{"title":"Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography.","authors":"Thomas M Leibiger, Lie Min, Kelvin H Lee","doi":"10.1016/j.omtm.2024.101383","DOIUrl":null,"url":null,"abstract":"<p><p>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 (<i>N</i> <sub><i>total</i></sub> = 2,746), and HCP profiles showed universally present species (<i>N</i> <sub><i>universal</i></sub> = 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.</p>","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":"32 4","pages":"101383"},"PeriodicalIF":4.6000,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11650319/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Therapy-Methods & Clinical Development","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.omtm.2024.101383","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/12/12 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍：
The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella.
Topics of particular interest within the journal''s scope include:
Gene vector engineering and production,
Methods for targeted genome editing and engineering,
Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells,
Methods for gene and cell vector delivery,
Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine,
Analysis of gene and cell vector biodistribution and tracking,
Pharmacology/toxicology studies of new and next-generation vectors,
Methods for cell isolation, engineering, culture, expansion, and transplantation,
Cell processing, storage, and banking for therapeutic application,
Preclinical and QC/QA assay development,
Translational and clinical scale-up and Good Manufacturing procedures and process development,
Clinical protocol development,
Computational and bioinformatic methods for analysis, modeling, or visualization of biological data,
Negotiating the regulatory approval process and obtaining such approval for clinical trials.
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