Purpose

Silicone oil droplets in biopharmaceutical products can originate from sources such as siliconized surfaces of primary packaging materials, potentially triggering the formation of protein–silicone oil particles. To better understand this phenomenon, there is a need for particle detection devices that cannot only distinguish between protein particles and silicone oil droplets but also determine particle sizes ranging from nanometers to micrometers.

Method

In this study, we conducted a systematic assessment of imaging flow cytometry (IFC) using the FlowSight® instrument. Our first step was to investigate specific instrument settings using protein particle samples spiked with silicone oil for particle classification. Based on these findings, we established suitable, harmonized working templates. Next, we evaluated the instrument’s accuracy and precision for particle sizes within the range of 0.5 to 100 µm and their respective concentrations. Finally, we investigated any constraints in particle concentration within this size range.

Results

This study demonstrates that IFC can effectively distinguish protein particles from silicone oil droplets when the latter is labeled with a specific fluorescent dye. Our findings suggest that fluorescently labeled particles ≥ 0.5 µm can be reliably detected. Through our research, we determined the particle concentration limits for each particle size in the range of 0.5 to 10 µm, with a precision deviation of less than 15%. However, our study also revealed that IFC exhibited insufficient accuracy for the tested particle concentrations within this size range. Additionally, we showed that the measurements were significantly influenced by the instrument settings.

Conclusion

Although we addressed numerous new aspects to enhance the experimental procedure of IFC measurements, we conclude that IFC is not an ideal technique for quantifying sub-visible particles. Instead, it should be employed to provide supportive characterization data in conjunction with commonly used sub-visible particle detection methods. If distinguishing between protein particles and silicone oil droplets is essential, IFC is an option, as long as the fluorescent dye is carefully selected.

Graphical Abstract

目的

生物制药产品中的硅油滴可能源自初级包装材料的硅化表面等来源，可能引发蛋白质硅油颗粒的形成。为了更好地理解这种现象，需要一种颗粒检测装置，不仅可以区分蛋白质颗粒和硅油滴，还可以确定从纳米到微米的颗粒尺寸。

方法

在本研究中，我们使用 FlowSight® 仪器对成像流式细胞仪 (IFC) 进行了系统评估。我们的第一步是使用掺有硅油的蛋白质颗粒样品来研究特定的仪器设置，以进行颗粒分类。根据这些发现，我们建立了合适的、统一的工作模板。接下来，我们评估了仪器对 0.5 至 100 µm 范围内的粒径及其各自浓度的准确度和精密度。最后，我们研究了该尺寸范围内颗粒浓度的任何限制。

结果

这项研究表明，当后者用特定的荧光染料标记时，IFC 可以有效地区分蛋白质颗粒和硅油滴。我们的研究结果表明，可以可靠地检测到 ≥ 0.5 µm 的荧光标记颗粒。通过研究，我们确定了每个粒径的颗粒浓度限值在 0.5 至 10 µm 范围内，精度偏差小于 15%。然而，我们的研究还表明，IFC 在此尺寸范围内测试的颗粒浓度的准确性不足。此外，我们还表明测量结果受到仪器设置的显着影响。

结论

尽管我们解决了许多新问题来增强 IFC 测量的实验程序，但我们得出的结论是，IFC 并不是量化亚可见粒子的理想技术。相反，它应该与常用的亚可见粒子检测方法一起提供支持性的表征数据。如果必须区分蛋白质颗粒和硅油滴，只要仔细选择荧光染料，IFC 就是一种选择。

图形概要