Targeting early apoptotic genes in batch and fed-batch CHO cell cultures
Danny Chee Furng Wong1, Kathy Tin Kam Wong, Peter Morin Nissom
1Bioprocessing Technology Institute, Biomedical Sciences Institutes, Agency for Science, Technology and Research, A*STAR, 20 Biopolis Way, #06-01, Centros, Singapore 138668.
Abstract:
Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed. Compared to parental cells, the CHO GT cell lines showed apoptosis resistance as they significantly delayed and/or suppressed initiator caspase-8 and -9 and executioner caspase-3 activities during culture. FBC of CHO GT cell lines reached significantly higher maximum viable cell densities (up to 9 x 10(6) cells/mL) compared with the parental cell line (5 x 10(6) cells/mL). The recombinant interferon gamma (IFN-gamma) yields were increased by up to 2.5-fold. Furthermore, it was observed that the IFN-gamma was more highly sialylated.
Insights
Engineered Chinese hamster ovary (CHO) GT cell lines exhibit enhanced apoptosis resistance and improved recombinant protein production. These gene-targeted cell lines demonstrate higher viable cell densities and increased yields of interferon gamma (IFN-gamma).
Area of Science:
- Biotechnology
- Cell Biology
- Genetic Engineering
Background:
- Apoptosis, or programmed cell death, can limit productivity in mammalian cell cultures.
- Identifying and modulating key apoptosis-related genes is crucial for enhancing cell culture performance.
Purpose of the Study:
- To develop and characterize Chinese hamster ovary (CHO) Gene Targeted (GT) cell lines with improved apoptosis resistance.
- To evaluate the impact of gene targeting on cell growth, apoptosis suppression, and recombinant protein production.
Main Methods:
- Transcriptional profiling using microarray identified four key apoptosis signaling genes: Fadd, Faim, Alg-2, and Requiem.
- CHO GT cell lines were created: two overexpressing anti-apoptotic genes (Faim, Fadd DN) and two with knockdown of pro-apoptotic genes (Alg-2, Requiem) via small interfering RNA (siRNA).
- Comparative batch culture (BC) and fed-batch culture (FBC) analyses were performed against parental CHO cells.
Main Results:
- CHO GT cell lines demonstrated significant resistance to apoptosis, delaying or suppressing initiator (caspase-8, -9) and executioner (caspase-3) activities.
- Fed-batch cultures of CHO GT cell lines achieved substantially higher maximum viable cell densities (up to 9 x 10^6 cells/mL) compared to parental cells (5 x 10^6 cells/mL).
- Recombinant interferon gamma (IFN-gamma) yields increased up to 2.5-fold, with enhanced sialylation observed.
Conclusions:
- Targeting early apoptosis signaling genes effectively enhances apoptosis resistance in CHO cells.
- Engineered CHO GT cell lines offer a promising platform for significantly improving cell density and recombinant protein production.
- The enhanced CHO GT cell lines provide a robust system for high-yield manufacturing of biologics like IFN-gamma.
