(307b) Block Co-Polymer P188 Contributes to Increased Viability and Harvest Duration for High Density Perfusion Cell Culture

Recently, demand for medicines more complicated than traditional monoclonal antibodies (mAbs) have been on the rise. Some Chinese hamster ovary (CHO) cells engineered to produce these complex molecules have a relatively low cell specific productivity compared to CHO cells engineered to produce mAbs. Therefore, to meet the demand for these molecules, perfusion cell culture has been implemented to achieve high cell densities and more productivity with a small footprint. Even with perfusion supplying fresh nutrients and removing waste, cells in culture become stressed over time and experience a decrease in viability. Accumulation of dead cells in perfusion culture can be problematic as it can lead to filter fouling, ultimately resulting in either increased costs due to filter replacement or termination of the run. Cell viability can be increased by increasing the perfusion rate, but at the expense of additional media costs or with added facility fit constraints. Therefore, it is beneficial to increase cell survival without increasing perfusion rate.

Poloxamer 188 (P188), also called pluronic F-68, is a relatively inexpensive media component that has been traditionally added to media to protect cells from interactions with sparging bubbles.^1,2 While P188 addition adds significant benefit compared to media without it, decrease in cell-bubble interactions becomes limited for P188 concentrations greater than 1g/L.¹ However, cells cultured in media with higher levels of P188 than that developed for fed-batch processes at AstraZeneca showed an increase in specific productivity (q_p), viability and a significant decrease in host cell protein in high-density perfusion cell culture.

One explanation for these findings is that there are more cells per volume and more bubbles due to increased oxygen demand, increasing the probability for cell-bubble interaction. However, mechanisms other than protection from sparging, are demonstrated in the literature albeit not all in the context of bioprocessing. These include P188 sealing of the plasma and mitochondrial outer membranes.³ Cells under stress can undergo apoptosis or necrosis which involve a loss of membrane integrity.⁴ The membrane sealing capability of P188 may allow cells to prevent membrane permeability leading to cell death.

Flow cytometric analysis revealed that culture with low P188 perfusion media had a higher percentage of necrotic cells and more cells with elevated mitochondrial superoxide than culture with high P188 media. In the literature, levels of mitochondrial reactive oxygen species (mROS) have been shown to increase following mitochondrial membrane permeabilization, subsequently triggering cell death pathways.⁵ This mechanism suggests that P188 internalized by cells during culture may seal an impaired mitochondrial membrane preventing necrosis, increasing cell viability during culture.

High density cell culture exposes cells lines developed for production of complex molecules to not only high amounts of sparging to meet oxygen demand, but also high levels of intracellular stress, increasing the risk of mitochondrial dysfunction. A high level of cell necrosis leads to a buildup of cell debris that results in filter fouling and early termination of the run as well as decreased productivity. Additional P188 in the perfusion media can extend the run and increase productivity without significantly increasing manufacturing cost.

1. Ma et. Al., 2004, “Quantitative studies of cell-bubble interactions and cell damage at different pluronic F-68 and cell concentrations”, Biotechnology Progress, Vol. 20, No. 4, pp. 1183-91.
2. Tharmalingam, T., Ghebeh, H., Wuerz, T., Butler, 2008, “Pluronic enhances the robustness and reduces the cell attachment of mammalian cells”, Molecular biotechnology, Vol. 39, No. 2, pp. 167-77.
3. Wang et. Al., 2017, “Mitochondrial mechanisms of neuronal rescue by F-68, a hydrophilic Pluronic block co-polymer, following acute substrate deprivation”, Neurochemistry International, Vol. 109, p. 126-140
4. Zhang, Y., Chen, X., Gueydan, C., Han, J., 2018, “Plasma membrane changes during programmed cell deaths”, Cell Research, Vol. 28, pp. 9-21.
5. Dussmann, H., Kogel, D., Rehm, M., Prehn, J., 2003, “Mitochondrial membrane permeabilization and superoxide production during apoptosis”, Journal of Biological Chemistry, Vol. 278, No. 15, pp. 12645-12649.