Evaluation of solvent systems for optimized extractables studies of single use bioprocessing solutions

- Characterization of the extractables profile for single-use systems was performed to know potential leachables under operational conditions.
- A key issue in the design of extractables studies is the extraction solvent composition (polarity) and pH.
- The composition of the model solvent for extractable profiling of single-use bags was determined by a DoE approach.
- The study was focused on the determination of the antioxidant Irgafos 168® and its degradation products.

Despite their advantages, there is concern that single-use systems used in biopharmaceutical manufacture might release potentially toxic substances during standard unit operations that negatively impact cell growth. Characterization of the extractables profile for single-use systems is necessary to know which compounds potentially become leachables under operational cell culture conditions. A key issue in the design of extractables studies is the composition of the model solvent, in particular its pH and polarity. In this study, a new approach, based on design of experiments (DoE), has been applied to determine the composition of the model solvent for extractable profiling of single-use bags (SUBs). Particular focus was placed on the determination of the degradation products of the antioxidant Irgafos 168®, due to evidence that some of these degradation products have cytotoxic effects on CHO cells. Results indicated that 2-propanol:water is the most appropriate solvent for the extraction of highly hydrophobic compounds with polar groups and/or acid-base properties from SUBs. The described DoE approach simplifies the number of experiments, evaluates all possible solvent water mixtures to select the best extraction solvent based on polarity, establishes the influence of each variable and provides information about variable interaction, which represents an important improvement over current best practice. The developed approach was applied to seven SUBs from different vendors and production dates facilitating the identification of potentially non-satisfactory films for cultivation of CHO cell lines under process conditions.