Doyle Drbohlav Laura M, Sharma Anant Navanithan, Gopalrathnam Ganapathy, Huang Lihua, Bradley Scott
Department of Pharmaceutical Chemistry, University of Kansas, Simons Biosciences Research Laboratories, 2095 Constant Ave. Lawrence, KS 66047.
Bioproduct Pharma Design, Lilly Research Laboratories, Eli Lilly and Company, 1400 West Raymond St., Indianapolis, IN 46221;
PDA J Pharm Sci Technol. 2019 Jul-Aug;73(4):320-330. doi: 10.5731/pdajpst.2018.009639. Epub 2019 Feb 15.
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, Fe, 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. 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, Fe, 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.
在我们之前发表的工作中，我们报道了聚山梨醇酯80（PS80）在组氨酸缓冲液中短暂暴露于不锈钢后会迅速氧化，以及柠檬酸盐和乙二胺四乙酸（EDTA）防止这种氧化的能力。我们当前研究的重点是通过研究温度、光照和不锈钢的影响以及柠檬酸盐和EDTA的作用，从机理上理解PS80的氧化过程。此外，还在三种不同的缓冲系统中研究了PS80的氧化：组氨酸、柠檬酸盐和磷酸盐。当玻璃容器中含PS80的缓冲液暴露于50°C的高温下时，30天后在组氨酸或柠檬酸盐缓冲系统中均未观察到PS80氧化；然而，在磷酸盐缓冲系统中14天内就观察到了PS80氧化。这些结果表明，温度不会引发组氨酸或柠檬酸盐缓冲系统中的PS80氧化，但在磷酸盐缓冲系统中可能是一个因素。当含有PS80的三种缓冲系统暴露于20%、50%或100%的国际协调会议（ICH）Q1B光照条件下，随后在50°C黑暗中孵育时，磷酸盐缓冲系统中的PS80在7天内发生氧化，而组氨酸和柠檬酸盐缓冲系统中的PS80分别在14天和35天后才出现氧化产物。磷酸盐缓冲系统中的PS似乎最易受光照影响，因为组氨酸和柠檬酸盐缓冲系统中的PS80氧化程度较低，且组氨酸缓冲系统中的氧化速度比柠檬酸盐缓冲系统更快。最后，当向含有PS80的组氨酸缓冲液中加入金属离子铁时，证明了柠檬酸盐和EDTA不仅能作为螯合剂，还能作为自由基猝灭剂/清除剂。虽然核磁共振（NMR）或电子顺磁共振（EPR）无法明确鉴定自由基的存在，但PS80氧化产物的观察表明它们是存在的。在我们之前发表的工作中，我们报道了聚山梨醇酯80（PS80）在组氨酸缓冲液中短暂暴露于不锈钢后会迅速氧化，以及柠檬酸盐和乙二胺四乙酸（EDTA）防止这种氧化的能力。我们当前研究的重点是通过研究温度、光照和不锈钢的影响以及柠檬酸盐和EDTA的作用，从机理上理解PS80的氧化过程。此外，还在三种不同的缓冲系统中研究了PS80的氧化：组氨酸、柠檬酸盐和磷酸盐。温度研究表明，组氨酸或柠檬酸盐缓冲系统中的PS80氧化不是由温度引发的，但在磷酸盐缓冲系统中可能是一个因素。磷酸盐缓冲系统中的PS80似乎最易受光照影响，因为组氨酸和柠檬酸盐缓冲系统中的PS80氧化程度较低，组氨酸缓冲系统的氧化速度比柠檬酸盐缓冲系统更快。最后，当向含有PS80的组氨酸缓冲液中加入金属离子铁时，证明了柠檬酸盐和EDTA不仅能作为螯合剂，还能作为自由基猝灭剂/清除剂。虽然NMR和EPR都无法确定自由基的存在，但PS80氧化产物的观察表明它们是存在的。
