Improving the reproducibility of mixing-time experiments in stirred single-use bioreactors.

Mixing time is a key metric for characterizing bioreactors, as it governs bulk homogenization and correlates with the underlying flow field and shear exposure. Having knowledge of turbulent flow regimes is particularly important for shear-sensitive (mammalian) cells, which are often cultivated in single-use systems. To characterize such systems in terms of their mixing-time behavior, colorimetric pH-shift assays can be employed. However, operator-dependent variability during the reagent addition often limits the comparability of such experiments. To address this limitation and enhance reproducibility, a compact linear actuator was developed that enables automation and, thus, standardization of the addition process. The device was designed using computer-aided design software, fabricated via 3D printing, and subsequently integrated into mixing-time experiments. It is compatible with both stirred single-use bioreactors and glass bioreactors. For automated experiments using the linear actuator, neither the mixing time nor the replicate variance increased in the single-use bioreactor relative to the glass bioreactor under non-aerated conditions, demonstrating strong comparability between the two systems. The use of the linear actuator increased measurement precision, as evidenced by a 13.6% reduction in the coefficient of variation. This improvement resulted from the injection profiles, which were consistent in their immersion depth, immersion angle, tracer volume, and injection speed. Accordingly, this approach provides a low-cost, plug-and-play method to improve the reproducibility of mixing-time experiments, thereby facilitating bioreactor characterization, technology transfer, and validation of computational fluid dynamics models. KEY POINTS: Automated acid/base dosing improves reproducibility versus manual addition. Variation of the local mixing is detectable through spatially resolved analysis. Enables robust characterization of both stirred single-use and glass bioreactors.