Effect of disaccharide-polyol systems on the thermal stability of freeze-dried Mycobacterium bovis

Abstract Live attenuated Mycobacterium bovis ( M. bovis ), marketed as Bacille Calmette-Guerin is the only FDA-approved vaccine against tuberculosis. The prerequisite of cold chain storage between 2 and 8 °C hinders the global vaccination effort. The study aims to investigate the effect of trehalose, sucrose and glycerol combinations in enhancing the stability of M. bovis . The bacilli were formulated in various ratios of trehalose-glycerol, sucrose-glycerol, trehalose-sucrose-glycerol systems (test samples) and sodium glutamate (control), freeze-dried and stored for 28 days at 4 °C, 25 °C and 37 °C. Bacteria viability at pre-, post-freeze-drying and after storage were quantified by its density in colony-forming unit per milliliter (CFU/mL) as obtained through the pour plate method. Formulations were characterized using differential scanning calorimetry. Structural collapsed cakes were found on all freeze-dried formulations because of the low T g ’. Comparing between binary and ternary formulations, trehalose-sucrose-glycerol was found to be a superior lyoprotectant. Upon storage, the viability of bacteria in disaccharide-polyol formulations was highest when stored at 4 °C followed by 25 °C. The lowest viability was found after storage at 37 °C. While the ternary disaccharide-polyol system may be used as a thermoprotectant up to 25 °C, sodium glutamate has a superior thermoprotective effect at temperature above 25 °C.