A comparison of SWATH-MS methods for measurement of residual host cell proteins in adeno-associated virus preparations

Abstract

Introduction: 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.

Methods: 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, 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.

Results: 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.

Discussion: This work highlights a SWATH-MS method with data collection and processing specifically tailored for rAAV residual HCP analysis.

Keywords: DIA-NN; SWATH-MS; adeno-associated virus (AAV); data independent acquisition (DIA); host cell proteins (HCPs); liquid chromatography-tandem mass spectrometry (LC-MS/MS); mass spectrometry.

FIGURE 1

DIA LC-MS/MS data acquisition and analysis overview showing the four workflows tested. Protein requirement and acquisition time are shown for triplicate data acquisition of one sample.

FIGURE 2

Total HCP identifications for the four different DIA LC-MS/MS workflows (A). Protein identification in triplicate injections was required for inclusion in each method group. Protein identifications averaged across the sample set (N = 10) for each method (B). Non-parametric Wilcoxon Signed-Rank statistical tests were applied to determine if the means of paired groups were significantly different. ** indicates statistical significance at p < 0.01.

FIGURE 3

Average precursor identifications and precursor identification CV across triplicate injections for all four SWATH-MS methods across the sample set (N = 10). ** indicates statistical significance at p < 0.01. ns indicates that the differences were not statistically significant at a 95% confidence interval.

FIGURE 4

Individual HCP quantitation (ng/µg) for the 961 HCPs commonly quantified with both the DDA and in silico spectral libraries in Skyline across all samples (N = 10) (A). Individual HCP quantitation (ng/µg) for the 1,758 HCPs commonly quantified with both Sciex instruments using DIA-NN data processing and the in silico spectral library across all samples (N = 10) (B). Linear regression trendlines of the non-transformed data are shown on each subplot, with equations of fit and goodness of fit r ².

FIGURE 5

Box plots showing CV values of HCP qunatitation from triplicate data acquisition with each SWATH-MS method (A). Median CV for each method was compared, with each point representing an individual sample (N = 10) (B). No statistically significant differences in median HCP quantitation CV were noted across the methods. Box plots are truncated to improve visualization of the 25th–75th percentile region.

FIGURE 6

Normalized protein abundance (ng/µg) calculated for each sample across the four SWATH-MS methods. The 10 highest-abundance residual HCPs previously identified for rAAV purification using POROS™ CaptureSelect™ AAVX affinity chromatography were evaluated.

FIGURE 7

Normalized protein abundance (ng/µg) comparisons between Skyline and DIA-NN for the 10 highest-abundance residual HCPs previously identified (Leibiger et al., 2024a). Skyline-IS-6600 was comapred to DIA-NN-6600 (A) and DIA-NN-7600 (B). Data for all samples is plotted together, with color coding designations of rAAV serotypes or EGFP control material. Linear regression was performed across the full datasets. Linear regression trendlines of the non-transformed data are shown on each subplot, with equations of fit and goodness of fit r ².