Abstract
The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly 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 of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. 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, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 μM. 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.
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Acknowledgments
The authors thank Dr. Kristina Howard for help with the flow cytometry measurements and Alan Carlin for critical reading of the manuscript and useful suggestions on presentation style.
Funding
This study was intramurally funded by the Center for Drug Evaluation and Research, USFDA, for “Improved Understanding of Bioprocessing” and “Product Quality and Biopharmaceutics of Complex Dosage Forms”. RG, SAK, BG, BMBB, and TC received ORISE fellowships from CDER.
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RG, SAK, AM, BMBB, JW, and CNM performed the experiments. BG and TC assisted with bioprocessing and purification. RG, CNM, SAK, AM, JW, and BMBB analyzed the data. RG, CNM, SAK, BMBB, SY, MA, CNC, JW, and PFJ wrote the manuscript. RG, AM, and CNM conceptualized the work.
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Graham, R., Ketcham, S., Mohammad, A. et al. Zinc supplementation improves the harvest purity of β-glucuronidase from CHO cell culture by suppressing apoptosis. Appl Microbiol Biotechnol 104, 1097–1108 (2020). https://doi.org/10.1007/s00253-019-10296-1
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DOI: https://doi.org/10.1007/s00253-019-10296-1