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

Abstract

In our previous published work, we reported rapid Polysorbate80 (PS80) oxidation in histidine buffer after brief stainless steel exposure 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 impact of temperature, light, stainless steel and the role of citrate and EDTA. Additionally, PS80 oxidation was studied in three different buffer sytems: histidine, citrate, and phosphate. When the PS80 containing buffers were exposed to elevated temperature of 50 °C in glass containers, no PS80 oxidation was observed in either histidine and citrate buffer systems after 30 days; however, PS80 oxidation was observed in phosphate buffer within 14 days. This study demonstrated that temperature does not initiate PS80 oxidation in histidine or citrate buffer systems, but may be a factor in phosphate buffer. When the 3 buffer systems containing PS80 were exposed to 20, 50, and 100 % ICH Q1B light conditions and subsequently incubated at 50°C (dark), the PS80 in phosphate buffer underwent oxidation within 7 days, whereas the PS80 in histidine and citrate buffer systems showed oxidation products only after 14 and 35 days, respectively. While PS80 in phosphate buffer seemed to be most vulnerable to light, PS80 in both histidine and citrate buffers underwent oxidation to a lesser extent, with the histidine buffer showing rapid oxidation compared to citrate buffer. Finally, the ability of citrate and EDTA to act not only as chelators, but also as a radical quencher/scavengers was demonstrated when metal ions such as Fe (2+) were spiked into histidine buffer containing PS80. While radicals could not be unambiguously identified by NMR or EPR, the observation of PS80 oxidation products indicate their presence.