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Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Published on: April 16, 2014
Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO
Haimanti Dorai1, Dawn Ellis, Yun Seung Keung
1Pharmaceutical Development, Centocor R&D, Radnor, PA 19087, USA. hdorai@its.jnj.com
Abstract:
A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.
Insights
Over-expressing anti-apoptotic genes in Chinese Hamster Ovary cells enhances biotherapeutic production. This method rapidly identifies cell lines with improved viability and higher antibody yields, reducing waste products like lactate.
Area of Science:
- Biotechnology
- Cell Biology
- Genetic Engineering
Background:
- Chinese Hamster Ovary (CHO) cells are crucial for biotherapeutic manufacturing.
- Enhancing CHO cell robustness is key to improving production efficiency and product yield.
- Apoptosis (programmed cell death) limits cell longevity and productivity in CHO cultures.
Purpose of the Study:
- To develop superior CHO host cell lines for biomanufacturing by over-expressing anti-apoptotic genes.
- To utilize high-throughput screening of caspase activity for rapid selection of apoptosis-resistant cell lines.
- To evaluate the impact of anti-apoptotic gene expression on cell viability, antibody production, and metabolic by-products.
Main Methods:
- Over-expression of single or combined anti-apoptotic genes (e.g., Bcl-2Δ, Bcl-XL, E1B-19K, Aven, XIAPΔ) in CHO cells.
- High-throughput screening of caspase 3/7 activity to identify transfectants with reduced apoptosis.
- Assessment of integrated viable cell count (IVCC), transient antibody production, and stable clone selection.
- Bioreactor studies to compare product titer and lactate accumulation in engineered vs. control cell lines.
Main Results:
- Over-expression of anti-apoptotic genes significantly reduced caspase 3/7 activity.
- Maximal improvement in IVCC was achieved with single genes (Bcl-2Δ, Bcl-XL) or combinations (E1B-19K, Aven, XIAPΔ).
- Engineered cell lines showed higher transient antibody production and increased numbers of high-yield stable clones.
- A stable clone expressing Bcl-2Δ (BΔ31-1) achieved 180% higher product titer than the control, with significantly reduced lactate accumulation.
Conclusions:
- The high-throughput caspase screening method effectively identifies CHO host cell lines with enhanced anti-apoptotic characteristics.
- Over-expressing anti-apoptotic genes is a viable strategy to improve CHO cell line performance for biotherapeutic manufacturing.
- Engineered CHO cells exhibit improved viability, higher antibody yields, and favorable metabolic profiles, leading to more efficient bioprocesses.
