Effect of peptide concentration and temperature on leuprolide stability indimethyl sulfoxide

The effects of temperature and concentration on leuprolide degradation in d imethyl sulfoxide (DMSO) were explored. Leuprolide degradation products wer e analyzed by reverse phase high-performance liquid chromatography (RP-HPLC ), size exclusion chromatography (SEC) and structurally characterized by ma ss 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. Leupro lide degradation products were identified by mass spectrometry and could ge nerally be attributed to isomerization, hydrolysis, oxidation, or aggregati on. The hydrolytic degradation products consisted primarily of backbone cle avage C-terminal to Trp(3), Ser(4), Tyr(5), Leu(6) and Leu(7), and oxidatio n of Trp(3) and beta-elimination of Ser(4) were identified. Leuprolide degr adation at 50 degrees C, 65 degrees C and 80 degrees C proceeded in an expo nential fashion (E-a = 22.6 +/- 1.2 kcal/mol); however, leuprolide degradat ion plateau'd after approximately 6 months at 37 degrees C. Upon closer exa mination. degradation product peak areas were seen to vary with temperature . For example, aggregation products did not increase with time at 37 degree s C, but aggregation peak intensities increased sharply with time at 80 deg rees C. Increasing the temperature also increased the proportion of leuprol ide degrading via isomerization/hydrolytic pathways, and decreased the prop ortion degrading via oxidation. These variations suggested that solvent die lectric, free Hf in an aprotic solvent, oxygen solubility, impurities and r esidual moisture may play a role. Leuprolide solubilized in DMSO yields ade quate stabililty for a 1 year implantable osmotic delivery system, where us e of a dry aprotic solvent results in conditions similar to solid state sta bility. (C) 1999 Elsevier Science B.V. All rights reserved.