A Comparison of SWATH-MS Methods for Measurement of Residual Host Cell Proteins in Adeno-Associated Virus Preparations

Analysis of residual host cell proteins (HCPs) in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. To address these challenges, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) data was acquired on four AAV serotypes (2, 5, 8, and 9) produced with HEK293 cells and purified using POROS™ CaptureSelect™ AAVX affinity chromatography with two different analysis software (Skyline and DIA-NN) and instruments (Sciex TripleTOF 6600 and ZenoTOF 7600). An improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods well-suited for rAAV HCP analytics was developed by comparing number of protein and precursor identification, HCP quantification and quantification coefficient of variance (CV).