processing for large-scale manufacturing by substantially reducing the turbidity of the supernatant and reducing the need for filtration.
THE RESULTS
In the first part of this case study, titer recovery was evaluated for a 35-liter batch of HEK293 cells that were processed at 2000 x g and a feed rate of 2 liters per minute. The UniFuge Pilot had high recovery that approached the theoretical maximum. Figure 1 compares the viral titers, showing only a 2.34% difference in viral titer recovery using the UniFuge Pilot compared with a benchtop centrifuge.
Next, turbidity was evaluated for 2 batches of HEK293 cells that were processed at variable feed rates at 3000 x g. Figure 2 shows the average clarification efficiency across the range of feed rates. Under all conditions, the clarification efficiency was high and compared well with the theoretical maximum.
DISCUSSION
The UniFuge Pilot’s low shear operation clarifies HEK293 cells to yield a low-turbidity supernatant with high viral titer. These performance results give bioprocessing experts confidence in the UniFuge Pilot as a low-shear clarification method for largescale, high-yield production of lentiviral vector that streamlines downstream purification.
REFERENCES
Moreira AS, Cavaco DG, Faria TQ, Alves PM, Carrondo MJT, Peixoto C. Advances in lentivirus purification. Biotechnol J. 2021;16(1):e2000019. doi:10.1002/biot.202000019
Perry C, Rayat ACME. Lentiviral vector bioprocessing. Viruses. 2021;13(2):268. doi:10.3390/v13020268
Saballus M, Kampmann M. Fluidized bed centrifugation of precipitated and flocculated cell cultures: An intensified clarification approach for monoclonal antibodies. J Biotechnol. 2022;352:16-25. doi:10.1016/j.jbiotec.2022.05.004
ABOUT THE UNIFUGE FAMILY
The UniFuge family provides low-shear separation, high-recovery performance, and fast processing times in three scalable models: UFMini, UniFuge Pilot, and U2k. With a variety of bowl sizes and flow rates ranging from 29 ml/min to 20 L/min, the UniFuge family of single-use separation systems offer both scalability and process efficiency in an aseptic closed system. The UniFuge Pilot was the industry pioneer in single-use centrifuge separation technologies. In 2021 CARR Biosystems released the UFMini to better support customers with a need for smaller scale processing using single-use technology.
ABOUT CARR BIOSYSTEMS
Founded in 1993 in Medfield, Massachusetts, CARR Centritech introduced the Powerfuge and continued to develop advanced separation systems for the Life Sciences and Specialty Chemical markets with the Viafuge, Centritech, and UniFuge models. In 2022, CARR Centritech transitioned to CARR Biosystems with continued emphasis on developing high-quality products to meet safety and quality standards throughout a variety of applications across the globe.
