{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T02:35:31Z","timestamp":1793414131225,"version":"4.5.0"},"reference-count":26,"publisher":"American Chemical Society (ACS)","issue":"34","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Recombinant adeno-associated virus vectors (rAAVs) are the most important vectors for in vivo gene therapies, yet their safety relies on low levels of residual host cell proteins (HCPs). While mass spectrometry-based proteomics enables sensitive and untargeted HCP profiling, it faces challenges with matrix interferences from purification buffers and the high dynamic range between abundant viral capsids and trace HCPs. Although sample preparation methods that address these challenges are well-established for antibody products, their adaptation to rAAV purification stages and products remains largely unexplored. In this study, we systematically evaluated three widely used proteomic sample preparation workflows\u2500In-Solution, FASP, and SP3\u2500across different stages of rAAV purification. In addition, each workflow was tested under both standard denatured digestion conditions and a \u201cnative\u201d digestion strategy designed to reduce dynamic range by preserving capsid integrity while selectively digesting HCPs. This comparison identified the native FASP protocol as the most effective sample preparation method for overcoming matrix interference and dynamic range challenges, consistently outperforming other workflows in HCP identification across purification stages. Further optimization of the native FASP workflow enhanced its performance, achieving the highest host cell (HC) proteome depth, particularly in highly purified drug substance samples. This optimized sample preparation strategy provides a robust and easy-to-use framework for deep characterization of the host cell proteome in rAAV samples. By enabling deeper insights into the HCP profile, this approach supports improved understanding of the rAAV purification process and facilitates the development of targeted strategies to enhance product quality and safety.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c01709","type":"journal-article","created":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T01:01:15Z","timestamp":1786755675000},"page":"24855-24864","source":"Crossref","is-referenced-by-count":1,"title":["Evaluation and Optimization of Different Digestion\nStrategies for In-Depth Proteomic Characterization of Residual Host\nCell Proteins in rAAV-Based Gene Therapy Products"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-1995-5067","authenticated-orcid":true,"given":"Kathrin","family":"Viode\u0301","sequence":"first","affiliation":[{"name":"Roche Diagnostics GmbH , , ,","place":["Penzberg, Germany, 82377"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timo","family":"Bohlig","sequence":"additional","affiliation":[{"name":"Roche Diagnostics GmbH , , ,","place":["Penzberg, Germany, 82377"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabienne","family":"Intlekofer","sequence":"additional","affiliation":[{"name":"Roche Diagnostics GmbH , , ,","place":["Penzberg, Germany, 82377"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberto","family":"Falkenstein","sequence":"additional","affiliation":[{"name":"Roche Diagnostics GmbH , , ,","place":["Penzberg, Germany, 82377"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Thomann","sequence":"additional","affiliation":[{"name":"Roche Diagnostics GmbH , , ,","place":["Penzberg, Germany, 82377"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"2026090306462323400_cit1","doi-asserted-by":"publisher","first-page":"16002","DOI":"10.1038\/mtm.2016.2","article-title":"Manufacturing of recombinant adeno-associated viral\nvectors for clinical trials","volume":"3","author":"Cle\u0301ment","year":"2016","journal-title":"Mol. Ther. Methods\nClin. Dev."},{"issue":"6","key":"2026090306462323400_cit2","doi-asserted-by":"publisher","first-page":"2479","DOI":"10.1016\/j.ymthe.2025.04.045","article-title":"Current clinical applications\nof AAV-mediated gene therapy","volume":"33","author":"Byrne","year":"2025","journal-title":"Mol. Ther."},{"issue":"5","key":"2026090306462323400_cit3","doi-asserted-by":"publisher","first-page":"1903","DOI":"10.1016\/j.ymthe.2025.03.064","article-title":"AAV vector\ndevelopment, back to the future","volume":"33","author":"Suarez-Amaran","year":"2025","journal-title":"Mol. Ther."},{"key":"2026090306462323400_cit4","doi-asserted-by":"publisher","first-page":"107453","DOI":"10.1016\/j.pharmthera.2019.107453","article-title":"Adeno-associated viral\nvector-mediated immune responses: Understanding barriers to gene delivery","volume":"207","author":"Nidetz","year":"2020","journal-title":"Pharmacol. Ther."},{"issue":"4","key":"2026090306462323400_cit5","doi-asserted-by":"publisher","first-page":"503","DOI":"10.1038\/gt.2009.157","article-title":"High AAV vector purity\nresults in serotype- and tissue-independent enhancement of transduction\nefficiency","volume":"17","author":"Ayuso","year":"2010","journal-title":"Gene Ther."},{"issue":"11","key":"2026090306462323400_cit6","doi-asserted-by":"publisher","first-page":"4186","DOI":"10.1002\/bit.27905","article-title":"Assessment of quality\nattributes for adeno-associated viral vectors","volume":"118","author":"Tustian","year":"2021","journal-title":"Biotechnol. Bioeng."},{"issue":"6","key":"2026090306462323400_cit7","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1167\/iovs.66.6.38","article-title":"Investigating\nthe Immunogenic Potential of Variations in Host Cell Protein Levels\nin Clinical-Grade AAV8 Products","volume":"66","author":"Seitz","year":"2025","journal-title":"Invest. Ophthalmol.\nVisual Sci."},{"issue":"1","key":"2026090306462323400_cit8","doi-asserted-by":"publisher","first-page":"80","DOI":"10.3390\/biomedicines2010080","article-title":"Product-Related\nImpurities in Clinical-Grade Recombinant AAV Vectors: Characterization\nand Risk Assessment","volume":"2","author":"Wright","year":"2014","journal-title":"Biomedicines"},{"key":"2026090306462323400_cit9","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1016\/j.copbio.2021.06.026","article-title":"Analytics of host\ncell proteins (HCPs): lessons from biopharmaceutical mAb analysis\nfor Gene therapy products","volume":"71","author":"Bracewell","year":"2021","journal-title":"Curr. Opin. Biotechnol."},{"key":"2026090306462323400_cit10","doi-asserted-by":"publisher","first-page":"108575","DOI":"10.1016\/j.biotechadv.2025.108575","article-title":"Host cell protein-mediated\nadjuvanticity and immunogenicity\nrisks of biotherapeutics","volume":"81","author":"Panikulam","year":"2025","journal-title":"Biotechnol. Adv."},{"issue":"8","key":"2026090306462323400_cit11","doi-asserted-by":"publisher","first-page":"684","DOI":"10.2174\/1389201016666150505122228","article-title":"Scalable downstream\nstrategies for purification of recombinant adeno-associated virus\nvectors in light of the properties","volume":"16","author":"Qu","year":"2015","journal-title":"Curr. Pharm.\nBiotechnol."},{"issue":"2","key":"2026090306462323400_cit12","doi-asserted-by":"publisher","first-page":"382","DOI":"10.1002\/bit.26466","article-title":"Comparison of platform\nhost cell protein ELISA to process-specific host cell protein ELISA","volume":"115","author":"Gunawan","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"2026090306462323400_cit13","doi-asserted-by":"publisher","first-page":"116009","DOI":"10.1016\/j.jpba.2024.116009","article-title":"A simple and sensitive differential digestion\nmethod to analyze adeno-associated virus residual host cell proteins\nby LC-MS","volume":"242","author":"Hu","year":"2024","journal-title":"J. Pharm. Biomed. Anal."},{"issue":"42","key":"2026090306462323400_cit14","doi-asserted-by":"publisher","first-page":"16510","DOI":"10.1021\/acs.analchem.4c00893","article-title":"Native Digestion\nand Shotgun Proteomics for Host Cell Protein Profiling of Adeno-Associated\nViruses","volume":"96","author":"Lodge","year":"2024","journal-title":"Anal. Chem."},{"key":"2026090306462323400_cit15","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.aca.2019.08.032","article-title":"Filter\nAided Sample Preparation - A tutorial","volume":"1090","author":"Wis\u0301niewski","year":"2019","journal-title":"Anal.\nChim. Acta"},{"issue":"1","key":"2026090306462323400_cit16","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1038\/s41596-018-0082-x","article-title":"Single-pot, solid-phase-enhanced sample preparation\nfor proteomics experiments","volume":"14","author":"Hughes","year":"2019","journal-title":"Nat. Protoc."},{"issue":"6","key":"2026090306462323400_cit17","doi-asserted-by":"publisher","first-page":"1470","DOI":"10.1016\/j.xphs.2023.12.018","article-title":"Ubiquitin: Characterization of a Host Cell Protein\nCovalently Attached to a Monoclonal Antibody Product by LC-MS\/MS","volume":"113","author":"Kufer","year":"2024","journal-title":"J. Pharm. Sci."},{"key":"2026090306462323400_cit18","doi-asserted-by":"publisher","first-page":"276","DOI":"10.1016\/j.ejpb.2023.06.019","article-title":"SP3-based host cell\nprotein monitoring in AAV-based gene therapy\nproducts using LC-MS\/MS","volume":"189","author":"Smith","year":"2023","journal-title":"Eur. J. Pharm. Biopharm."},{"issue":"1","key":"2026090306462323400_cit19","doi-asserted-by":"publisher","first-page":"265","DOI":"10.1038\/s42003-021-01797-9","article-title":"Surfactant-assisted\none-pot sample preparation for label-free single-cell proteomics","volume":"4","author":"Tsai","year":"2021","journal-title":"Commun. Biol."},{"issue":"5","key":"2026090306462323400_cit20","doi-asserted-by":"publisher","first-page":"1890","DOI":"10.1021\/acs.analchem.3c03884","article-title":"Analysis of Host Cell\nProteins in\nAAV Products with ProteoMiner Protein Enrichment Technology","volume":"96","author":"Zhang","year":"2024","journal-title":"Anal. Chem."},{"key":"2026090306462323400_cit21","doi-asserted-by":"publisher","first-page":"15213","DOI":"10.1021\/acs.analchem.4c02400","article-title":"In-Depth Host Cell\nProtein Analysis and Viral Protein Impurity Monitoring in Adeno-Associated\nVirus-Based Gene Therapy Products Using Optimized Wide Window Data-Dependent\nAcquisition Method","volume":"96","author":"Huo","year":"2024","journal-title":"Anal. Chem."},{"issue":"7","key":"2026090306462323400_cit22","doi-asserted-by":"publisher","first-page":"3170","DOI":"10.1128\/JVI.02192-06","article-title":"Innate immune\nresponse to adenoviral\nvectors is mediated by both Toll-like receptor-dependent and -independent\npathways","volume":"81","author":"Zhu","year":"2007","journal-title":"J. Virol."},{"issue":"4","key":"2026090306462323400_cit23","doi-asserted-by":"publisher","first-page":"101623","DOI":"10.1016\/j.omtm.2025.101623","article-title":"Characterization\nof difficult-to-remove host cell proteins\nin adeno-associated virus downstream processing","volume":"33","author":"Leibiger","year":"2025","journal-title":"Mol. Ther. Methods Clin. Dev."},{"issue":"9","key":"2026090306462323400_cit24","doi-asserted-by":"publisher","first-page":"2104","DOI":"10.1021\/acs.jproteome.2c00145","article-title":"Protein Contaminants\nMatter: Building\nUniversal Protein Contaminant Libraries for DDA and DIA Proteomics","volume":"21","author":"Frankenfield","year":"2022","journal-title":"J. Proteome Res."},{"issue":"4","key":"2026090306462323400_cit25","doi-asserted-by":"publisher","first-page":"101383","DOI":"10.1016\/j.omtm.2024.101383","article-title":"Quantitative\nproteomic analysis of residual host cell\nprotein retention across adeno-associated virus affinity chromatography","volume":"32","author":"Leibiger","year":"2024","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"2026090306462323400_cit26","doi-asserted-by":"publisher","first-page":"1579098","DOI":"10.3389\/fbioe.2025.1579098","article-title":"A comparison\nof SWATH-MS methods for measurement of\nresidual host cell proteins in adeno-associated virus preparations","volume":"13","author":"Leibiger","year":"2025","journal-title":"Front. Bioeng. Biotechnol."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c01709","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/34\/24855\/66580315\/acs.analchem.6c01709.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/34\/24855\/66580315\/acs.analchem.6c01709.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:46:26Z","timestamp":1788432386000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/34\/24855\/5257677\/Evaluation-and-Optimization-of-Different-Digestion"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,14]]},"references-count":26,"journal-issue":{"issue":"34","published-online":{"date-parts":[[2026,8,14]]},"published-print":{"date-parts":[[2026,9,1]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c01709","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,14]]}}}