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

Abstract

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 G/G 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.

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer

“…If the assay for residual host cell DNA is designed to only test enough material to be able to meet the specification, it is possible that information about process variability contained in the sample matrix might be lost. Finally, this work provides another example demonstrating that there are many raw materials used in the production and purification of protein therapeutics, any of which could serve as the source of an unknown impurity 19–21 or leachable compound 22,23 that may impact process performance or product quality. Even the most robust raw material control strategies may not be able to catch such unexpected issues, highlighting the need for robust complementary quality systems for control and monitoring of product quality and investigation and response to deviations.…”

Suspect and non-target screening of chemicals in household cleaning products, and their toxicity assessment

“…Oleamide, is one of the surfactant, has a variety of industrial uses including as a slip agent, a lubricant, and a corrosion inhibitor [80]. Oleamide is known to be leached out of polypropylene plastics [81]. It is reported that, in rats, the oral LD50 of undiluted oleamide DEA was 12.4 mL/kg [82].…”

Transformation of Biopharmaceutical Manufacturing Through Single-Use Technologies: Current State, Remaining Challenges, and Future Development

“…The second compound found to inhibit cell growth is 3,5-dinitro-bisphenol A (BPA), first reported in 2018 from polycarbonate shake flasks (111). Using water-based extraction studies, the authors showed that some polycarbonate flasks exhibit seeding density-dependent growth inhibition of CHO cells, with different CHO strains demonstrating varying sensitivities.…”