(191ak) Challenges in Single-Use Bioprocessing Systems: Evaluating the Cytotoxicity of a Leachate from Plastic Single-Use Bioreactors

The rapid expansion of single-use systems for bioprocessing is largely due to the ease of use and availability in pre-sterilized forms. While plastics used in biopharmaceutical production have gained acceptance for regular use, particularly in pilot-scale or experimental facilities, the additives used as stabilizers or to improve the processing of these plastics have a tendency to migrate or leach over time. One such additive is Irgafos 168®, or tris(2,4-di-tert-butylphenyl)phosphite, which is used as an antioxidant stabilizer. When undergoing radiation treatment during sterilization this is converted to bis(2,4-di-tertibutylphenyl)phosphate, or bDtBPP. bDtBPP has been found to leach from single-use bioprocessing bags into growth media, and our work has focused on determining its impact on cell growth for cell lines used in biopharmaceutical production. Specifically, we selected a Chinese Hamster Ovary cell line (CHO-K1), which was grown in both adherent and suspended form, to determine LC50 values of bDtBPP and other compounds used in plastics. Specifically for bDtBPP, the LC50 values were found to be higher for adherent cells (where albumin likely masks some of the toxic effects), with a drop in viable cell density observed at lower concentrations than cell viability (% live). This second observation indicates that the bDtBPP impacts metabolism and cell reproduction at low concentrations prior to cell death being prominent, so this may impact production rates of biopharmaceuticals. Our work seeks to establish a protocol for evaluating cytotoxicity of leachates, while at the same time gaining a better understanding the toxic behavior of plastic additives (and their degradants) when used in single-use bioprocessing systems.