Lin Zhang1,2, Sandeep Yadav3, Barthélemy Demeule3, Y John Wang3, Olivier Mozziconacci1, Christian Schӧneich4. 1. Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, 66047, USA. 2. Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana, 46285, USA. 3. Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California, 94080, USA. 4. Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, 66047, USA. schoneic@ku.edu.
Abstract
PURPOSE: To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. METHODS: Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. RESULTS: The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via "acyl-cleavage" of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation. CONCLUSIONS: The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.
PURPOSE: To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. METHODS: Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. RESULTS: The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via "acyl-cleavage" of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation. CONCLUSIONS: The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.
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