Bristol-Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jul 1;878(21):1865-70. doi: 10.1016/j.jchromb.2010.04.039. Epub 2010 May 4.
In this paper, we discuss an improved high-performance liquid chromatography (HPLC) method for the quantitation of polysorbate 80 (polyoxyethylenesorbitan monooleate), a commonly used stabilizing excipient in therapeutic drug solutions. This method is performed by quantitation of oleic acid, a hydrolysis product of polysorbate 80. Using base hydrolysis, polysorbate 80 releases the oleic acid at a 1:1 molar ratio. The oleic acid can then be separated from other polysorbate 80 hydrolysis products and matrices using reversed phase HPLC. The oleic acid is monitored without derivatization using the absorbance at 195 nm. The method was validated and also shown to be accurate for the quantitation of polysorbate 80 in a high protein concentration monoclonal antibody drug product. For the measured polysorbate 80 concentrations, the repeatability was less than 6.2% relative standard deviation of the mean (% RSD) with the day-to-day intermediate precision being less than 8.2% RSD. The accuracy of the oleic acid quantitation averaged 94-109% in different IgG(1) and IgG(4) drug solutions with variable polysorbate 80 concentrations. In this study, polyoxyethylene, a by-product of the polysorbate 80 hydrolysis was also identified. This peak was not identified by previous methods and also increased proportionally to the polysorbate 80 concentration. We have developed and qualified a method which uses common equipment found in most laboratories and is usable over a range of monoclonal antibody subclasses and protein concentrations.
本文讨论了一种改进的高效液相色谱（HPLC）方法，用于定量聚山梨酯 80（聚氧乙烯山梨糖醇单油酸酯），这是治疗药物溶液中常用的稳定赋形剂。该方法通过定量分析聚山梨酯 80 的水解产物油酸来进行。使用碱性水解，聚山梨酯 80 以 1:1 摩尔比释放油酸。然后，使用反相 HPLC 将油酸与其他聚山梨酯 80 水解产物和基质分离。使用 195nm 处的吸光度对油酸进行监测，无需衍生化。该方法经过验证，也可准确测定高蛋白浓度单克隆抗体药物产品中的聚山梨酯 80。对于所测量的聚山梨酯 80 浓度，重复性小于平均值的 6.2%相对标准偏差（%RSD），日间中间精密度小于 8.2%RSD。在不同 IgG(1)和 IgG(4)药物溶液中，具有可变聚山梨酯 80 浓度的油酸定量的准确度平均为 94-109%。在本研究中，还鉴定了聚山梨酯 80 水解的副产物聚氧乙烯。该峰以前的方法未鉴定，并且与聚山梨酯 80 浓度成比例增加。我们已经开发并合格了一种方法，该方法使用大多数实验室中常见的设备，并且可用于多种单克隆抗体亚类和蛋白质浓度。
