ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Industrial Biotechnology
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1579098
A Comparison of SWATH-MS Methods for Measurement of Residual Host Cell Proteins in Adeno-Associated Virus Preparations
Provisionally accepted
- University of Delaware, Newark, United States
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Analysis of residual host cell proteins (HCPs) 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. in silico), 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, HCP 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 in silico 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.
Keywords: Host cell protein, adeno-associated virus (AAV), Mass Spectrometry, SWATH-MS, DIA-NN, Data independent acquisition (DIA), Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Received: 18 Feb 2025; Accepted: 23 Apr 2025.
Copyright: © 2025 Leibiger, Lie and Lee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Kelvin H. Lee, University of Delaware, Newark, United States
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