Lyophilization is a process of drying where the solution is deeply frozen and then the solvent is removed from it by the processes of sublimation and desorption. Given the fact that lyophilization is time and energy consuming process, its optimizaton is essential. Research show, that with a choice of appropriate excipients, such as stabilizers and bulking agents, protein formulation can be exposed to higher temperatures of primary drying. Even though product appearance is acceptable and activity of the active substance and short time of reconstitution are preserved. The choice of process parameters (temperature and pressure) is based on knowledge of critical temperatures of formulation, among which we classify glass transition temperature of the maximally freeze-concentrated solution (Tg'), temperature of eutectic melting (Teu) and collapse temperature (Tc), which can be subdivided into temperature at which the first changes in the structure are observed with the freeze-drying microscopy (Tconset) and temperature where full collapse is observed (Tcfull). For a long time a fact, that drying above Tcfull will result in full collapse of product, was applied, but newer studies show, that the addition of appropriate bulking agents and stabilizers provides acceptable cake appearance even when product temperature (Tp) during primary drying exceeds Tcfull. The purpose of this thesis is to study influence of Tp on product collapse with increasing of pressure and shelf temperature of primary drying (agressive conditions) and to define the highest accaptable temperature and pressure of primary drying. Measured Tps at the end of each cycle were compared to critical temperature of the formulation, which were defined by differental scanning calorimetry (DSC) and freeze-drying microscopy (FDM). With the purpose to evaluate impact of excipients on product collapse, three formulations with protein substance (monoclonal antibody) were lyophilized. One with 6% m/V of amorphous succrose and two partially crystaline with 2% m/V succrose and 4% m/V glycine and mannitol respectively. At the end of every cycle products were visually evaluated, reconstitution time and particle size were determinded and scanning electron microscopy (SEM) was preformed to estimate structure morphology. Initiating of agressive conditions of drying led to increased Tp, which was higher than Tg' in every agressive cycle. Even though Tp of formulation with succrose exceeded Tcfull already in the first agressive cycle, product collapse was seen when shelf temperature and pressure were further increased (agressive cycle 6), Tp was then 5,7 °C above Tcfull. The possible reason for this is, that Tc measured with FDM is only a good estimation of temperature, at which collapse is achieved during process of drying. The collapse of other formulations was not seen, because of presence of the crystal component (glycine/manitol). Using more agressive cycles primary drying time was reduced for 80% according to the conservative cycle and thereby time- and cost-efficiency of the lyophilization process were successfully increased.
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