Background/aim: Vaccines are often lyophilized in order to retain their stability and efficacy for a longer period of time. However, the same lyophilization process may also cause a major degradation of the vaccine, especially during early phases of manufacturing, leading to a loss of potency of the product. Many viral diseases, such as rabies, are acute and fatal unless the vaccine is administered prior to exposure or the onset of symptoms in the case of postexposure treatment. Materials and methods: We investigated the effect of lyophilization on the stability of the virus structure during rabies vaccine manufacturing using dynamic light scattering and transmission electron microscopy. Results: Our results indicate that some viruses lose their stability and efficacy in the course of lyophilization if the pH of the cell culture medium is controlled by solvated CO2 because the structure of the rabies virus is very sensitive to the solution pH: the virus either aggregates or its shape is deformed at low solution pH, whereas at high pH empty capsid shells are formed. Conclusion: Based on our findings, we developed a new formulation for the rabies vaccine that is stable in different buffers owing to the prevention of pH upshift upon lyophilization.