Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography
Date
2024-11-30
Journal Title
Journal ISSN
Volume Title
Publisher
Molecular Therapy - Methods and Clinical Development
Abstract
To better understand host cell protein (HCP) retention in adeno-associated virus (AAV) downstream processes, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to quantitatively profile residual HCPs for four AAV serotypes (AAV2, -5, -8, and -9) produced with HEK293 cells and purified using POROS CaptureSelect AAVX affinity chromatography. A broad range of residual HCPs were detected in affinity eluates after purification (Ntotal = 2,746), and HCP profiles showed universally present species (Nuniversal = 1,117) and species unique to one or more AAV serotype. SWATH-MS revealed that HCP persistence was dominated by high-abundance conserved species (HACS), which appeared across all serotype conditions studied. Due to the notable contribution of these species to overall residual HCP levels, physical and functional characteristics of HACS were examined to determine trends that coincide with persistence. Subnetwork interaction mapping and Gene Ontology function enrichment analysis revealed extensive physical interactions between these proteins and significant enrichment for biological processes, molecular functions, and reactome pathways related to protein folding, nucleic acid binding, and cellular stress. The abundant and conserved nature of these HCPs and their functions offers a new perspective for mechanistic evaluations of impurity retention for AAV downstream processes.
Graphical abstract available at: https://doi.org/10.1016/j.omtm.2024.101383
Description
This article was originally published in Molecular Therapy - Methods and Clinical Development. The version of record is available at: https://doi.org/10.1016/j.omtm.2024.101383.
© 2024 The Author(s). Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords
adeno-associated virus, AAV, host cell protein, HCP, AAVX affinity chromatography, liquid chromatography-tandem mass spectrometry, LC-MS/MS, proteomics
Citation
Leibiger, Thomas M., Lie Min, and Kelvin H. Lee. “Quantitative Proteomic Analysis of Residual Host Cell Protein Retention across Adeno-Associated Virus Affinity Chromatography.” Molecular Therapy - Methods & Clinical Development 32, no. 4 (December 2024): 101383. https://doi.org/10.1016/j.omtm.2024.101383.