Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USA.
J Chromatogr A. 2011 Apr 15;1218(15):2138-45. doi: 10.1016/j.chroma.2010.09.057. Epub 2010 Sep 29.
Polysorbate 20 (polyoxyethylenesorbitan monolaurate) and polysorbate 80 (polyoxyethylenesorbitan monooleate) used in protein drug formulations are complex mixtures that have been difficult to characterize. Here, two HPLC methods are used with evaporative light scattering detection (ELSD) and mass spectrometry (MS) to characterize polysorbate from commercial vendors. The first HPLC method used a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion exchange and reversed-phase sorbent) with a step gradient to quantify both the total polyoxyethylene sorbitan ester and polyoxyethylene sorbitan (POE sorbitan, a non-surfactant) in polysorbate. The results indicated POE sorbitan was present from 16.0 to 27.6 and 11.1 to 14.5% (w/w) in polysorbate 20 and 80, respectively. The second HPLC method used a reversed-phase stationary phase (Zorbax SB-300 C(8)) with a shallow gradient to separate, identify, and quantify the multiple ester species present in polysorbate. For all lots of polysorbate 20 analyzed, only 18-23% of the material was the expected structure, polyoxyethylenesorbitan monolaurate. Up to 40% and 70% (w/w) di- and triesters were found in polysorbate 20 and polysorbate 80 respectively. Likewise, polyoxyethylenesorbitan monooleate accounted for only 20% of polysorbate 80. A variability of 3-5% was observed for each ester species between multiple lots of polysorbate 20. The reversed-phase method was then used to determine the rate of hydrolysis for each polyoxyethylene sorbitan ester of polysorbate 20 in basic solution at room temperature. Increasing rates of hydrolysis were observed with decreasing aliphatic chain lengths in polysorbate 20.
聚山梨酯 20（聚氧乙烯山梨醇单月桂酸酯）和聚山梨酯 80（聚氧乙烯山梨醇单油酸酯）用于蛋白质药物制剂，它们是复杂的混合物，难以进行表征。在这里，我们使用两种高效液相色谱法（HPLC）与蒸发光散射检测（ELSD）和质谱（MS）相结合，对来自商业供应商的聚山梨酯进行了表征。第一种 HPLC 方法使用混合模式固定相（Waters Oasis MAX，混合模式阴离子交换和反相固定相），采用逐步梯度，定量测定聚山梨酯中的总聚氧乙烯山梨醇酯和聚氧乙烯山梨醇（非表面活性剂 POE 山梨醇）。结果表明，聚山梨酯 20 中的 POE 山梨醇含量为 16.0%至 27.6%（w/w），聚山梨酯 80 中的 POE 山梨醇含量为 11.1%至 14.5%（w/w）。第二种 HPLC 方法使用反相固定相（Zorbax SB-300 C(8)），采用浅梯度，分离、鉴定和定量聚山梨酯中存在的多种酯类物质。对所有分析的聚山梨酯 20 批次，只有 18-23%的物质是预期的结构，即聚氧乙烯山梨醇单月桂酸酯。在聚山梨酯 20 和聚山梨酯 80 中，分别发现了高达 40%和 70%（w/w）的二酯和三酯。同样，聚氧乙烯山梨醇单油酸酯在聚山梨酯 80 中仅占 20%。在多个批次的聚山梨酯 20 之间，每种酯类物质的差异为 3-5%。然后，使用反相方法在室温碱性溶液中测定聚山梨酯 20 中每种聚氧乙烯山梨醇酯的水解速率。随着聚山梨酯 20 中脂肪链长度的降低，水解速率逐渐增加。
