Thermally Stressed Solid-State Stability of Semaglutide: Understanding the Influence of Temperature on Protein Content, Secondary Structure, Phase Transition, and Chemical Degradation.

OBJECTIVE: Semaglutide (SMG), a clinically relevant peptide-based therapeutic whose physical and chemical stability are critical concerns during manufacturing and storage. Although the stability of SMG in solution has been extensively studied, its solid-state behaviour remains unclear. This study aimed to systematically evaluate the impact of thermal stress on the solid-state physicochemical stability of SMG. METHODS: The solid-state stability of SMG was assessed using complementary analytical techniques, including Fourier transform-infrared (FT-IR) spectroscopy, circular dichroism (CD), differential scanning calorimetry (DSC), hot-stage microscopy (HSM), reverse-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). RESULTS: FT-IR and CD analyses demonstrated that SMG retains its native α-helical conformation up to 60°C. However, the α-helical content decreased from 49.07% to 43.75% at 60°C and further to 0.2% at 80°C, indicating extensive conformational transitions at elevated temperatures that compromise receptor binding and in vivo performance. DSC and HSM confirmed that SMG remains amorphous under all tested conditions and revealed three major thermal events: residual water loss, enthalpy recovery associated with physical ageing, and thermal decomposition. The overlap of enthalpy recovery with the glass transition phase limited the determination of Tg by conventional DSC; however, modulated DSC enabled the separation of these events, establishing a Tg of 169°C. RP-HPLC and LC-HRMS analyses showed a temperature-dependent degradation and impurity formation. CONCLUSION: The solid-state stability study identified temperature as a critical factor influencing SMG stability and emphasises the importance of stringent process control in the development of SMG-based formulations.