Optimization of Heat-Resistance Technology for a Duck Hepatitis Lyophilized Live Vaccine

Abstract

In this study, to improve the quality of a live attenuated vaccine for duck viral hepatitis (DHV), the lyophilization of a heat-resistant duck hepatitis virus vaccine was optimized. The optimized heat protectors were made of 10% sucrose, 1.2% pullulan, 0.5% PVP, and 1% arginine, etc., with a titer freeze-drying loss of ≤0.50 Lg. The vaccine product's valence measurements demonstrated the following: the vaccine could be stored at 2-8 °C for 18 months with a virus titer loss ≤0.91 Lg; at 37 °C for 10 days with a virus valence loss ≤0.89 Lg; and at 45 °C for 3 days with a virus titer loss ≤0.90 Lg. Regarding safety, no deaths occurred in two-day-old ducklings immunized with a 10 times dose vaccine; their energy, diet, and weight gain were all normal, demonstrating that the DHV heat-resistant vaccines were safe for ducklings and did not cause any immune side effects. Duck viral hepatitis freeze-dried vaccine began to produce antibodies at 7 d after immunization, reached above 5.0 on 14 d, and reached above 7.0 on 21 d, showing a continuous upward trend. This indicates that duck viral hepatitis vaccine has a good immunogen level. The optimization of the freeze-drying process saves costs and also improves the quality of the freeze-drying products, which provides important theoretical and technical support for the further study of vaccine products.

Keywords: DHV; optimization of the freeze-drying process; stabilizers; thermostability.

Figure 1

The rapid freeze-drying process. Each formulation was mixed 1:1 with a DHV antigen, and then dispended into 7 mL sterile glass vials. The lyophilized procedures were set as below. Samples were frozen to −40 °C at a rate of 1.13 °C/min. Primary drying was performed at −25 °C. Secondary drying was performed at 28 °C. Vials were rubber-stoppered under vacuum and tightly sealed with an aluminum cap under normal air pressure.

Figure 2

Screening results for the stabilizer formulations. (a) The titer loss of each stabilizer formulation during lyophilization. The lyophilization loss is the difference between the vaccine titers before and after the lyophilization of the formulations. (b) The heat loss of each stabilizer formulation. The heat loss was the titer reductions for the lyophilized stabilizer formulations before and after incubating at 37 °C for 10 days. The statistical analysis in the results was evaluated by SPSS Statistics 17.0 and Prism 6.0.

Figure 3

The thermal stability of the vaccine. A7 was tested at 2–8 °C (a); the A7 and commercial vaccines at 37 °C (b) and 45 °C (c) were tested in three lots (001, 002, and 003). The freeze-dried DHV vaccine without added stabilizer was assessed as the negative control group.

Figure 3

The thermal stability of the vaccine. A7 was tested at 2–8 °C (a); the A7 and commercial vaccines at 37 °C (b) and 45 °C (c) were tested in three lots (001, 002, and 003). The freeze-dried DHV vaccine without added stabilizer was assessed as the negative control group.

Figure 4

(a) SEM of commercial vaccines; (b) SEM of freeze-dried vaccines with formulation A7. The structure of the freeze-dried samples of the A7 protective agent is the same as that of commercial seedlings from SEM.

Figure 5

Antibody test results of the freeze-dried vaccines and commercial vaccines.