Characterization of Protein Aggregates, Silicone Oil Droplets, and Protein-Silicone Interactions Using Imaging Flow Cytometry
Characterization of Protein Aggregates, Silicone Oil Droplets, and Protein-Silicone Interactions Using Imaging Flow Cytometry
复制标题
DOI：
10.1016/j.xphs.2019.05.018
复制
发表时间：
2020-01-01
影响因子：
3.8
通讯作者：
Probst, Christine
中科院分区：
文献类型：
--
作者：
Probst, Christine
在生产和储存过程中，蛋白质治疗剂暴露于硅化表面有可能诱导蛋白质聚集或产生具有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价蛋白质硅油相互作用。对于小于5 μ m的颗粒，工业上建立的方法难以区分蛋白质聚集体和硅油滴。此外，用于获取低于5 μ m范围内颗粒的新兴技术受到以下因素的限制：低采样体积、窄尺寸范围、堵塞等并发症以及无法评估蛋白质-硅胶相互作用。在本研究中，成像流式细胞术作为区分蛋白质聚集体和硅油滴以及定量蛋白质-硅油相互作用的新方法进行了评价。开发了一种使用低浓度外源染料的简单快速的荧光标记方案。结果表明，成像流式细胞术可用于区分荧光标记的蛋白质聚集体和硅油液滴，准确度大于95%，无论大小如何，并且可通过检查图像数据或使用从图像数据中提取的特征定量评估蛋白质-硅油相互作用。(C)2020年，任作家。由爱思唯尔公司出版美国药学协会（American Pharmacopoeia Association）（R）这是一个在CC BY-NC-ND许可证下的开放获取文章（http：//creativecommons.org/licenses/by-nc-nd/4.0/）。
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 mu m. In addition, emerging techniques for accessing particles in the sub-5 mu m 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. (C) 2020 The Authors. Published by Elsevier Inc. on behalf of the American Pharmacists Association (R). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).