A Mechanistic Understanding of Polysorbate 80 Oxidation in Histidine and Citrate Buffer Systems—Part 2

Abstract

In our previously published work, we reported rapid polysorbate 80 (PS80) oxidation in a histidine buffer after brief exposure to stainless steel and the ability of citrate and EDTA to prevent this oxidation. The focus of our current study was to mechanistically understand PS80 oxidation by studying the impacts of temperature, light, and stainless steel and the role of citrate and EDTA. Additionally, PS80 oxidation was studied in three different buffer systems: histidine, citrate, and phosphate. When the PS80-containing buffers in glass containers were exposed to the elevated temperature of 50°C, no PS80 oxidation was observed in either the histidine or the citrate buffer systems after 30 days; however, PS80 oxidation was observed in the phosphate buffer system within 14 days. These results demonstrated that temperature does not initiate PS80 oxidation in the histidine or the citrate buffer systems, but it may be a factor in the phosphate buffer system. When the three buffer systems containing PS80 were exposed to 20%, 50%, or 100% ICH Q1B light conditions and subsequently incubated in the dark at 50°C, the PS80 in the phosphate buffer system underwent oxidation within 7 days, whereas the PS80 in the histidine and the citrate buffer systems showed oxidation products only after 14 and 35 days, respectively. PS80 in the phosphate buffer system seemed to be the most vulnerable to light as PS80 in both the histidine and the citrate buffer systems underwent oxidation to a lesser extent, with faster oxidation occurring in the histidine buffer system than in the citrate buffer system. Finally, the ability of citrate and EDTA to act as not only chelators but also radical quenchers/scavengers was demonstrated when a metal ion, Fe²⁺, was spiked into the histidine buffer containing PS80. While radicals could not be unambiguously identified by NMR or EPR, the observation of PS80 oxidation products indicated their presence.

LAY ABSTRACT: In our previously published work, we reported rapid polysorbate 80 (PS80) oxidation in a histidine buffer after brief exposure to stainless steel and the ability of citrate and EDTA to prevent this oxidation. The focus of our current study was to mechanistically understand PS80 oxidation by studying the impacts of temperature, light, and stainless steel and the role of citrate and EDTA. Additionally, PS80 oxidation was studied in three different buffer systems: histidine, citrate, and phosphate. The temperature study demonstrated that PS80 oxidation in the histidine or the citrate buffer systems is not initiated by temperature, but may be a factor in the phosphate buffer system. PS80 in the phosphate buffer system seemed to be the most vulnerable to light, as PS80 in both the histidine and the citrate buffer systems underwent oxidation at a lower level, with the histidine buffer system showing more rapid oxidation than the citrate buffer system. Finally, the ability of citrate and EDTA to act as not only chelators but also radical quenchers/scavengers was demonstrated when a metal ion, Fe²⁺, was spiked into the histidine buffer containing PS80. While neither NMR nor EPR could definitively identify the presence of free radicals, the observation of PS80 oxidation products indicates that they were present.