Semaglutide (SGL), a long-acting GLP-1 (Glucagon-like peptide) receptor agonist, is a 31-amino acid peptide modified with a C18 fatty diacid for albumin binding. Peptides are fragile and susceptible to degradation during formulation, storage, and transportation. The degradation of peptides resulted in the formation of impurities that may impact safety, efficacy, immunogenicity, and regulatory compliance. The present study examines the effects of pH, temperature, buffer species, and molarity on the stability of SGL. Reverse-phase ultra-performance liquid chromatography (RP-UPLC) was used to separate impurities, followed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) for their molecular weight. The stress stability studies were conducted in thermal stress conditions at 25 °C, 40 °C, 60 °C (for 28 days), and 80 °C (for 7 days). The influence of pH, buffer strength, and buffer species on the degradation of SGL was investigated at 25 °C and 40 °C. The degradation of SGL resulted in thirteen known impurities, and their fragments were identified using LC-MS analysis. Six impurities, such as impurity 4 (m/z = 2717.21), impurity 5 (m/z = 4129.64), impurity 7 (m/z = 3762.28), impurity 8 (m/z = 3456.94), impurity 12 (m/z = 2967.5), and impurity 13 (m/z = 839), were formed across all the testing conditions. These impurities were relatively stable when compared to other formed impurities. The influence of pH on the thermal stability of SGL was demonstrated. The impurities, such as impurity 2 (m/z = 845.13), impurity 9 (m/z = 3397.76), impurity 10 (m/z = 701.0), and impurity 11 (m/z = 4125.7), were absent across all pH conditions, but these impurities were found when water was used as a solvent. The study demonstrated that the pH was a key factor for the thermal degradation of SGL. The degradation pathways were elucidated based on the mass data for known masses. The solution-state thermal stress studies were performed to select the buffer for formulating long-acting PLGA formulations. Moreover, the solution state stress stability data could be helpful for optimization of the pharmaceutical process, in vivo stability of SGL in muscles, storage and transportation of finished products, and determining the shelf-life.
