Scientific Publications by FDA Staff

Appl Microbiol Biotechnol 2020 Feb;104(3):1097-108

Zinc supplementation improves the harvest purity of beta-glucuronidase from CHO cell culture by suppressing apoptosis.

Graham RJ, Ketcham S, Mohammad A, Bandaranayake BMB, Cao T, Ghosh B, Weaver J, Yoon S, Faustino PJ, Ashraf M, Cruz CN, Madhavarao CN

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-muM concentrations to determine the impact on the production and quality of beta-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, beta-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 beta-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 muM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of beta-glucuronidase from zinc-supplemented bioreactors.