Khossravi Mehrnaz, Kao Yung-Hsiang, Mrsny Randall J, Sweeney Theresa D
Pharmaceutical R & D, Genentech Inc., South San Francisco, California 94080, USA.
Pharm Res. 2002 May;19(5):634-9. doi: 10.1023/a:1015306112979.
Polysorbate 20 is a commonly used excipient in biopharmaceutical formulations, some of which may have an enzymatic activity. The action(s) of polysorbate 20 in biopharmaceutical formulations as a stabilizer require this surfactant to maintain its intact structure. This manuscript evaluates a new analytic method for the analysis of polysorbate 20 degradation in the format of a biopharmaceutical formulation and makes a comparison with several established methods of analysis.
Polysorbate 20 samples were degraded in a controlled environment utilizing the enzyme pancreatic lipase to generate degradants that included lauric acid and the sorbitan polyoxyethylene side chain. A new method was developed with sufficient sensitivity to analyze the degraded solutions. Lauric acid was derivatized with the fluorescent reagent 9-anthryldiazomethane to form 9-anthrylmethylethyl ester. The derivatized lauric acid was separated by reversed-phase chromatography and detected by fluorescence or UV spectroscopy. Three established methods utilized to measure polysorbate 20 were evaluated for their ability to detect degraded polysorbate 20. These methods were: (1) fluorescence analysis with N-phenyl-1-naphthylamine fluorescent dye; (2) UV spectroscopy with ammonium cobaltothiocyanate colorimetric reagent; and (3) nuclear magnetic resonance (NMR).
Polysorbate 20 incubation with lipase resulted in degraded polysorbate 20 as determined by the derivatized lauric acid assay. The UV spectroscopy assay utilizing ammonium cobaltothiocyanate reagent was not able to detect the degradation of polysorbate 20 in the samples. The fluorescence method of analysis detected polysorbate 20 degradation as an approximate 50% decrease in micelles in comparison to standard nondegraded polysorbate 20 solutions. NMR analysis resulted in similar proton peak areas for both degraded and nondegraded polysorbate 20 samples. NMR spectra did contain minor differences between the samples.
It is essential to choose the appropriate method of polysorbate 20 evaluation to assess the content, stability, and compatibility of a formulation. Current established methods to assess polysorbate 20 may overlook and do not necessarily monitor the potential degradation of this surfactant, which results in the formation of lauric acid. Because this type of degradation may occur in a formulation by an enzymatically active biopharmaceutical, a new method of analysis has been established.
聚山梨酯20是生物制药制剂中常用的辅料，其中一些制剂可能具有酶活性。聚山梨酯20在生物制药制剂中作为稳定剂发挥作用需要这种表面活性剂保持其完整结构。本文评估了一种以生物制药制剂形式分析聚山梨酯20降解的新分析方法，并与几种既定的分析方法进行了比较。
利用胰脂肪酶在可控环境中使聚山梨酯20样品降解，生成包括月桂酸和脱水山梨醇聚氧乙烯侧链在内的降解产物。开发了一种具有足够灵敏度的新方法来分析降解后的溶液。月桂酸用荧光试剂9-蒽基重氮甲烷衍生化，形成9-蒽基甲基乙酯。衍生化的月桂酸通过反相色谱分离，并用荧光或紫外光谱检测。评估了三种用于测定聚山梨酯20的既定方法检测降解的聚山梨酯20的能力。这些方法是：（1）用N-苯基-1-萘胺荧光染料进行荧光分析；（2）用硫氰酸钴铵比色试剂进行紫外光谱分析；（3）核磁共振（NMR）。
通过衍生化月桂酸测定法确定，聚山梨酯20与脂肪酶孵育导致聚山梨酯20降解。使用硫氰酸钴铵试剂的紫外光谱分析无法检测样品中聚山梨酯20的降解。荧光分析方法检测到聚山梨酯20降解，与标准未降解聚山梨酯20溶液相比，胶束减少约50%。核磁共振分析得出降解和未降解聚山梨酯20样品的质子峰面积相似。样品之间的核磁共振谱确实存在微小差异。
选择合适的聚山梨酯20评估方法对于评估制剂的含量、稳定性和相容性至关重要。目前用于评估聚山梨酯20的既定方法可能会忽略且不一定能监测到这种表面活性剂的潜在降解，这种降解会导致月桂酸的形成。由于这种类型的降解可能在具有酶活性的生物制药制剂中发生，因此建立了一种新的分析方法。
