Abstract
Squalene-based nanoemulsions are widely used as adjuvants in vaccine formulations, but their stability can be affected by environmental factors such as pH, oxidative stress, temperature, and light. We have produced a squalene nanoemulsion (CTVad1) to support the clinical development of new vaccines. This study aimed to develop and validate an HPLC method for squalene quantification in SpiN-Tec, a recombinant protein vaccine against COVID-19. We also aimed to evaluate the stability of the CTVad1 adjuvant and SpiN-Tec under controlled storage conditions. A reversed-phase HPLC method was developed and validated, and comprehensive forced degradation studies were performed on the raw material and SpiN-Tec under acidic, basic, oxidative, thermal, and photolytic conditions to demonstrate the stability-indicating capability of the method. Physicochemical, morphological, and biological characteristics were assessed, and stability studies of both the vaccine and the CTVad1 adjuvant were performed under accelerated and long-term conditions. The HPLC method was selective, precise, accurate, linear, and robust. Squalene raw material degraded under all tested conditions, whereas formulation in nano-sized globules improved its stability, with degradation observed only under hydrogen peroxide and light exposure. CTVad1 remained stable over time, exhibiting only minor, non-critical changes within the specification limits in both accelerated and long-term stability studies, regardless of the glass packaging used (clear or amber). In addition, the SpiN-Tec vaccine maintained its physicochemical and biological integrity under all tested conditions, with all evaluated parameters remaining within the established specification ranges. Our findings demonstrate that proper formulation, packaging, and storage conditions can preserve squalene stability in nanoemulsion-based vaccines, ensuring the quality, safety, and efficacy of the SpiN-Tec vaccine and its adjuvant throughout shelf life.