Analytical Chemistry, Regeneron Pharmaceuticals, Tarrytown, NY, 10591, USA.
Formulation Group, Regeneron Pharmaceuticals, Tarrytown, NY, 10591, USA.
Anal Biochem. 2022 Jan 15;637:114472. doi: 10.1016/j.ab.2021.114472. Epub 2021 Nov 18.
Polysorbates (PSs) are surfactants commonly added to therapeutic protein drug product formulations to protect proteins from denaturation and aggregation during storage, transportation, and delivery. However, enzymatic hydrolysis of PSs has been recognized as the primary route of PS degradation in monoclonal antibody formulations, resulting in the release of free fatty acids that drive undesired particulate formation. Here, we present a rapid lipase activity assay with optimized incubation conditions for accurate quantitation of free fatty acids without a fatty acid extraction step. This assay can detect low levels of PS degradation (0.000024% PS20 degradation) within 1 day with minimal sample preparation. The levels of released free fatty acids were found to strongly correlate with the degree of PS20 degradation. The case study described herein suggests that this approach can detect low levels of PS20 degradation caused by sub-ppm lipase levels within 1 day, compared with the duration of 14 days needed for PS degradation assays based on two-dimensional liquid chromatography-charge aerosol detection.
聚山梨酯（PS）是一种常用的表面活性剂，添加到治疗性蛋白药物产品配方中，以保护蛋白质在储存、运输和给药过程中免于变性和聚集。然而，酶水解聚山梨酯已被认为是单克隆抗体配方中聚山梨酯降解的主要途径，导致释放游离脂肪酸，从而驱动不希望的颗粒形成。在这里，我们提出了一种快速的脂肪酶活性测定方法，优化了孵育条件，可在无需脂肪酸提取步骤的情况下准确定量游离脂肪酸。该测定法可在 1 天内检测到低水平的 PS 降解（PS20 降解 0.000024%），样品制备量最少。结果表明，释放的游离脂肪酸水平与 PS20 降解程度密切相关。本文所述的案例研究表明，与基于二维液相色谱-荷质比检测的 PS 降解测定法需要 14 天相比，该方法可在 1 天内检测到由亚ppm 级脂肪酶水平引起的 PS20 低水平降解。
