Related Experiment Videos
Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures
B T Tey1, R P Singh, L Piredda
1Centre for Bioprocess Engineering, School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, UK.
Abstract:
The influence of Bcl-2 expression on the suppression of apoptosis during the cultivation of an NS0 cell line expressing a chimeric antibody was investigated. Following selection of transfectants in medium containing G418, Western analysis revealed evidence of some up-regulation of endogenous Bcl-2 expression even in the control vector transfectants. Cultivation of the two cell lines in suspension batch cultures clearly demonstrated the enhanced robustness of the bcl-2 vector transfected cells. Suppression of apoptosis resulted in an approximately 20% increase in maximum viable cell number, and a doubling in culture duration compared to the control transfected cells. However, despite the significant affect on viability, Bcl-2 expression did not result in an increase in final antibody titre in comparison with the control cell line. Exposure of cells to various nutrient limited conditions further emphasised the influence of Bcl-2 on cell survival. After 3 days of exposure to serum, glucose, glutamate and asparagine deprivation, the viable cell number and viability were significantly higher in the bcl-2 transfected cell line. When control cells were deprived of all amino acids, there was a complete loss of viability and viable cell number within 3 days. By contrast, the bcl-2 transfected cell line retained greater than 75% of the initial viable cell number and about 70% viability. In response to exposure to 8 mM thymidine (a cytostatic agent) the control cell line underwent complete loss of viability and viable cell number after 6 days. This compared with 18 days for complete loss of viability in the bcl-2 transfected cell line. As under batch culture conditions, there was no difference between the two cell lines in final antibody titre, which indicated that MAb synthesis is limited by nutrient availability during the latter stages of culture in both cases. When fed batch cultures were carried out using a concentrated essential amino acid feed, the bcl-2 cell line exhibited a 60% increase in maximum viable cell number and a 50% increase in culture duration, when compared to the control cell line. Moreover, the bcl-2 cell line exhibited a greater than 40% increase in maximum antibody titre.
Insights
Bcl-2 expression enhances NS0 cell robustness by suppressing apoptosis, increasing cell viability and culture duration. This leads to higher antibody titers, particularly in fed-batch cultures with essential amino acid feeding.
Area of Science:
- Biotechnology
- Cell Biology
- Bioprocessing
Background:
- Apoptosis, or programmed cell death, is a critical factor limiting cell viability in biopharmaceutical production.
- The B-cell lymphoma 2 (Bcl-2) protein family plays a key role in regulating apoptosis.
- Understanding how to modulate apoptosis can improve cell culture performance and therapeutic protein production.
Purpose of the Study:
- To investigate the impact of Bcl-2 expression on apoptosis suppression in NS0 cells during chimeric antibody production.
- To evaluate the effect of Bcl-2 on cell robustness, viability, and antibody production under various culture conditions.
Main Methods:
- NS0 cell lines were transfected with a vector expressing Bcl-2.
- Western analysis was used to confirm Bcl-2 expression.
- Cell cultures were performed under batch and fed-batch conditions, including nutrient-limited and cytostatic agent exposure.
- Viable cell number, viability, and antibody titer were measured.
Main Results:
- Bcl-2 expression significantly suppressed apoptosis, leading to a ~20% increase in maximum viable cell number and doubled culture duration in batch cultures.
- Bcl-2 transfected cells showed enhanced survival under nutrient deprivation (serum, glucose, amino acids) and cytostatic agent (thymidine) stress.
- While Bcl-2 did not increase antibody titer in standard batch cultures, fed-batch cultures with essential amino acid feed resulted in a >40% increase in maximum antibody titer.
Conclusions:
- Bcl-2 expression is a viable strategy to enhance NS0 cell robustness and improve culture performance.
- Modulating apoptosis via Bcl-2 can significantly increase cell viability and culture duration, leading to improved antibody production, especially under optimized feeding strategies.