Lyophilization of a viral vaccine: Strategies for surrogate use in analytical characterization of cake properties and considerations on virus infectivity.

Lyophilization has the potential to increase the stability of viral vectors and reduce the dependence on an uninterrupted cold chain. However, freezing and drying stress can inactivate viruses, resulting in reduced infectivity. Development of virus formulations presents additional challenges due to biosafety level restrictions that increase the complexity of sample handling. Particularly, when working with virus-containing lyophilized powders, the characterization of lyophilized cake properties by Karl-Fischer titration, differential scanning calorimetry, and X-ray-powder diffraction requires stringent safety precautions. Our feasibility study aimed to evaluate the use of the virus-free formulation buffers as surrogates for the analytical characterization of lyophilized cake properties, based on the hypothesis that the impact from the low mass fraction of the virus is negligible. We used the ChAdOx1 nCoV-19 vaccine as model viral vector; we formulated it with selected excipient combinations and applied different freezing protocols, including shelf-ramped freezing, quench freezing, and annealing for lyophilization to generate both amorphous and (partially) crystalline cake morphologies. Our results indicate that virus-free formulation buffers can be used as convenient surrogates for the evaluation of cake appearance, residual moisture content and crystallinity. In addition, we show that the extent of preservation of viral infectivity after lyophilization analyzed by TCID50 assay depends on both the formulation and the freezing protocol.