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Chemical Identity and Mechanism of Action and Formation of a Cell Growth Inhibitory Compound from Polycarbonate Flasks

Peng, Jinlin, Zhao, Yaopeng, Hong, Yulong, Burkhalter, Robert S., Hogue, Carrie L., Tran, Elizabeth, Wei, Lai, Romeo, Lori, Dolley-Sonneville, Paula, Melkoumian, Zara, Liang, Xinmiao, Fang, Ye
Analytical chemistry 2018 v.90 no.7 pp. 4603-4610
agonists, biopharmaceuticals, carbonates, cell culture, cell cycle, cell growth, cell proliferation, culture flasks, free radicals, growth retardation, liquid chromatography, mass spectrometry, mechanism of action, nitric oxide, nuclear magnetic resonance spectroscopy
This paper reports the chemical identity and mechanism of action and formation of a cell growth inhibitory compound leached from some single-use Erlenmeyer polycarbonate shaker flasks under routine cell culture conditions. Single-use cell culture vessels have been increasingly used for the production of biopharmaceuticals; however, they often suffer from issues associated with leachables that may interfere with cell growth and protein stability. Here, high-performance liquid-chromatography preparations and cell proliferation assays led to identification of a compound from the water extracts of some polycarbonate flasks, which exhibited subline- and seeding density-dependent growth inhibition of CHO cells in suspension culture. Mass spectroscopy, nuclear magnetic resonance spectroscopy, and chemical synthesis confirmed that this compound is 3,5-dinitro-bisphenol A. Cell cycle analysis suggests that 3,5-dinitro-bisphenol A arrests CHO-S cells at the G₁/Gₒ phase. Dynamic mass redistribution assays showed that 3,5-dinitro-bisphenol A is a weak GPR35 agonist. Analysis of the flask manufacturing process suggests that 3,5-dinitro-bisphenol A is formed via the combination of molding process with γ-sterilization. This is the first report of a cell culture/assay interfering leachable compound that is formed through γ-irradiation-mediated nitric oxide free radical reaction.