Effect of peptide concentration and temperature on leuprolide stability in dimethyl sulfoxide

The effects of temperature and concentration on leuprolide degradation in dimethyl sulfoxide (DMSO) were explored. Leuprolide degradation products were analyzed by reverse phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) and structurally characterized by mass spectrometry. Leuprolide solution stability in DMSO was characterized at 50, 100, 200, 400 mg/ml at 37-80 degrees C for 2 months to 3 years. Leuprolide degradation products were identified by mass spectrometry and could generally be attributed to isomerization, hydrolysis, oxidation, or aggregation. The hydrolytic degradation products consisted primarily of backbone cleavage C-terminal to Trp(3), Ser(4), Tyr(5), Leu(6) and Leu(7), and oxidation of Trp(3) and beta-elimination of Ser(4) were identified. Leuprolide degradation at 50 degrees C, 65 degrees C and 80 degrees C proceeded in an exponential fashion (E(a)=22. 6+/-1.2 kcal/mol); however, leuprolide degradation plateau'd after approximately 6 months at 37 degrees C. Upon closer examination, degradation product peak areas were seen to vary with temperature. For example, aggregation products did not increase with time at 37 degrees C, but aggregation peak intensities increased sharply with time at 80 degrees C. Increasing the temperature also increased the proportion of leuprolide degrading via isomerization/hydrolytic pathways, and decreased the proportion degrading via oxidation. These variations suggested that solvent dielectric, free H(+) in an aprotic solvent, oxygen solubility, impurities and residual moisture may play a role. Leuprolide solubilized in DMSO yields adequate stabililty for a 1 year implantable osmotic delivery system, where use of a dry aprotic solvent results in conditions similar to solid state stability.