(498d) Zinc Supplementation Improves the Harvest Purity of ?-Glucuronidase in CHO Cell Culture By Suppressing Apoptosis

The variability of trace metals in cell culture media is a potential manufacturing concern because it can affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production. To better understand the influences of excess supplementation, zinc and copper were initially supplemented at 20 times the control medium concentration in parallel bioreactors to determine the impact on the production and quality of β-Glucuronidase, a lysosomal enzyme. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was also tested to induce a depletion of trace metals in bioreactor culture medium to examine deficiency (n=2, each treatment, and no-supplementation as control). Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, exhibiting a higher viability compared to both the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the cell harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with varying concentrations of zinc, up to 120 times the level of the base medium concentration. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-Glucuronidase from zinc supplemented bioreactors. In summary, a threshold zinc concentration seems necessary to increase the longevity of CHO cells and it can be a significant factor to maintain cell homeostasis in the longer duration bioreactor operations with perfusion.