Improving extracellular vesicles production through a Bayesian optimization-based experimental design.

With the growing demand and diversity of biological drugs, developing optimal processes for their accelerated production with minimal resource utilization is a pressing challenge. Typically, such optimization involves multiple target properties, such as production yield, biological activity, and product purity. Therefore, strategic experimental design techniques that can characterize the parameter space while simultaneously arriving at the optimal process satisfying multiple target properties are required. To achieve this, we propose the use of a multi-objective batch Bayesian optimization (MOBBO) algorithm and illustrate its successful application for the production of extracellular vesicles (EVs) from a 3D culture of mesenchymal stem cells (MSCs) considering three objectives, namely to maximize the vesicle-to-protein ratio, maximize the enzymatic activity of the MSC-EV protein CD73, and minimize the amount of calregulin impurities. We show that the optimal combination of the process parameters to address the intended objectives could be achieved with only 32 experiments. For the four parameters considered (i.e., microcarrier concentration, seeding density, centrifugation time, and impeller speed), this number of experiments is comparable to or lower than the classical design of experiments (DoE) and the traditional one-factor-at-a-time (OFAT) approach. We illustrate how the algorithm adaptively samples in the process parameter space, selectively excluding unfavorable regions, thus minimizing the number of experiments required to reach optimal conditions. Finally, we compare the obtained solutions to the literature data and present possible applications of the collected data for other modeling activities such as Quality by Design, process monitoring, control, and scale-up.