Manufacturing process development and analytical characterization of an OMVs-based vaccine candidate applying the DoE approach

In the context of the growing need for safer and more effective vaccine platforms, Outer Membrane Vesicles (OMVs) emerge as a promising solution. Naturally released by bacteria and mimicking the structure and composition of the bacterial membrane, they have the ability to elicit a specific immune response. Within this framework, both the bacterial fermentation phase and the subsequent purification phase play a crucial role in the development of the final product. This work provides the significant advancements that have been made through the strategic implementation of design-of-experiment (DOE) methodologies in the development of a OMVs based candidate vaccine In the initial phase, the growth medium of a Gram-negative bacterium belonging to the Neisseriaceae family and releasing OMVs was optimized identifying the key components with the DOE approach. Subsequently, the same mathematical-statistical approach enabled the identification of the most effective purification buffer compositions to be used in a Tangential Flow Filtration (TFF) process to obtain a contaminant-free product. In the final part, a phenol-free protocol was developed to isolate and purify Lipopolysaccharide, a crucial component of the bacterial wall, from the obtained OMVs with a high degree of purity. This high purity is a fundamental requirement for potential structural and immunological analyses.