Dual Effect of Histidine on Polysorbate 20 Stability: Mechanistic Studies.

PURPOSE: L-Histidine (L-His) and polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing. METHODS: The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products. RESULTS: Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with 13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates. CONCLUSION: L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.

Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry.

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.