Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zürich, 8093 Zürich, Switzerland.
Cilag GmbH International, a Division of Johnson & Johnson TDS-Biologics, Analytical Development, 8200 Schaffhausen, Switzerland.
Mol Pharm. 2024 Nov 4;21(11):5761-5771. doi: 10.1021/acs.molpharmaceut.4c00754. Epub 2024 Oct 23.
Polysorbates 20 and 80 (PS20 and PS80) are commonly used in the formulations of biologics to protect against interfacial stresses. However, these surfactants can degrade over time, releasing free fatty acids, which assemble into solid particles or liquid droplets. Here, we apply a droplet microfluidic platform to analyze the interactions between antibodies and oleic acid, the primary free fatty acid resulting from the hydrolysis of PS80. We show that antibodies adsorb within seconds to the polar oleic acid-water interface, forming a viscoelastic protein layer that leads to particle formation upon mechanical rupture. By testing two different monoclonal antibodies of pharmaceutical origin, we show that the propensity to form a rigid viscoelastic layer is protein-specific. We further demonstrate that intact PS80 is effective in preventing antibody adsorption at the oleic acid-water interface only at low antibody concentrations and low pH, where oleic acid is fully protonated. Importantly, introduction of the amino acid l-arginine prevents the formation of the interfacial layer and protein particles even at high antibody concentrations (180 mg mL). Overall, our findings indicate that oleic acid droplets in antibody formulations can lead to the formation of protein particles via an interface-mediated mechanism. Depending on the conditions, intact PS80 alone might not be sufficient to protect against antibody aggregation. Additional mitigation strategies include the optimization of protein physicochemical properties, pH, and the addition of arginine.
聚山梨酯 20 和 80（PS20 和 PS80）常用于生物制剂的配方中，以保护其免受界面张力的影响。然而，这些表面活性剂会随着时间的推移而降解，释放出游离脂肪酸，这些脂肪酸会组装成固体颗粒或液滴。在这里，我们应用液滴微流控平台来分析抗体与油酸之间的相互作用，油酸是 PS80 水解产生的主要游离脂肪酸。我们发现，抗体在几秒钟内就会吸附到极性的油酸-水界面上，形成一个粘弹性的蛋白质层，当受到机械破坏时会形成颗粒。通过测试两种不同的、源自药物的单克隆抗体，我们表明形成刚性粘弹性层的倾向是蛋白质特异性的。我们进一步证明，完整的 PS80 只有在低抗体浓度和低 pH 值（此时油酸完全质子化）下，才能有效防止抗体在油酸-水界面上的吸附。重要的是，引入氨基酸 l-精氨酸甚至在高抗体浓度（180mg/mL）下也能防止界面层和蛋白质颗粒的形成。总的来说，我们的研究结果表明，抗体制剂中的油酸液滴可能通过界面介导的机制导致蛋白质颗粒的形成。根据具体情况，完整的 PS80 本身可能不足以防止抗体聚集。其他缓解策略包括优化蛋白质的物理化学性质、pH 值以及添加精氨酸。
