Thompson Benjamin R, Pham Kiet G, Phan Minh D, Sanchez-Puga Pablo, Gutfreund Philipp, Wang Tingting, Qian Ken K, Heinrich Frank, Liu Yun, Wagner Norman J
Department of Chemical & Biomolecular Engineering, Center for Neutron Science, University of Delaware, Newark, Delaware 19716, United States.
NIST Center for Neutron Research, National institute of Standards and Technology, Gaithersburg, Maryland 20899, United States.
ACS Appl Mater Interfaces. 2025 Jul 16;17(28):40116-40128. doi: 10.1021/acsami.5c07162. Epub 2025 Jul 1.
Monoclonal antibodies (mAbs), due to their amphiphilicity, adsorb strongly at the air-water (a/w) interface, such that interfacial stresses can lead to the formation of visible and subvisible particles in solution that adversely affect the therapeutic efficacy and immunogenicity of protein formulations. Furthermore, prior work in our group shows that the interfacial shear elastic modulus of mAbs adsorbed at the a/w interface correlates strongly with the long-term (3 year) stability in solution, providing a possible means to accelerate the testing of formulations. To mitigate interface-induced instability in formulation and delivery, the biopharmaceutical industry employs excipients, such as nonionic surfactants, to rapidly adsorb to and protect against mAb adsorption at interfaces. However, the molecular interactions at the a/w interface during mechanical deformation are not established despite their technological importance. To address this need, the adsorption of a mAb (studied previously) at the a/w interface in the presence and absence of the nonionic surfactant Poloxamer 188 (P188) is examined through measurements of interfacial pressure and rheology, Brewster angle microscopy (BAM), and neutron reflectometry (NR). We find that mAb adsorbs rapidly to the air-water interface, but the evolution of the surface pressure and interfacial shear moduli continues long after the surface excess has reached a plateau. NR measurements quantify changes in the molecular layer when undergoing dilatational stresses, which may occur during the storage and transport of mAbs. The injection of P188 into the subphase below an established mAb interface increases the surface pressure and decreases interfacial shear moduli, but mAbs remain at the interface with a modified surface topology. Comparisons to adsorption from a premixed mAb-P188 solution highlight the importance of the order of addition in therapeutic formulations, and the results in totality provide molecular evidence for the effect of the a/w interface on mAb interactions and stability.
单克隆抗体（mAb）由于具有两亲性，会在空气 - 水（a/w）界面强烈吸附，以至于界面应力会导致溶液中形成可见和亚可见颗粒，这会对蛋白质制剂的治疗效果和免疫原性产生不利影响。此外，我们小组之前的研究表明，吸附在a/w界面的mAb的界面剪切弹性模量与溶液中的长期（3年）稳定性密切相关，这为加速制剂测试提供了一种可能的方法。为了减轻制剂和给药过程中由界面引起的不稳定性，生物制药行业使用辅料，如非离子表面活性剂，来快速吸附并防止mAb在界面吸附。然而，尽管其在技术上具有重要性，但在机械变形过程中a/w界面处的分子相互作用尚未明确。为满足这一需求，通过测量界面压力和流变学、布鲁斯特角显微镜（BAM）和中子反射率（NR），研究了一种mAb（之前已研究过）在有和没有非离子表面活性剂泊洛沙姆188（P188）存在的情况下在a/w界面的吸附情况。我们发现mAb会迅速吸附到空气 - 水界面，但在表面过量达到平稳后，表面压力和界面剪切模量仍会持续变化。NR测量量化了在经历膨胀应力时分子层的变化，这种应力可能在mAb的储存和运输过程中出现。将P188注入已形成的mAb界面下方的亚相中会增加表面压力并降低界面剪切模量，但mAb仍留在界面上，表面拓扑结构发生了改变。与从预混合的mAb - P188溶液中吸附的比较突出了在治疗制剂中添加顺序的重要性，并总体结果为a/w界面在mAb相互作用和稳定性方面的影响提供了分子证据。
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