{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:58:40Z","timestamp":1790945920077,"version":"4.1.0"},"reference-count":47,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Zelle Biotechnology Pvt. Ltd."}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Accurate characterization of host cell proteins (HCPs) is critical for biologics development, yet analytical outcomes are strongly influenced by the digestion strategy and chromatographic design. Here, we systematically evaluated digestion parameters, including digestion mode, enzyme ratio, and incubation time, together with LC-MS gradient architecture using a CHO null harvest as a controlled model system to isolate workflow-dependent analytical effects from biological variability. Specifically, the study was designed to determine how these variables jointly influence HCP detection and the physicochemical and biological characteristics of the detected protein populations. By comparing reducing and nonreducing digestion approaches across multiple trypsin ratios and incubation times, we found that under low-enzyme conditions, the primary constraint on HCP recovery is insufficient proteolysis rather than chromatographic undersampling. Reconfiguration of the gradient architecture altered peptide sampling efficiency and MS\/MS utilization, enabling controlled comparison of digestion workflows under equivalent acquisition constraints. While higher enzyme ratios increased overall HCP identifications, subcellular localization analysis revealed that low-enzyme native digestion preferentially detected secreted and extracellular proteins with limited access to intracellular species. Extension of native digestion partially restored intracellular protein detection but did not fully recapitulate the coverage achieved by higher enzyme workflows. These findings demonstrate that digestion accessibility and chromatographic spacing impose workflow-dependent effects that shape both the depth and biological origin of detected HCPs, underscoring the need to consider the digestion strategy and chromatographic design together in HCP characterization workflows.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00097","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T01:01:26Z","timestamp":1789088486000},"page":"5200-5211","source":"Crossref","is-referenced-by-count":0,"title":["Integrated\nEffects of Digestion Strategy and LC Gradient\nArchitecture Define Host Cell Protein Detectability in CHO Null Harvests"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-8983-4157","authenticated-orcid":true,"given":"Paresh","family":"Tank","sequence":"first","affiliation":[{"name":"Amity University , , Mumbai \u2212 Pune Expressway, Bhatan, Post-Somathne, Panvel , , ,","place":["Mumbai, Maharashtra, India, 410 206"]},{"name":"Zelle Biotechnology Pvt. Ltd. , , A-7 M.I.D.C. Mira Industrial Area, Western Express Highway, Mira Road , , ,","place":["Thane, Maharashtra, India, 401 104"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatima","family":"D\u2019Souza","sequence":"additional","affiliation":[{"name":"Zelle Biotechnology Pvt. Ltd. , , A-7 M.I.D.C. Mira Industrial Area, Western Express Highway, Mira Road , , ,","place":["Thane, Maharashtra, India, 401 104"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kayalvizhi","family":"Nagarajan","sequence":"additional","affiliation":[{"name":"Periyar University , , , ,","place":["Salem, Tamil Nadu, India, 636011"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Krishnan","family":"Muthukalingan","sequence":"additional","affiliation":[{"name":"Central University of Tamil Nadu , , , ,","place":["Thiruvarur, Tamil Nadu, India, 610005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aruni Wilson Santhosh","family":"Kumar","sequence":"additional","affiliation":[{"name":"Amity University , , Mumbai \u2212 Pune Expressway, Bhatan, Post-Somathne, Panvel , , ,","place":["Mumbai, Maharashtra, India, 410 206"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neelamegam","family":"Ramesh Kumar","sequence":"additional","affiliation":[{"name":"Amity University , , Mumbai \u2212 Pune Expressway, Bhatan, Post-Somathne, Panvel , , ,","place":["Mumbai, Maharashtra, India, 410 206"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,10]]},"reference":[{"key":"2026100208412722200_cit1","doi-asserted-by":"publisher","first-page":"1722","DOI":"10.1038\/s41587-022-01582-x","article-title":"Biopharmaceutical benchmarks 2022","volume":"40","author":"Walsh","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"2026100208412722200_cit2","doi-asserted-by":"publisher","first-page":"1439","DOI":"10.1208\/s12248-016-9948-4","article-title":"Evaluating\nImmunogenicity\nRisk Due to Host Cell Protein Impurities in Antibody-Based Biotherapeutics","volume":"18","author":"Jawa","year":"2016","journal-title":"AAPS J."},{"key":"2026100208412722200_cit3","doi-asserted-by":"publisher","first-page":"2870","DOI":"10.1002\/bit.27808","article-title":"\u201cHigh-risk\u201d host cell proteins (HCPs):\nA multi-company collaborative view","volume":"118","author":"Jones","year":"2021","journal-title":"Biotechnol.\nBioeng."},{"key":"2026100208412722200_cit4","doi-asserted-by":"publisher","first-page":"3558","DOI":"10.1016\/j.xphs.2021.06.033","article-title":"Identification and\nCharacterization of Polysorbate-Degrading Enzymes in a Monoclonal\nAntibody Formulation","volume":"110","author":"Graf","year":"2021","journal-title":"J. Pharm. Sci."},{"key":"2026100208412722200_cit5","doi-asserted-by":"publisher","DOI":"10.1002\/btpr.3128","article-title":"Identification and Characterization of a Residual Host Cell Protein\nHexosaminidase B Associated with N-Glycan Degradation during the Stability\nStudy of a Therapeutic Recombinant Monoclonal Antibody Product","volume":"37","author":"Li","year":"2021","journal-title":"Biotechnol. Prog."},{"key":"2026100208412722200_cit6","doi-asserted-by":"publisher","first-page":"120","DOI":"10.1016\/j.ejpb.2023.04.021","article-title":"A fast and sensitive\nhigh-throughput assay to assess polysorbate-degrading hydrolytic activity\nin biopharmaceuticals","volume":"187","author":"Gupta","year":"2023","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"2026100208412722200_cit7","doi-asserted-by":"publisher","first-page":"2128","DOI":"10.1016\/j.xphs.2024.05.013","article-title":"Identification of Acyl-Protein Thioesterase-1 as a\nPolysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation\nUsing Activity-Based Protein Profiling","volume":"113","author":"S\u030cprager","year":"2024","journal-title":"J. Pharm.\nSci."},{"key":"2026100208412722200_cit8","doi-asserted-by":"publisher","first-page":"1418","DOI":"10.1111\/bcp.13938","article-title":"Adverse immunostimulation caused by impurities: The\ndark side of biopharmaceuticals","volume":"85","author":"Reijers","year":"2019","journal-title":"Br. J. Clin.\nPharmacol."},{"key":"2026100208412722200_cit9","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1007\/s11095-019-2586-7","article-title":"Impact\nof a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic\nMonoclonal Antibodies","volume":"36","author":"Rane","year":"2019","journal-title":"Pharm. Res."},{"key":"2026100208412722200_cit10","unstructured":"European Medicines Agency.\n          \n          ICH Q6B: Test\nProcedures\nand Acceptance Criteria for Biotechnological\/Biological Products\u2500Step\n5; European Medicines Agency, 1999. https:\/\/www.ema.europa.eu\/en\/documents\/scientific-guideline\/ich-q-6-b-test-procedures-and-acceptance-criteria-biotechnologicalbiological-products-step-5_en.pdf (Accessed June 2026)."},{"key":"2026100208412722200_cit11","unstructured":"International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use.\n          \n          Development and\nManufacture of Drug Substances (Chemical Entities and Biotechnological\/Biological\nEntities), Q11; 2012. https:\/\/database.ich.org\/sites\/default\/files\/Q11Guideline.pdf (Accessed June 2026)."},{"key":"2026100208412722200_cit12","doi-asserted-by":"publisher","first-page":"117051","DOI":"10.1016\/j.jpba.2025.117051","article-title":"Host cell\nprotein quantitation by LC-MS. Experimental\ndemonstration, qualification, and comparison of methods in USP 1132.1","volume":"265","author":"Chrone","year":"2025","journal-title":"J. Pharm. Biomed. Anal."},{"key":"2026100208412722200_cit13","unstructured":"United States Pharmacopeia.\n          \n          \u27e81132.1\u27e9\nResidual Host Cell Protein Measurement in Biopharmaceuticals by Liquid\nChromatography-Mass Spectrometry. https:\/\/www.uspnf.com (Accessed\nJune 2026)."},{"key":"2026100208412722200_cit14","doi-asserted-by":"publisher","first-page":"2297450","DOI":"10.1080\/19420862.2023.2297450","article-title":"Antibodies to watch\nin 2024","volume":"16","author":"Crescioli","year":"2024","journal-title":"mAbs"},{"key":"2026100208412722200_cit15","doi-asserted-by":"publisher","first-page":"382","DOI":"10.1002\/bit.26466","article-title":"Comparison\nof platform host cell\nprotein ELISA to process-specific host cell protein ELISA","volume":"115","author":"Gunawan","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"2026100208412722200_cit16","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":"2026100208412722200_cit17","doi-asserted-by":"publisher","first-page":"828","DOI":"10.1002\/btpr.2640","article-title":"Experience with\nhost cell protein impurities in biopharmaceuticals","volume":"34","author":"Vanderlaan","year":"2018","journal-title":"Biotechnol. Prog."},{"key":"2026100208412722200_cit18","doi-asserted-by":"publisher","first-page":"1783","DOI":"10.1021\/acs.analchem.5c07715","article-title":"Fe-N3S Single-Atom\nNanozyme with Asymmetric Coordination for Ultra-Low-Background Colorimetric\nImmunoassays","volume":"98","author":"Yu","year":"2026","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit19","doi-asserted-by":"publisher","first-page":"19783","DOI":"10.1021\/acs.analchem.5c03445","article-title":"Two-Dimensional Dissipative\nPaper-Based Immunoassay with Oxygen Vacancy-Enriched CeO2 Nanozymes for Portable Cancer Screening","volume":"97","author":"Yu","year":"2025","journal-title":"Anal.\nChem."},{"key":"2026100208412722200_cit20","doi-asserted-by":"publisher","first-page":"1955432","DOI":"10.1080\/19420862.2021.1955432","article-title":"Host cell\nprotein detection gap risk mitigation: Quantitative IAC-MS for ELISA\nantibody reagent coverage determination","volume":"13","author":"Waldera-Lupa","year":"2021","journal-title":"mAbs"},{"key":"2026100208412722200_cit21","doi-asserted-by":"publisher","first-page":"1955811","DOI":"10.1080\/19420862.2021.1955811","article-title":"Host cell protein profiling\nof commercial therapeutic protein drugs as a benchmark for monoclonal\nantibody-based therapeutic protein development","volume":"13","author":"Molden","year":"2021","journal-title":"mAbs"},{"key":"2026100208412722200_cit22","doi-asserted-by":"publisher","DOI":"10.1002\/btpr.2983","article-title":"A novel approach to evaluate ELISA antibody coverage\nof host cell proteins\u2500combining ELISA-based immunocapture and\nmass spectrometry","volume":"36","author":"Pilely","year":"2020","journal-title":"Biotechnol. Prog."},{"key":"2026100208412722200_cit23","doi-asserted-by":"publisher","first-page":"726","DOI":"10.1016\/j.jpha.2021.05.002","article-title":"Detection and quantitation of host\ncell proteins in monoclonal antibody drug products using automated\nsample preparation and data-independent acquisition LC-MS\/MS","volume":"11","author":"Strasser","year":"2021","journal-title":"J. Pharm. Anal."},{"key":"2026100208412722200_cit24","doi-asserted-by":"publisher","first-page":"347","DOI":"10.1038\/nature19949","article-title":"Mass-spectrometric exploration of\nproteome structure and function","volume":"537","author":"Aebersold","year":"2016","journal-title":"Nature"},{"key":"2026100208412722200_cit25","doi-asserted-by":"publisher","first-page":"1275","DOI":"10.4155\/bio-2021-0123","article-title":"Biopharmaceutical\nquality control with mass spectrometry","volume":"13","author":"Liu","year":"2021","journal-title":"Bioanalysis"},{"key":"2026100208412722200_cit26","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201900276","article-title":"Data-Independent Acquisition\nMass Spectrometry-Based Proteomics and Software Tools: A Glimpse in\n2020","volume":"20","author":"Zhang","year":"2020","journal-title":"Proteomics"},{"key":"2026100208412722200_cit27","doi-asserted-by":"publisher","first-page":"991","DOI":"10.1016\/j.tibtech.2025.07.026","article-title":"Advanced mass\nspectrometry techniques for monitoring biopharmaceutical host cell\nproteins","volume":"44","author":"Tank","year":"2026","journal-title":"Trends Biotechnol."},{"key":"2026100208412722200_cit28","doi-asserted-by":"publisher","first-page":"9716","DOI":"10.1021\/acs.analchem.9b01259","article-title":"Evaluation of Peptide\nFractionation and Native Digestion as Two Novel Sample Preparation\nWorkflows to Improve HCP Characterization by LC-MS\/MS","volume":"91","author":"Kufer","year":"2019","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit29","doi-asserted-by":"publisher","first-page":"4383","DOI":"10.1021\/acs.analchem.0c03931","article-title":"Simple and Sensitive Method for Deep\nProfiling of Host Cell Proteins in Therapeutic Antibodies by Combining\nUltra-Low Trypsin Concentration Digestion, Long Chromatographic Gradients,\nand BoxCar Mass Spectrometry Acquisition","volume":"93","author":"Nie","year":"2021","journal-title":"Anal.\nChem."},{"key":"2026100208412722200_cit30","doi-asserted-by":"publisher","first-page":"723","DOI":"10.1021\/acs.analchem.1c03095","article-title":"Versatile LC-MS-Based Workflow with Robust 0.1 ppm\nSensitivity for Identifying Residual HCPs in Biotherapeutic Products","volume":"94","author":"Yang","year":"2022","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit31","doi-asserted-by":"publisher","first-page":"5436","DOI":"10.1021\/acs.analchem.7b00304","article-title":"A Novel Sample Preparation for Shotgun\nProteomics Characterization\nof HCPs in Antibodies","volume":"89","author":"Huang","year":"2017","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit32","doi-asserted-by":"publisher","first-page":"A0152","DOI":"10.5702\/massspectrometry.A0152","article-title":"Quantitation\nof Host Cell Proteins\nby Capillary LC\/IMS\/MS\/MS in Combination with Rapid Digestion on Immobilized\nTrypsin Column Under Native Conditions","volume":"13","author":"Tomioka","year":"2024","journal-title":"Mass\nSpectrom."},{"key":"2026100208412722200_cit33","doi-asserted-by":"publisher","DOI":"10.1002\/btpr.3343","article-title":"Analytical characterization of host-cell-protein-rich\naggregates in monoclonal antibody solutions","volume":"39","author":"Herman","year":"2023","journal-title":"Biotechnol. Prog."},{"key":"2026100208412722200_cit34","doi-asserted-by":"publisher","first-page":"2375798","DOI":"10.1080\/19420862.2024.2375798","article-title":"Illuminating\na biologics development challenge: systematic characterization of\nCHO cell-derived hydrolases identified in monoclonal antibody formulations","volume":"16","author":"Maier","year":"2024","journal-title":"mAbs"},{"key":"2026100208412722200_cit35","doi-asserted-by":"publisher","first-page":"10947","DOI":"10.1021\/acs.analchem.5c02358","article-title":"A Bimetallic Nanozyme\nSynergistic Effect-Driven Enzyme Cascade Nanoreactor for Instant Immunoassay","volume":"97","author":"Wu","year":"2025","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit36","doi-asserted-by":"publisher","first-page":"7526","DOI":"10.1021\/acs.analchem.5c00743","article-title":"S-Modified\nMOF Nanozyme Cascade System with Multi-Enzyme Activity for Dual-Mode\nAntibiotic Assay","volume":"97","author":"Wang","year":"2025","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit37","doi-asserted-by":"publisher","first-page":"12999","DOI":"10.1021\/acs.analchem.4c00977","article-title":"diaPASEF Enables High-Throughput\nProteomic Analysis of Host Cell\nProteins for Biopharmaceutical Process Development","volume":"96","author":"Tsukidate","year":"2024","journal-title":"Anal. Chem."},{"key":"2026100208412722200_cit38","doi-asserted-by":"publisher","first-page":"1017","DOI":"10.1038\/s41592-025-02663-w","article-title":"Unifying the analysis\nof bottom-up proteomics data with CHIMERYS","volume":"22","author":"Frejno","year":"2025","journal-title":"Nat. Methods"},{"key":"2026100208412722200_cit39","doi-asserted-by":"publisher","first-page":"8605","DOI":"10.1038\/s41467-024-51870-0","article-title":"Detection of host cell microprotein impurities in antibody\ndrug products","volume":"15","author":"Tzani","year":"2024","journal-title":"Nat. Commun."},{"key":"2026100208412722200_cit40","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1038\/s41597-020-00594-z","article-title":"A comprehensive\nCHO SWATH-MS spectral library for robust quantitative profiling of\n10,000 proteins","volume":"7","author":"Sim","year":"2020","journal-title":"Sci. Data"},{"key":"2026100208412722200_cit41","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.jbiotec.2024.02.001","article-title":"SWATH-MS as\na strategy for CHO host cell protein identification and quantification\nsupporting the characterization of mAb purification platforms","volume":"384","author":"Carvalho","year":"2024","journal-title":"J. Biotechnol."},{"key":"2026100208412722200_cit42","doi-asserted-by":"publisher","first-page":"115475","DOI":"10.1016\/j.ab.2024.115475","article-title":"Qualification\nof a LC-HRMS platform method for biosimilar development using NISTmab\nas a model","volume":"688","author":"Tank","year":"2024","journal-title":"Anal. Biochem."},{"key":"2026100208412722200_cit43","doi-asserted-by":"publisher","first-page":"923","DOI":"10.1021\/acs.jproteome.0c00664","article-title":"Optimized Sample Preparation and\nData Processing of\nData-Independent Acquisition Methods for the Robust Quantification\nof Trace-Level Host Cell Protein Impurities in Antibody Drug Products","volume":"20","author":"Pythoud","year":"2021","journal-title":"J. Proteome Res."},{"key":"2026100208412722200_cit44","doi-asserted-by":"publisher","first-page":"114369","DOI":"10.1016\/j.ejpb.2024.114369","article-title":"Integrated\nstrategy\nfor deep profiling of host cell proteins in downstream processing\nof therapeutic monoclonal antibodies: Novel approach to isolate and\ndigest host cell proteins","volume":"201","author":"Zarei","year":"2024","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"2026100208412722200_cit45","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1038\/s41596-018-0082-x","article-title":"Single-pot,\nsolid-phase-enhanced sample preparation\nfor proteomics experiments","volume":"14","author":"Hughes","year":"2019","journal-title":"Nat. Protoc."},{"key":"2026100208412722200_cit46","doi-asserted-by":"publisher","first-page":"389","DOI":"10.1038\/s41597-020-00724-7","article-title":"A comprehensive\nspectral assay library to quantify the Escherichia coli proteome by\nDIA\/SWATH-MS","volume":"7","author":"Midha","year":"2020","journal-title":"Sci. Data"},{"key":"2026100208412722200_cit47","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2026.05.011","article-title":"Redefining\nbiologics\nsafety through advanced analytics: MS-based host cell protein profiling","volume-title":"Trends In Biotechnology","author":"Tank","year":"2026"}],"container-title":["Journal of Proteome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jproteome.6c00097","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5200\/68478591\/acs.jproteome.6c00097.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5200\/68478591\/acs.jproteome.6c00097.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:51:47Z","timestamp":1790945507000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jprobs\/article\/25\/10\/5200\/5428048\/Integrated-Effects-of-Digestion-Strategy-and-LC"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,10]]},"references-count":47,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,10]]},"published-print":{"date-parts":[[2026,10,2]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jproteome.6c00097","relation":{},"ISSN":["1535-3893","1535-3907"],"issn-type":[{"value":"1535-3893","type":"print"},{"value":"1535-3907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,10]]}}}