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Simultaneous targeting of Requiem & Alg-2 in Chinese hamster ovary cells for improved recombinant protein production
Yiping Lim1, Athanasios Mantalaris, Miranda G S Yap
1Department of Chemical Engineering and Chemical Technology, Imperial College London, London, SW7 2AZ, UK.
Abstract:
Apoptosis is known to be the main cause of cell death in the bioreactor environment, leading to the loss of recombinant protein productivity. In a previous study, transcriptional profiling was used to identify and target four early apoptosis-signaling genes: FADD, FAIM, Alg-2, and Requiem. The resulting cell lines had increased viable cell numbers and extended culture viability, which translated to increased protein productivity. Combinatorial targeting of two genes simultaneously has previously been shown to be more effective than targeting one gene alone. In this study, we sought to determine if targeting Requiem and Alg-2 was more effective than targeting Requiem alone. We found that targeting Requiem and Alg-2 did not result in extended culture viability, but resulted in an increase in maximum viable cell numbers and cumulative IVCD under fed-batch conditions. This in turn led to an approximately 1.5-fold increase in recombinant protein productivity.
Insights
Targeting apoptosis genes Requiem and Alg-2 in bioreactors increased cell numbers and recombinant protein production by 1.5-fold, enhancing bioprocess efficiency.
Area of Science:
- Biotechnology
- Cell Biology
- Bioprocess Engineering
Background:
- Apoptosis (programmed cell death) reduces recombinant protein productivity in bioreactors.
- Previous work identified four key apoptosis-signaling genes (FADD, FAIM, Alg-2, Requiem) for targeting.
- Simultaneous gene targeting proved more effective than single-gene targeting.
Purpose of the Study:
- To investigate if combinatorial targeting of Requiem and Alg-2 enhances bioprocess outcomes compared to targeting Requiem alone.
- To evaluate the impact on viable cell numbers, culture viability, and recombinant protein productivity.
Main Methods:
- Utilized transcriptional profiling to identify apoptosis-related genes.
- Engineered cell lines with targeted gene modifications.
- Conducted fed-batch bioreactor cultures to assess cell growth and protein production.
Main Results:
- Targeting both Requiem and Alg-2 did not extend culture viability but increased maximum viable cell numbers.
- Cumulative integrated cell-viability (IVCD) improved under fed-batch conditions.
- Recombinant protein productivity saw an approximate 1.5-fold increase.
Conclusions:
- Combinatorial targeting of Requiem and Alg-2 enhances cell growth and productivity in bioreactors.
- This strategy offers a viable approach to improve recombinant protein yields.
- Further research can optimize gene combinations for maximal bioprocess efficiency.
