PUB - Publikationen an der Universität Bielefeld

The application of Single-Use (SU) bioprocessing technology in the biopharmaceutical market is fully established due to their manifold advantages. Therefore, the demand for biocompatible SU materials is increasing to ensure consistent cell growth and patient safety. Biocompatibility must be demonstrated according to USP <87> and ISO 10993-5, respectively, before marketing. However, the standards were originally developed for medical devices and thus, prescribed test conditions do not represent cell culture processes in the biopharmaceutical industry. Consequently, the American Society for Testing and Materials (ASTM) published a standard guide for cell growth assessment of SU material (designation E3231-19), representing current best practices for biocompatibility testing of bioprocessing materials [1]. This thesis aimed at refining a CHO-based assay for biocompatibility assessment based on the ASTM guideline E3231-19 using an industrial CHO DG44 cell line. Hence, eligibility of alternative cell lines for biocompatibility testing was evaluated and the effects of leachables on these cell lines were investigated. The well-known leachable bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP), a degradation product of the phosphite antioxidant Irgafos®168, served as a model compound for the different approaches throughout this study. In the first part, degradation pathways from phosphite antioxidants upon gamma irradiation were assessed. Two novel degradation products could be identified having the same structure motif as bDtBPP. Subsequent toxicity testing with CHO and HEK293T cell lines indicated a structure-activity-relationship probably mediated by the alkyl chains of the substances. In the second part of this thesis the effects of a leachable mixture on a CHO antibody production process were assessed. Even though the mixture caused a cell growth inhibition, cell-specific productivity was not affected. Additionally, an interaction study indicates that toxicity of one leachable can be mitigated by the presence of another one. In the third part the impact of leachables on a CAR-T manufacturing process was investigated. Combination of biocompatibility studies of SU tubing with toxicity studies of leachables indicate a negligible effect of bDtBPP on lentivirus production, while extracts of tubing materials caused growth inhibition in either HEK293T or autologous T-cells. In the final part of this thesis the affinity of CHO and T-cells to different leachables was assessed. Results of biosorption experiments indicate an increased affinity towards hydrophobic leachables and the highest adsorption coefficient was determined for bDtBPP for both cell types.