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.

Vacuum freeze-drying, commonly referred to as lyophilization, is one of the primary processes in vaccine industrial manufacturing. It offers significant advantages, including a marked extension of the shelf-life of dried products, effective preservation of biological activity, and user convenience. However, in the production of animal freeze-dried vaccines, the diverse nature of antigens, the complexity of heat-stable protectant formulations, and variations in manufacturing equipment pose substantial challenges to process design for animal freeze-dried vaccines. Commercially available animal freeze-dried vaccines commonly suffer from antigen potency loss, insufficient product stability, low production efficiency and high manufacturing costs. Freeze-drying process optimization can markedly improve the quality of freeze-dried vaccines, with key evaluation indicators including vaccine stability, shelf-life, sample morphology, and residual moisture content. Focusing on multiple types of animal vaccines, this review summarizes research advances in improving the quality of animal freeze-dried vaccines from the perspectives of modifying material properties, enhancing freezing-stage regulation, increasing the specific surface area for drying, and optimizing heat conduction. Finally, future research strategies for animal freeze-dried vaccines are prospected. Although numerous approaches have been proposed to enhance the quality of animal freeze-dried vaccines, comprehensive considerations from multiple dimensions are still required to provide more holistic theoretical support for cost reduction and efficiency improvement in the production of animal freeze-dried vaccines.

Background: Pseudorabies vaccines based on the live attenuated PRV Bartha-K61 strain remain essential for controlling pseudorabies in swine, but poor thermal stability during storage and transport limits field use. Objectives: This study aimed to develop a thermostable freeze-dried live pseudorabies vaccine through the integrated optimization of formulation and lyophilization and to preliminarily assess its humoral immunogenicity in piglets. Methods: Trehalose, mannitol, and glycine were screened as candidate lyoprotectants by single-factor experiments, followed by Box–Behnken response surface optimization using viral titer retention as the response. Critical thermal parameters were determined to establish a formulation-specific lyophilization cycle. Product quality was evaluated by post-lyophilization titer, cake appearance, residual moisture, and electron microscopy, and stability was compared with a commercial freeze-dried vaccine at 2–8 °C, 25 °C, 37 °C, and 45 °C. Conclusions: The optimized formulation, ST005, contained 9.5% trehalose, 2.0% mannitol, and 1.5% glycine. It showed a collapse-related critical temperature of approximately −34.0 °C and a dried-product glass transition temperature of 69.1 °C, produced an intact cake with residual moisture below 3.0%, and preserved viral morphology after lyophilization. Titer loss remained below 1.0 log10 TCID₅₀/mL for 24 months at 2–8 °C, 9 months at 25 °C, 21 days at 37 °C, and 9 days at 45 °C, outperforming the commercial comparator. In PRV-seronegative piglets, stored ST005 induced robust gB-specific and neutralizing antibody responses after storage under refrigerated, ambient, and accelerated conditions.