Out of the plethora of chemical species extractable at low levels from the materials of construction of single-use bioprocess containers, we have identified one particularly conspicuous compound and shown it to be highly detrimental to cell growth. The compound, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), is derived from the breakdown of tris(2,4-di-tert-butylphenyl)phosphite (trade name Irgafos 168®), a common antioxidant additive present in many formulations of polyethylene (one of the polymers commonly used as the material contacting process fluids in bioprocess containers). Cell growth experiments using several mammalian cell lines and growth media spiked with bDtBPP show harmful effects at concentrations well below the parts-per-million range. Cellular response to bDtBPP is rapid, and results in a significant decrease in mitochondrial membrane potential. The migration of bDtBPP from polyethylene-based films is shown to be time- and temperature-dependent. Further, experiments suggest that exposure of oxidized Irgafos 168 to ionizing radiation (such as gamma irradiation) is an important condition for the generation of significant amounts of leachable bDtBPP. LAY ABSTRACT: Biopharmaceuticals are drugs manufactured using cells that are genetically engineered to produce a therapeutic protein. A current trend in biomanufacturing is the replacement of hard-plumbed stainless steel vessels (where these cells are grown) with specialized, pre-sterilized, disposable plastic bags. While this move has significant environmental and cost benefits, the effect of plastics on the biomanufacturing process is not yet completely understood. Here we show that if a chemical compound formed by the breakdown of a common antioxidant additive to plastics leaches into the cell culture liquid, the growth of mammalian cells is strongly inhibited. Some of the factors that promote the generation of this compound, and the conditions that favor migration of the compound into process fluids, are explored here.

中文翻译：

---

一次性生物处理容器中对细胞生长有害的可浸出化合物的鉴定

在从一次性生物处理容器的构造材料中可以提取的少量化学物质中，我们鉴定出一种特别引人注目的化合物，并显示出它对细胞生长非常有害。化合物双（2,4-二叔丁基苯基）磷酸酯（bDtBPP）是由三（2,4-二叔丁基苯基）亚磷酸酯（商品名Irgafos168®）分解而得的存在于许多聚乙烯配方中的添加剂（通常是一种与生物工艺容器中的工艺流体接触的材料）。使用几种哺乳动物细胞系和掺有bDtBPP的生长培养基进行的细胞生长实验表明，浓度远低于百万分之几时，有害作用。细胞对bDtBPP的反应很快，并导致线粒体膜电位显着降低。bDtBPP从聚乙烯基薄膜的迁移显示出与时间和温度有关。此外，实验表明，氧化的Irgafos 168暴露于电离辐射（例如γ辐射）是产生大量可浸出的bDtBPP的重要条件。摘要：生物药物是使用经过基因改造以产生治疗性蛋白质的细胞制造的药物。生物制造的当前趋势是用专门的，预先消毒的一次性塑料袋代替硬管不锈钢容器（这些细胞在其中生长）。尽管此举具有明显的环境和成本优势，但塑料对生物制造过程的影响尚未完全了解。在这里，我们表明，如果通过将常见的抗氧化剂添加到塑料中而分解而形成的化合物浸出到细胞培养液中，则哺乳动物细胞的生长会受到强烈抑制。在此探讨了一些促进该化合物生成的因素，以及有利于该化合物向工艺流体中迁移的条件。