Development of new mass spectrometry based methods for conformational characterization of Alum adjuvant adsorbed antigens: Application to the Bexsero vaccine

Alessandro Vadi PhD Thesis Abstract A panel of three MS based techniques was developed and consistently applied for the characterization of Alum adsorbed antigens. The efficacy of these techniques was confirmed by their application to the NHBA-NUbp Alum maturation effect case of study. Alum maturation effect, intended as NHBA-NUbp in vitro relative potency (IVRP) response variation during time, was selected as a critical attribute for the antigen analytical characterization. Alum maturation effect understanding had the availability of a wide panel of analytical tools selected by the dedicated project. Mass spectrometry was one of the platforms able to study NHBA-NUpb monovalent formulated samples directly without perturbing the interphases adsorption. Three Mass Spec techniques were selected and applied for their ability to probe by enzymatic and chemical modifications the conformational characteristics of the adsorbed antigen. The techniques applied to mass spectrometry were: Limited proteolysis, Hydrogen-Deuterium exchange (HDx), and Hydroxyl Radical Footprinting (HRF). For what is in our knowledge, those methodologies have never been applied to conformational studies on adjuvant adsorbed proteins: method development was therefore challenged by the difficulty of combining common sample treatment found in literature for each technique with the presence of colloidal aluminum hydroxide (Alum). In conclusion we believe to have provided new extremely valuable tools for conformational characterization of adjuvant adsorbed antigens.