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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.