Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography

Simple item page
Author(s)Leibiger, Thomas M.
Author(s)Min, Lie
Author(s)Lee, Kelvin H.
Date Accessioned2025-01-15T19:18:25Z
Date Available2025-01-15T19:18:25Z
Publication Date2024-11-30
DescriptionThis 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/).
AbstractTo 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
SponsorThis work was supported in part by financial assistance award 70NANB17H002 from the US Department of Commerce, National Institute of Standards and Technology. Microscopy access was supported by grants from NIH-NIGMS (National Institute of General Medical Sciences) (P20 GM103446), the NIGMS (P20 GM139760) and the State of Delaware. The authors thank Shannon Modla in the Delaware Biotechnology Institute’s Bio-Imaging Center for assistance with nsTEM imaging. The authors are grateful to Sartorius for providing the Octet AAVX biosensors. The graphical abstract figure was created using BioRender.
CitationLeibiger, 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.
ISSN2329-0501
URLhttps://udspace.udel.edu/handle/19716/35723
Languageen_US
PublisherMolecular Therapy - Methods and Clinical Development
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
Keywordsadeno-associated virus
KeywordsAAV
Keywordshost cell protein
KeywordsHCP
KeywordsAAVX affinity chromatography
Keywordsliquid chromatography-tandem mass spectrometry
KeywordsLC-MS/MS
Keywordsproteomics
TitleQuantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography
TypeArticle
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography.pdf
Size:
3.8 MB
Format:
Adobe Portable Document Format
Description:
Main article
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
2.22 KB
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
Open Access Publications - Department of Chemical and Biomolecular Engineering