Leibiger Thomas M, Min Lie, Lee Kelvin H
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, United States.
Front Bioeng Biotechnol. 2025 May 2;13:1579098. doi: 10.3389/fbioe.2025.1579098. eCollection 2025.
Analysis of residual host cell proteins in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. One strategy to address these challenges is through development of improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods with greater sensitivity and reduced sample requirement.
In this work, we compare the performance of four sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) methods for identification and quantitation of residual HCPs in rAAV2, -5, -8, and -9 preparations produced with human embryonic kidney 293 (HEK293) cells and purified using immunoaffinity chromatography. Key SWATH-MS parameters including spectral library construction (data dependent vs. ), data processing software (DIA-NN vs. Skyline), and mass spectrometer instrument (Sciex TripleTOF 6600 vs. Sciex ZenoTOF 7600) were assessed. Method attributes including sample requirement and processing time, and method outputs including protein and precursor identifications, host cell protein quantitation comparisons across methods, and quantitation coefficients of variance (CV) were considered to help establish a SWATH-MS workflow well-suited for rAAV HCP analytics.
A 78% increase in HCP identifications, 80% reduction in sample requirement, and 70% reduction in instrument runtime was achieved with an spectral library, data processing in DIA-NN, and data collection with the Sciex ZenoTOF 7600 instrument (DIA-NN-7600 method) compared to a previously established method using a DDA-derived spectral library, data processing in Skyline, and data collection with the Sciex TripleTOF 6600 instrument (Skyline-DDA-6600 method). Additionally, the DIA-NN-7600 method shows median HCP quantitation CV below 10% for triplicate data acquisitions, and comparable quantitation to other methods for a panel of highly abundant residual HCPs previously identified in rAAV downstream processing.
This work highlights a SWATH-MS method with data collection and processing specifically tailored for rAAV residual HCP analysis.
由于腺相关病毒（AAV）制剂中样品的可得性低且复杂性高，对其中残留宿主细胞蛋白的分析具有挑战性。应对这些挑战的一种策略是开发具有更高灵敏度和更低样品需求的改进型液相色谱 - 串联质谱（LC-MS/MS）方法。
在本研究中，我们比较了四种全理论碎片离子质谱的顺序窗口采集（SWATH-MS）方法在鉴定和定量用人胚肾293（HEK293）细胞生产并使用免疫亲和色谱纯化的rAAV2、-5、-8和-9制剂中残留宿主细胞蛋白（HCP）方面的性能。评估了关键的SWATH-MS参数，包括谱图库构建（数据依赖型与……）、数据处理软件（DIA-NN与Skyline）以及质谱仪仪器（Sciex TripleTOF 6600与Sciex ZenoTOF 7600）。考虑了方法属性，包括样品需求和处理时间，以及方法输出，包括蛋白质和前体鉴定、不同方法间宿主细胞蛋白定量比较以及定量变异系数（CV），以帮助建立适合rAAV HCP分析的SWATH-MS工作流程。
与先前建立的使用DDA衍生谱图库、在Skyline中进行数据处理以及使用Sciex TripleTOF 6600仪器进行数据采集的方法（Skyline-DDA-6600方法）相比，使用谱图库、在DIA-NN中进行数据处理以及使用Sciex ZenoTOF 7600仪器进行数据采集的方法（DIA-NN-7600方法）使HCP鉴定增加了78%，样品需求减少了80%，仪器运行时间减少了70%。此外，DIA-NN-7600方法在三次重复数据采集时显示出HCP定量CV中位数低于10%，并且对于先前在rAAV下游加工中鉴定出的一组高丰度残留HCP，其定量与其他方法相当。
本研究突出了一种专门为rAAV残留HCP分析量身定制的数据采集和处理的SWATH-MS方法。
