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To address multi-resistant bacterial infections, interest has regrown for phagotherapy as an alternative or complementary treatment. While compassionate use of bacteriophages already showed cases of remission (N. Dufour et al, 2017), clinical trials are still at their early stages (Górski et al, 2020).

To encourage clinical trials and ensure exploitable data, it is essential that the final product’s quality, efficacy, and safety are guaranteed. Having resort to risk-based approaches such as Quality by Design (QbD) is much encouraged by the Food and Drug Administration and other regulatory authorities (Yu LX et al., 2014). One of the objectives of the QbD is to define a design space in which the product quality is guaranteed. This design space can be achieved with a Design of Experiment (DoE) based optimization, which helps identifying the optimal conditions as well as critical process parameters.

In this purpose, the process development department of Clean Cells has worked on implementing a robust and flexible DoE for upstream process development (USP). The following main objectives were to be achieved:

• To reach the highest phage titers within a reduced time frame
• To gain knowledge on robustness and repeatability
• To be able to select the most interesting condition depending on any specific constraints (technical, cost, robustness, time)
• To be applicable to any other phage

In this work, an optimization of the phage production process was carried out on T7 and T4 bacteriophages, both specific to Escherichia coli. The capabilities of the models to predict the phage titers were proven to be effective, which indicates that this approach can be used for further projects and applications. An improvement of the final titer was achieved on both phages, with an increase from 9.5 to 11.2 log₁₀PFU/mL for T7 and 7.9 to 11.2 log₁₀PFU/mL for T4. A design space for each phage was defined in which the phage production appeared to be robust and the phage quality guaranteed. With this data, the scale-up strategy could be secured as well as the manufacturing process.