Harvest of HEK Cells in Extracellular Expression Systems and for n-1 Seed Train Intensification

Abstract

In the production of viral vectors and proteins, cell harvest is a critical processing step. In this study, we evaluate the performance of CARR Biosystems’® UFMini single-use centrifuge in the clarification of Expi293F cells by measuring supernatant turbidity and viable cell recovery (VCR). The system was evaluated at feed flowrates of 165 mL/min, 330 mL/min, and 496 mL/min with a separation force of 3000g. The average supernatant turbidity result was 2.28 NTU, indicating that the vast majority of the Expi293F cells were collected in the cell concentrate with minimal cell breakthrough into the supernatant. Additionally, the average VCR result of 98% indicates that the UFMini successfully cleared cellular debris and exhibited low shear stress during centrifugation processing. The CARR Biosystem’s UFMini single-use centrifuge successfully purified and concentrated the Expi293F cell feed while exhibiting low shear stress with minimal impact to cell viability.

The results demonstrate that the UFMini can be utilized to optimize clarification processing efficiency by providing a user-friendly single-use centrifugation process that maintains cell viability and produces a highly clarified effluent that can help to reduce burden on downstream processes.

Background

The biopharmaceutical industry is rapidly growing due to the increasing demand for efficient and cost-effective manufacturing processes for complex proteins and therapeutics. To meet this demand, biopharmaceutical manufacturers are under pressure to develop innovative technologies that can maintain high product recovery while reducing manufacturing costs. Centrifuges capable of viable whole-cell harvest can be useful for n-1 seed train intensification and in reducing cellular debris and host cell proteins (HCPs) in processes where proteins or viral vectors are expressed outside the cell. Single-use centrifugation technology has emerged as a preferred choice due to its cost-effectiveness, efficiency, and sustainability, providing an alternative to traditional manufacturing processes.

Expi293F™ cells, a derivative of HEK293 cells, have been engineered to express the Epstein-Barr virus nuclear antigen 1 (EBNA-1), leading to higher protein expression levels and more efficient biopharmaceutical manufacturing. These cells are capable of growing to high densities and producing proteins with post-translational modifications similar to those found in human proteins. As a result, they have become a widely used tool in the production of complex proteins that require post-translational modifications.

The selection of the appropriate expression system is also a critical factor in the production of recombinant proteins or viral vectors used in gene therapy applications. Lentiviruses have gained attention due to their ability to transduce a wide range of mammalian cells, including non-dividing cells. The Thermo Fisher Scientific Expi293F cell line has emerged as a widely used tool in the industry for lentiviral vector production. The use of efficient expression systems coupled with effective clarification methods is crucial for the successful production of high-quality recombinant proteins or viral vectors.

In addition to lentiviruses, adeno-associated virus (AAV) has gained significant attention in gene therapy applications due to its low immunogenicity and ability to efficiently transduce both dividing and non-dividing cells. AAV vectors have been successfully used to treat a variety of genetic disorders, including hemophilia and inherited retinal diseases. However, the production of AAV vectors remains challenging, with many current manufacturing processes relying on large-scale, laborintensive methods that are expensive and time-consuming. To overcome these challenges, researchers are exploring new technologies and processes, including single-use centrifugation technology, to increase the scalability and efficiency of AAV vector production.

In this study, we evaluated the performance of the CARR Biosystems UFMini single-use centrifuge for the clarification of Expi293F cells, with the goal of maintaining cell viability and producing high product recovery.

Materials

  • UniFuge® Mini (UFMini)
  • 1 UFMini Single-Use Module
  • 900 mL of [1x] [.0067M] Phosphate Buffered Saline (PBS)
  • Hach 2100Q Turbidity meter
  • Vi-Cell Blu Analyzer
  • 3 L of Expi293F cells

Methods

The study comprised three experimental runs, labeled HEK1, HEK2, and HEK3, in which 1 L of Expi293F cells was processed through the UFMini under specific experimental conditions. The harvest and clarification performance was evaluated through measurements of cell viability and supernatant turbidity using the Beckman Coulter Vi-CELL BLU analyzer and Hach2100Q Turbidity meter.

  1. Expi293F cells were grown to a cell density of 5.6 x 106 cells/mL using Albany’s Center for Biopharmaceutical Education and Training (CBET) facilities, materials, and instrumentation.
  2. The Expi293F cells were diluted with [1x] [.0067M] PBS to a final volume of 3 L and separated into 3 separate 1 L containers of Expi293F cells.
  3. 200 µL samples in triplicate were collected from the 1 L container of Expi293F cells.
  4. The CARR Biosystems UFMini was prepared for processing by installing the UFMini single-use module and tube-set. The “HEK1” experimental run conditions (Reference Table 1) were programmed into the UFMini.
  5. The UFMini bowl was prefilled with 270 mL [1x] [.0067M] PBS.
  6. 1 L of Expi293F cells were fed and centrifuged through the UFMini at 165 mL/min and 3000g, and a 30 mL sample was collected directly from the supernatant stream at the end of the centrifugation run*.
  7. 300 mL of Expi293F cell concentrate was discharged from the module.
  8. Data was collected by processing the Expi293F cell feed, cell concentrate, and supernatant samples through the Beckman Vi-CELL BLU analyzer and Hach2100Q Turbidity meter. Results were recorded and analyzed.
  9. Steps 3-8 were repeated twice, instead using the “HEK2” and “HEK3” run conditions for step 4.b.

 

 

 

 

 

 

Results

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The turbidity measurements for the Expi293F cell feed fractions ranged from 186-197 NTU, indicating a moderate level of particulate matter. The concentrate turbidity measurements ranged from 880-943 NTU, indicating a high degree of opacity due to the increased concentration of cells and particulate matter. On the contrary, the supernatant showed much lower turbidity values ranging from 2.01-2.38 NTU, indicating a high degree of transparency and a low concentration of cells and particulate matter. The Expi293F cell concentrate fractions were observed to be opaque, while the supernatant fractions were transparent, supporting the quantitative results measured using the Hach2100Q turbidity meter.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The VCR analysis provided valuable insight in the health and robustness of the HEK cell cultures. The runs HEK1, HEK2, and HEK3 had VCRs of 90, 106, and 98, respectively, with an average of 98%, indicating a successful harvesting and concentration of the Expi293F cells. The cell densities and viabilities of all three HEK cultures were similar, indicating appropriate culture conditions with high levels of viability and growth throughout the culture process.

 

 

 

 

 

 

 

Conclusions

For HEK1, HEK2, and HEK3, the supernatant turbidity readings averaged at 2.15 NTU with individual readings of 2.38 NTU, 2.06 NTU, and 2.01 NTU, respectively. After conducting postprocessing qualitative observation, the results revealed that the Expi293F cell concentrate had opacity, while the Expi293F supernatant was transparent. Additionally, no waste was detected in the UFMini Single-Use module, suggesting that the UFMini process efficiently separated the Expi293F cells with minimal cell breakthrough into the supernatant.

The VCR results were also collected for each of the three Expi293F centrifugation runs, which produced values of 90%, 106%, and 98% for HEK1, HEK2, and HEK3, respectively. The VCR average across all three runs was 98%. These results indicated that the UFMini cleared cellular debris and non-viable cells while recovering the vast majority of the cells in the concentrate stream with minimal shear stress during processing. Hence, the process of harvesting and concentrating the cells from the culture was successful, with a high percentage of viable cells recovered.

In addition, cell density and viability were consistent across all three HEK cultures, demonstrating similar values. Overall, the results of this experiment suggest that the CARR Biosystem’s UFMini Single-Use Centrifuge provides an efficient and successful clarification process with high recovery and minimal impact on cell viability.

Acknowledgments

The CARR Biosystems team is appreciative of the Albany CBET team’s support throughout completion of the experiment.

About the UniFuge Family

The UniFuge single-use 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.

The UFMini is the smallest scale single-use centrifuge in CARR Biosystems’ UniFuge family. The primary applications for the UFMini are in the harvest and washing of cells for use in cell therapy, cellular agriculture, and cell banking — as well as the clarification of cell cultures during production of Monoclonal Antibodies (mAb) and other therapeutic protein products. The UFMini single-use module provides a closed system for sterile processing and eliminates cross-contamination and the need for CIP and SIP cycles, while reducing change-over time between centrifugation runs. Furthermore, the UFMini, which fits on a standard-size laboratory bench, can easily scale up to the larger Unifuge Pilot, or to the even larger manufacturing scale U2k.

Fill out the form to download.

Contact Us

Stay Connected

Contact Us

Get in touch with a sales representative, request support or download our free white paper.

Contact Us