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Size Exclusion Chromatography with Dual Wavelength Detection as a Sensitive and Accurate Method for Determining the Empty and Full Capsids of Recombinant Adeno-Associated Viral Vectors

衣壳 重组DNA 病毒学 遗传增强 大小排阻色谱法 基因治疗载体 分子生物学 腺相关病毒 分辨率(逻辑) 病毒 色谱法 化学 生物 载体(分子生物学) 基因 病毒载体 计算机科学 生物化学 人工智能
作者
Meng He,Michelle Sorrentino,Denise Woodcock,Catherine R. O’Riordan,Vijender Dhawan,Marc F. J. M. Verhagen,Claire L. Davies
出处
期刊:Human Gene Therapy [Mary Ann Liebert, Inc.]
卷期号:33 (3-4): 202-212 被引量:24
标识
DOI:10.1089/hum.2021.123
摘要

Gene therapy has evolved over the past decade into a promising therapeutic class for treating many intractable diseases. Recombinant adeno-associated virus (AAV) is the most commonly used viral vector for delivering therapeutic genes. Independent of the manufacturing process for AAVs, the clinical materials are inherently heterogeneous and contain both empty and full capsids. Empty capsids can impact the safety and efficacy of AAV products and therefore their level needs to be controlled. Several analytical methods have been reported for this purpose. However, some of these methods have an insufficient assay range, or rely on instruments that cannot be readily implemented in a quality control (QC) environment. In this study, we describe a fast size exclusion chromatography (SEC) assay with dual-wavelength detection (SEC-DW) to directly determine the percent full capsids of AAV samples based on their peak area (PA) ratios. The two detection wavelengths selected to represent encapsidated transgenes and capsid proteins were 260 and 230 nm, respectively, instead of the conventionally used 260 and 280 nm. The use of 230 nm instead of 280 nm to monitor the contribution of the capsid protein results in a linear relationship between the PA260/PA230 ratio and the percent full capsids, unlike the nonlinear relationship observed when the PA260/PA280 ratio is used. As a result, the method exhibits a significantly extended assay range (up to 91% full capsids). The accuracy of the SEC-DW method was confirmed by comparing the results obtained against results from orthogonal high-resolution methods such as analytical ultracentrifugation (AUC) and cryo-electron microscopy and excellent agreement was obtained when common samples were analyzed using different methods. The SEC-DW method runs on a readily accessible high-performance liquid chromatography instrument platform, provides much higher assay throughput compared with AUC and electron microscopy, and can be implemented as a release method in a QC environment or used as a rapid screening tool to support process development and product understanding.
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