Formulation and filling of biological products occur in isolators to avoid contamination and ensure the maximum level of sterility. Isolators are commonly sanitized by vaporized hydrogen peroxide (VHP). Residuals of Hydrogen Peroxide (HP) - in the isolator - may contaminate the solution and trigger instability of the active pharmaceutical ingredients, excipients or entire drug product. The purpose of this work was to investigate the HP consumption during the freeze-drying of a vaccine and establish a thorough understanding of the factors influencing it. The protein formulation and its placebo were spiked with HP and after 2 hours freeze-dried. HP consumption during the lyophilization process was monitored as a function of the time and the freezing temperature.