Abstract：
OBJECTIVE: Polysorbates are among the most used surfactants in biopharmaceutical products containing proteins. Our work aims to develop a high-throughput fluorometric assay to further diversify the analytical toolbox for quantification of PSs.
METHODS: The assay leverages the micelle activated fluorescence signal from N-Phenyl-1-Naphthylamine (NPN). The development and optimization of assay parameters were guided by the pre-defined analytical target profile. Furthermore, NMR was used to probe the interaction between protein, PS80 and NPN in the measurement system and understand protein interference.
RESULTS: All assay parameters including excitation and emission wavelengths, standard curve, NPN concentration, and incubation time have been optimized and adapted to a microplate format, making it compatible with automated solutions that will be pursued in the near future to drive consistency and efficiency in our workflows. The specificity, accuracy, and precision of the assay have been demonstrated through a case study. Furthermore, NMR results provided additional insight into the change of the interaction dynamics between PS80 and NPN as the protein concentration increases. The results indicate minimal interaction between the protein and PS80 at lower concentration. However, when the concentration exceeds 75 mg/mL, there is a significant interaction between the protein and PS-80 micelle and monomer.
CONCLUSIONS: A high-throughput fluorometric assay has been developed for quantification of polysorbates in biopharmaceutical samples including in-process samples, drug substance and drug product. The assay reported herein could serve as a powerful analytical tool for polysorbate quantification and control, complementing the widely used liquid chromatography with charged aerosol detection method.
METHODS: The assay leverages the micelle activated fluorescence signal from N-Phenyl-1-Naphthylamine (NPN). The development and optimization of assay parameters were guided by the pre-defined analytical target profile. Furthermore, NMR was used to probe the interaction between protein, PS80 and NPN in the measurement system and understand protein interference.
RESULTS: All assay parameters including excitation and emission wavelengths, standard curve, NPN concentration, and incubation time have been optimized and adapted to a microplate format, making it compatible with automated solutions that will be pursued in the near future to drive consistency and efficiency in our workflows. The specificity, accuracy, and precision of the assay have been demonstrated through a case study. Furthermore, NMR results provided additional insight into the change of the interaction dynamics between PS80 and NPN as the protein concentration increases. The results indicate minimal interaction between the protein and PS80 at lower concentration. However, when the concentration exceeds 75 mg/mL, there is a significant interaction between the protein and PS-80 micelle and monomer.
CONCLUSIONS: A high-throughput fluorometric assay has been developed for quantification of polysorbates in biopharmaceutical samples including in-process samples, drug substance and drug product. The assay reported herein could serve as a powerful analytical tool for polysorbate quantification and control, complementing the widely used liquid chromatography with charged aerosol detection method.
摘要：
目的：聚山梨醇酯是含有蛋白质的生物制药产品中最常用的表面活性剂之一。我们的工作旨在开发一种高通量荧光测定法，以进一步使用于PS定量的分析工具箱多样化。
方法:该测定利用来自N-苯基-1-萘胺(NPN)的胶束激活的荧光信号。测定参数的开发和优化由预定义的分析靶标概况指导。此外，NMR用于探测蛋白质之间的相互作用，PS80和NPN在丈量体系中懂得卵白质的搅扰。
结果：所有测定参数，包括激发和发射波长，标准曲线,NPN浓度,和孵育时间已经过优化和适应微孔板格式，使其与将在不久的将来追求的自动化解决方案兼容，以推动我们工作流程的一致性和效率。特异性，准确度,并通过案例研究证明了分析的准确性。此外，NMR结果为PS80和NPN之间的相互作用动力学随蛋白质浓度增加的变化提供了额外的见解。结果表明在较低浓度下蛋白质与PS80之间的相互作用最小。然而,当浓度超过75mg/mL时，蛋白质与PS-80胶束和单体之间存在显著的相互作用。
结论：已经开发了一种高通量荧光测定法，用于定量生物制药样品中的聚山梨醇酯，包括过程中的样品，原料药和药品。本文报道的测定可以作为聚山梨酯定量和控制的强大分析工具，用带电气溶胶检测方法补充了广泛使用的液相色谱。
方法:该测定利用来自N-苯基-1-萘胺(NPN)的胶束激活的荧光信号。测定参数的开发和优化由预定义的分析靶标概况指导。此外，NMR用于探测蛋白质之间的相互作用，PS80和NPN在丈量体系中懂得卵白质的搅扰。
结果：所有测定参数，包括激发和发射波长，标准曲线,NPN浓度,和孵育时间已经过优化和适应微孔板格式，使其与将在不久的将来追求的自动化解决方案兼容，以推动我们工作流程的一致性和效率。特异性，准确度,并通过案例研究证明了分析的准确性。此外，NMR结果为PS80和NPN之间的相互作用动力学随蛋白质浓度增加的变化提供了额外的见解。结果表明在较低浓度下蛋白质与PS80之间的相互作用最小。然而,当浓度超过75mg/mL时，蛋白质与PS-80胶束和单体之间存在显著的相互作用。
结论：已经开发了一种高通量荧光测定法，用于定量生物制药样品中的聚山梨醇酯，包括过程中的样品，原料药和药品。本文报道的测定可以作为聚山梨酯定量和控制的强大分析工具，用带电气溶胶检测方法补充了广泛使用的液相色谱。
