Evaluation of Annealing parameters in Freeze-drying process

“The World Health Organization” estimates that from 2 to 3 million of lives are saved each year due to vaccination programs (Pollard and Bijker, 2021). This study focuses on Live Virus Vaccines (LVV), which use attenuated live viruses as antigens, to provide a long-term immunity (Robinson, 2016). LVV are not stable in liquid form, so require freeze-drying to preserve the stability and increase their shelf life. Freeze-drying consists of a freezing step in which the aqueous solution is brought to freeze before water is removed by an initial sublimation step (primary drying), followed by a desorption step of bound water (secondary drying). The optimization of this process is crucial in terms of cake appearance, virus stability and potency, residual moisture and cycle duration. Among the different methods, the treatment of annealing is an optimization technique, between freezing and primary drying. Specifically, after the freezing phase the frozen solution is heated to a product temperature above its glass transition temperature and kept in isotherm for a certain period influencing the critical product temperatures, thanks to the Ostwald ripening and glassy state relaxation (Kharatyan et al., 2022) phenomena. At the end, the temperature is brought back to the shelf’s freezing temperature and the freeze-drying process continues. The focus of this work is to evaluate the influence of annealing parameters, such as ramp rate, plateau temperature and plateau duration by assessing their impact on the different matrices under study. These analyses were conducted on the LVV buffer composition and on amorphous and crystalline excipients. The impact of annealing process on the glass transition temperature (Tg’), collapse temperature (Tc) and product resistance (Rp) was evaluated. Several methods were used to perform these analyses: Differential Scanning Calorimetry (DSC), Freeze-drying Microscopy (FDM) and the Pressure Rise Test (PRT). In addition, a visual inspection of the lyophilized cakes is performed to understand the effects on cake appearance and Karl Fischer equipment is used to define the impact on the moisture content. As a result, increasing annealing temperature and its plateau duration, the solution has more time to reorganize and, coupled with the increase in molecular mobility, reduces the stresses inside the structure, reaching a more orderly matrix characterized by a little bit higher Tg’ value. The rise in the crystallized portion and on the pore size of the sample, observable using the FDM, makes challenging to define a collapse temperature, being an amorphous property. The annealing ramp rate parameter doesn’t have a significant impact on the Tg’ and the Tc, using commercially applicable values (from 0,3 to 1,0°C/min). The collapse temperature is a kinetic phenomenon (Ohori and Yamashita, 2017); increasing the sublimation temperature ramp rate its value increases depending on the product analyzed, instead the sublimation pressure doesn’t impact it. These results are important leading to use more aggressive cycle parameters for primary drying. A series of PRT test has been done on the LVV placebo, expecting an impact of annealing on product resistance and cycle time. It wasn’t possible to determine a significative impact on the calculated Rp probably due to the complex formulation and the filling volume. Nevertheless, the annealed freeze-dried cakes present a better cake appearance and a more homogeneous moisture content inside a vials batch.