Lyophilization or freeze-drying is used to increase the stability of protein active ingredients. In this way, we slow down the chemical and physical processes, facilitate the handling of the drug and facilitate transport.
Since lyophilization is a cost-effective and time-consuming process, by designing the appropriate formulation, the cycle can be shortened to increase the cost-effectiveness of the process. Due to the burden of protein active ingredient on the low-temperature and low-pressure during the process the formulation must also contain, in addition to the active ingredient, excipients that will protect it from these effects and also later during the transport and storage.
Within the master's thesis, formulations with crystalline bulking agent glycine and various stabilizers were made. We used: sucrose, trehalose dihydrate, ?-cyclodextrin, sorbitol, PEG 4.000, PVP 10.000 and PVP 40.000. We studied whether the sucrose can be replaced by another stabilizer. Aqueous solutions of a mixture of glycine and stabilizer in 6 : 1, 5 : 2 and 1 : 6 ratios (at a concentration of 0,1 mg/mL or 0,2 mg/mL) were analyzed by differential dynamic calorimetry to measure the glass transition temperature of the maximum concentrated frozen solution (Tg'). Higher Tg' allows a shorter primary drying time and thus contribute to a shorter lyophilization cycle. The formulations were exposed to the aggressive lyophilization cycle (the product temperature during primary drying exceeded Tg') and the process was evaluated through the process graph and a visual examination of lyophilizates. The lyophilizates were also analyzed by differential dynamic calorimetry and the glass transition temperature (Tg) was measured. Finally, we measured the Tg of the lyophilizates containing model protein ingredient and Tg' after the reconstitution of lyophilizates, thereby checking the effect of the protein on the thermal properties of the formulations. Protein formulations were prepared with sucrose at concentrations of 20 and 30 mg/mL and with glycine and sucrose in ratios of 1 : 2 (at a concentration of 20 mg/mL) and 2 : 1 (at a concentration of 30 mg/mL).
The results showed that all of the analyzed formulations of the mixtures in which Tg' were detected they were higher than the mixture of glycine with sucrose. Similarly, aqueous solutions of all mixtures other than glycine and sorbitol in a ratio of 1 : 6 after lyophilization form an acceptable cake appearance. After measurement of Tg of lyophilizates, PVP 10.000 was found to be the most optimal excipient for the replacement of sucrose in lyophilization formulations, because it has higher Tg' than sucrose and high Tg of lyophilizates. Depending on the high measured Tg', ?-cyclodextrin could also be used in protein formulations, although Tg was not detected in the analysis of lyophilizates. We also did not detect Tg in mixtures of glycine and PEG 4,000, but lyophilizates of acceptable appearance suggest a stability of these formulations.
Measurements of the added protein-formulated formulations showed an increase in Tg and thus a prediction of better stability during storage, but at the same time decreased Tg', which could mean a longer lyophilization cycle, since a longer phase of primary drying is required, which may affect the stability of the formulations during the lyophilization process.
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