Engineering death resistance in CHO cells for improved perfusion culture

MacDonald, Michael A.; Nöbel, Matthias; Martı́nez, Verónica; Baker, Kym; Shave, Evan; Gray, Peter P.; Mahler, Stephen M.; Munro, Trent P.; Nielsen, Lars K.; Marcellin, Esteban

doi:10.1080/19420862.2022.2083465

Cited by 9 publications

(17 citation statements)

References 52 publications

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“…Key process parameters such as cell diameter, lactate and ammonia concentrations and osmolarity, followed the trends previously observed. (MacDonald, Nöbel, Martínez, et al, 2022). At the time of growth arrest, ammonia concentrations were 2.39 mM and 3.24 mM for replicate A and B, respectively and lactate concentrations were <2 mM for both cultures (Supporting Information Material S1: Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…The same parental cell line described previously served as a control (MacDonald, Nöbel, Martínez, et al, 2022). The control did not show any growth arrest and was controlled by a bleed set to maintain a constant VCD between Days 11 and 17.…”

Section: Resultsmentioning

confidence: 99%

“…In our preceding research, we investigated the performance of an apoptosis resistant CHO cell line, deficient of the BAK1, BAX, and BOK genes (MacDonald, Nöbel, Martínez, et al, 2022). Notably, we observed a growth arrest that was not induced by external factors.…”

Section: Introductionmentioning

confidence: 99%

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Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation

Nöbel,

Barry,

MacDonald

et al. 2023

Biotech & Bioengineering

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Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed‐batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase‐dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation

Nöbel,

Barry,

MacDonald

et al. 2023

Biotech & Bioengineering

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“…It has been reported that for the production of unstable therapeutic proteins, such as recombinant enzymes and blood coagulation factors, the perfusion process possesses more advantages (Bettinardi et al, 2020;MacDonald et al, 2021). In the perfusion mode, culture broth contained products and waste is perfused through the bioreactor under the perfusion controller, while the cells/products are retained or recycled back into the bioreactor (Ahn et al, 2008;MacDonald et al, 2021;MacDonald et al, 2022). Consequently, a consistent and steady culture condition with metabolized by-product rapidly removal results in a stable and uniform product of high quality under perfusion culture process (Schwarz et al, 2020;Yin et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Enhancing and stabilizing monoclonal antibody production by Chinese hamster ovary (CHO) cells with optimized perfusion culture strategies

Liang

Luo

2023

Front. Bioeng. Biotechnol.

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The perfusion medium is critical in maintaining high cell concentration in cultures for the production of monoclonal antibody by Chinese hamster ovary cells. In this study, the effects of perfusion culture strategies when using different media on the process stability, product titer, and product quality were investigated in 3-L bioreactor. The results indicated that continuous perfusion could maintain higher levels of cell density, product titer, and quality in comparison with those of the intermittent perfusion culture. Next, the perfusion culture conditions with different perfusion rates and temperature reduction methods were further optimized. When combining the high perfusion rates and delayed reduction of culture temperature at day 6, the product titer reached a higher level of 16.19 g/L with the monomer relative abundant of 97.6%. In this case, the main peak of the product reached 56.3% and the total N-glycans ratio was 95.2%. To verify the effectiveness of the optimized perfusion culture in a larger scale, a 200-L bioreactor was used to perform and the final product titer reached the highest level of 16.79 g/L at day 16. Meanwhile, the product quality (monomer abundant of 97.6%, main peak of 56.3%, and N-glycans ratio of 96.5%) could also be well maintained. This study provided some guidance for the high-efficient production of monoclonal antibody by CHO cells via optimized perfusion culture strategy.

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“…This strategy was shown to work for fed-batch processes 191,192 and would thus be an interesting follow-up study to progress with the intensification strategy proposed in this thesis. Also, for continuous perfusion process, apoptosis resistance via Bax and Bak knock-out adds value, as demonstrated by MacDonald et al 193 . Not only was the cell longevity extended, while maintaining productivity and quality, but also the needed cell bleed rate of the perfusion process was lower as cell growth was reduced and thus product losses can be avoided.…”

Section: An Ideal Cell Line For Intensified Processes Shouldmentioning

confidence: 91%

Process intensification for CHO cell biomanufacturing

Schulze

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Current CHO cell production processes require an optimized space-time-yield. Process intensification can support achieving this by enhancing the productivity and improving facility utilization. The use of perfusion at the last stage of the seed train (N-1) for high cell density inoculation of the fed-batch N-stage production culture is a relatively new approach with few industry applicable examples. Within this work, the impact of the cell-specific perfusion rate (CSPR) of the N-1 perfusion and the relevance of its control for the quality of generated inoculation cells was evaluated using an automated perfusion rate (PR) control based on online biomass measurements. Precise correlations (R² = 0.99) between permittivity and viable cell counts were found up to the high densities of 100×10 6 c•mL -1 . Cells from N-1 perfusion were cultivated at a high and low CSPR with 50 and 20 pL•(c•d) -1 , respectively. Lowered cell growth and an increased apoptotic reaction was found as a consequence of the latter due to nutrient limitations and reduced uptake rates. Subsequently, batch cultivations (N-stage) from the different N-1 sources were inoculated to evaluate the physiological state of the inoculum. Successive responses resulting from the respective N-1 condition were uncovered. While cell growth and productivity of approaches inoculated from high CSPR and a conventional seed were comparable, low CSPR inoculation suffered significantly in terms of reduced initial cell growth and impaired viability. This study underlines the importance to determine the CSPR for the design and implementation of an N-1 perfusion process in order to achieve the desired performance at the crucial production stage.

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Engineering death resistance in CHO cells for improved perfusion culture

Cited by 9 publications

References 52 publications

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation

Enhancing and stabilizing monoclonal antibody production by Chinese hamster ovary (CHO) cells with optimized perfusion culture strategies

Process intensification for CHO cell biomanufacturing

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