Hassan Alaa, Diederichs Tim, Garidel Patrick, Heerklotz Heiko
Institute of Pharmaceutical Sciences, Department of Pharmaceutics, University of Freiburg, Hermann-Herder-Str. 9, Freiburg 79104, Germany.
Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.
Mol Pharm. 2025 Jun 2;22(6):2917-2926. doi: 10.1021/acs.molpharmaceut.4c01268. Epub 2025 May 14.
Polysorbates (PSs) are key excipients for the colloidal stability of biopharmaceuticals with unique properties. A comprehensive understanding of the physicochemical properties of these multicomponent products is essential to address potential stability issues without compromising their functionality. Here, we demonstrate that polysorbate 80 HP (PS80) shows an anomalous clouding, i.e., a thermotropic liquid-liquid phase separation behavior, which cannot adequately be explained by the conventional interpretation assuming a pseudobinary system. In a binary two-phase system of surfactant and buffer, an increase in the total surfactant concentration increases the fraction of the surfactant-rich phase in the respective proportion (lever rule). PS80 within about 7 K of the lower critical solution temperature fails to comply with this; concentrations and compositions of the coexisting phases change with the total concentration. This renders the phases more alike and, at some point, eliminates phase separation. This significant deviation from the pseudobinary phase behavior can be resolved by conceptually dividing the numerous chemical species in PS80 into two independent pseudocomponents, PS80-I and -II. Ternary phase diagrams derived from this approach successfully explain the observed anomalous behavior. RP-UPLC-MS analysis indicated a concentration-dependent redistribution of the nonesterified components (NECs), suggesting, along with other evidence, that NECs are key constituents of component II. Specifically, free polyethylene glycol (PEG) and/or PEG-sorbitans seem to function as intrinsic cosurfactant(s) within PS80, modulating its wetting and clouding properties. The latter is important for interaction, association, and phase separation properties in biologics.
聚山梨酯（PSs）是具有独特性质的生物药物胶体稳定性的关键辅料。全面了解这些多组分产品的物理化学性质对于解决潜在的稳定性问题而不损害其功能至关重要。在此，我们证明聚山梨酯80 HP（PS80）表现出异常的浑浊现象，即热致液-液相分离行为，这无法用假设为假二元体系的传统解释充分解释。在表面活性剂和缓冲液的二元两相体系中，总表面活性剂浓度的增加会按相应比例增加富含表面活性剂相的比例（杠杆规则）。在较低临界溶液温度约7 K范围内的PS80并不符合此规律；共存相的浓度和组成随总浓度而变化。这使得各相更加相似，并且在某一时刻消除了相分离。通过将PS80中的众多化学物质概念性地分为两个独立的假组分PS80-I和-II，可以解决与假二元相行为的这一显著偏差。由此方法得出的三元相图成功地解释了观察到的异常行为。RP-UPLC-MS分析表明未酯化成分（NECs）存在浓度依赖性的重新分布，这与其他证据一起表明NECs是组分II的关键成分。具体而言，游离聚乙二醇（PEG）和/或聚乙二醇脱水山梨醇似乎在PS80中作为内在的共表面活性剂发挥作用，调节其润湿性和浑浊特性。后者对于生物制品中的相互作用、缔合和相分离特性很重要。
