{"doi":"10.1016/j.omtm.2025.101623","title":"Characterization of difficult-to-remove host cell proteins in adeno-associated virus downstream processing","abstract":"A scalable, serotype-agnostic, and fully chromatographic downstream platform was developed for the purification of adeno-associated virus (AAV) based on affinity chromatography (AC), anion-exchange chromatography (AEX), and multi-modal polishing chromatography (MMC). Quantitative proteomic profiling across each purification stage was performed using sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) to characterize residual host cell protein (HCP) retention for four AAV serotypes (AAV2, -5, -8, and -9) produced with suspension HEK293 cells. The downstream process showed cumulative vector genome (VG) yields ranging from 44.6 to 69.2% with full capsid enrichments ranging from 2.62- to 5.93-fold across all AAV samples. A total of 880, 99, and 21 HCPs were identified in all AAV samples after AC, AEX, and MMC, respectively. Vector-mediated mechanisms of retention are proposed (i.e., capsid or genome association) based on enrichment in protein- and nucleic acid-binding properties of difficult-to-remove HCPs, little clearance across multiple modes of separation, and divergence of HCP profiles for AAV-containing samples vs. null lysate material. The characterization of HCP retention described here advances the understanding of process-related impurity clearance in scalable AAV downstream systems, with implications for both purification process design and AAV interactions with host cell factors.","journal":"Molecular Therapy — Methods & Clinical Development","year":2025,"id":535234,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9579,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1057834,"name":"Lie Min","orcid":null,"position":1,"is_corresponding":false},{"id":346971,"name":"Kelvin H. Lee","orcid":"0000-0003-0908-2981","position":2,"is_corresponding":false},{"id":1250670,"name":"Thomas M Leibiger","orcid":null,"position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:51:56.297114Z","pmid":"41321396","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}