Shaking stress studies are typically performed during formulation development to test the liability of a drug product towards interfacial stress occurring during transport, especially if a liquid formulation is desired. We evaluated various shaking procedures using a polyA-surrogate solution and verified our findings by eGFP-LNP cell-expression experiments. Shaking on an orbital shaker in vertical and horizontal orientations at increasing speeds from 300 to 600 rpm resulted in decreasing levels of encapsulated nucleic acid content, larger LNP sizes, and decreasing PDI. We report that vertical and horizontal shaking of both polyA- and eGFP-LNPs led to white deposits on the inner glass vial surface, depending on time, rpm, and temperature. Increasing the fill volume/smaller headspace (0.3 versus 0.9 mL fill) did not mitigate this phenomenon in the studied configuration, and the use of hydrophobic primary packaging even accelerated the formation of white deposits. In contrast, we demonstrated that a lyophilized polyA-LNP dosage form was less susceptible to shaking and maintained cake integrity and product properties. Multiple vortexing steps resulted in an increase in LNP size, PDI, and a decrease in encapsulated polyA content. We conclude that shaking experiments of nucleic acid-loaded LNPs in their final configuration at intended transport conditions need to be considered during technical development.

中文翻译：

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别摇动它！ mRNA-脂质纳米粒子的机械应力测试

振动应力研究通常在制剂开发过程中进行，以测试药品对运输过程中发生的界面应力的责任，特别是在需要液体制剂的情况下。我们使用聚腺苷酸替代溶液评估了各种摇动程序，并通过 eGFP-LNP 细胞表达实验验证了我们的发现。在轨道摇床上以垂直和水平方向摇动，速度从 300 rpm 增加到 600 rpm，导致封装的核酸含量水平降低、LNP 尺寸增大和 PDI 降低。我们报告说，polyA-和 eGFP-LNP 的垂直和水平摇动会导致玻璃瓶内表面出现白色沉积物，具体取决于时间、转速和温度。增加填充体积/较小的顶部空间（0.3 毫升与 0.9 毫升填充）并没有减轻所研究配置中的这种现象，并且疏水性初级包装的使用甚至加速了白色沉积物的形成。相比之下，我们证明冻干聚 A-LNP 剂型不易摇动，并能保持饼的完整性和产品特性。多次涡旋步骤导致 LNP 大小、PDI 增加，以及封装的聚 A 含量减少。我们的结论是，在技术开发过程中需要考虑在预期运输条件下最终配置的核酸负载 LNP 的振荡实验。