Luminex Corporation, Seattle, Washington 98119.
J Pharm Sci. 2020 Jan;109(1):364-374. doi: 10.1016/j.xphs.2019.05.018. Epub 2019 May 25.
Protein therapeutic exposure to siliconized surfaces during manufacturing and storage has potential to induce protein aggregation or generate protein-silicone complexes that are potentially immunogenic. Consequently, assessing the stability and safety of protein therapeutics requires discrimination of protein and silicone oil particles and evaluation of protein-silicone oil interactions. Industry-established methods are challenged to distinguish protein aggregates from silicone oil droplets for particles smaller than 5 μm. In addition, emerging techniques for accessing particles in the sub-5 μm range are limited by low sampling volumes, narrow size ranges, complications such as clogging, and an inability to evaluate protein-silicone interactions. In this study, imaging flow cytometry was evaluated as a new method for discrimination of protein aggregates and silicone oil droplets, as well as for quantification of protein-silicone interactions. A simple and fast fluorescence labeling protocol using low concentrations of extrinsic dyes was developed. The results demonstrate that imaging flow cytometry can be used to discriminate fluorescently labeled protein aggregates and silicone oil droplets with greater than 95% accuracy, regardless of size, and protein-silicone oil interactions can be assessed qualitatively through inspection of image data or quantitatively using features extracted from the image data.
在制造和储存过程中，蛋白质治疗剂与硅化表面接触，有可能导致蛋白质聚集或产生潜在免疫原性的蛋白质-硅复合物。因此，评估蛋白质治疗剂的稳定性和安全性需要区分蛋白质和硅油颗粒，并评估蛋白质-硅油相互作用。对于小于 5 μm 的颗粒，行业中建立的方法难以将蛋白质聚集体与硅油液滴区分开来。此外，用于检测亚 5 μm 范围内颗粒的新兴技术受到采样体积小、尺寸范围窄、堵塞等并发症以及无法评估蛋白质-硅油相互作用的限制。在这项研究中，成像流式细胞术被评估为一种新的方法，用于区分蛋白质聚集体和硅油液滴，以及定量评估蛋白质-硅油相互作用。开发了一种简单快速的荧光标记方案，使用低浓度的外源性染料。结果表明，成像流式细胞术可以用于区分荧光标记的蛋白质聚集体和硅油液滴，准确率超过 95%，无论颗粒大小如何，并且可以通过检查图像数据进行定性评估蛋白质-硅油相互作用，或使用从图像数据中提取的特征进行定量评估。
