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在国际人用药品注册技术协调会Q14指南框架下重新审视细胞外囊泡的纳米流式细胞术定量分析

Revisiting the Nanoflow Cytometric Quantification of Extracellular Vesicles Under the Framework of ICH Q14 Guidelines.

作者信息

Li Ganghui, Cai Qizhe, Dong Yanan, Li Xiang, Qin Xi, Xue Miaomiao, Song Haifeng, Wang Yi

机构信息

China Pharmaceutical University Nanjing China.

Department of Ultrasound Beijing Chao-Yang Hospital Capital Medical University Beijing China.

出版信息

J Extracell Biol. 2025 May 8;4(5):e70050. doi: 10.1002/jex2.70050. eCollection 2025 May.

DOI:10.1002/jex2.70050
PMID:40342572
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12060124/
Abstract

Nanoflow cytometry (nanoFCM) is an increasingly important analytical procedure in every aspect of extracellular vesicle (EV) research, particularly in the development of EV-based therapeutics. The main objective of this study was to evaluate and optimise the key determinant factors of nanoFCM in the quantification analysis of EVs to ensure its consistency and reliability in the development of EV therapeutic drugs, thereby serving as a potential quality control measure. Our investigation followed the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q14 guideline. We revisited the day-to-day practice of nanoFCM measurement for HEK293 cell-derived and milk-derived EVs (mEVs), focusing on optimising particle quantification and identifying risk factors. Initial evaluation of the procedure revealed a considerable lack of consistency and reliability, which was then subjected to extensive optimisation. The key outcomes of this study include: (1) an optimised analytic procedure incorporating Tween-20, which significantly enhanced the precision and accuracy of the nanoFCM measurement and expanded the reportable range; (2) an analytical target profile (ATP) which provides a preliminary standard for future validation of nanoFCM procedures. Overall, this study serves as a foundation for future efforts towards the standardisation of analytical procedures for EV therapeutics.

摘要

纳米流式细胞术（nanoFCM）在细胞外囊泡（EV）研究的各个方面都是一种日益重要的分析方法，尤其是在基于EV的治疗药物研发中。本研究的主要目的是评估和优化nanoFCM在EV定量分析中的关键决定因素，以确保其在EV治疗药物研发中的一致性和可靠性，从而作为一种潜在的质量控制措施。我们的研究遵循了人用药品注册技术国际协调理事会（ICH）Q14指南。我们重新审视了nanoFCM对人胚肾293细胞衍生的EV和牛奶衍生的EV（mEV）进行测量的日常操作，重点是优化颗粒定量和识别风险因素。对该方法的初步评估显示出相当缺乏一致性和可靠性，随后对其进行了广泛的优化。本研究的关键成果包括：（1）一种结合吐温-20的优化分析方法，该方法显著提高了nanoFCM测量的精密度和准确性，并扩大了可报告范围；（2）一个分析目标概况（ATP），它为nanoFCM方法的未来验证提供了初步标准。总体而言，本研究为未来EV治疗药物分析方法标准化的工作奠定了基础。

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Isolation and quantification of human urinary exosomes using a Tween-20 elution solvent from polyester, capillary-channeled polymer fiber columns.使用聚酯、毛细管通道聚合物纤维柱中的吐温-20 洗脱溶剂从人尿中分离和定量外泌体。
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Invest Ophthalmol Vis Sci. 2024 Mar 5;65(3):6. doi: 10.1167/iovs.65.3.6.
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