{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T06:15:41Z","timestamp":1783491341109,"version":"3.55.0"},"reference-count":27,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["aiche.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotechnology Progress"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The presence of host cell proteins (HCPs) as process\u2010related impurities in monoclonal antibody (mAb) therapeutics presents risks, including immunogenicity, product instability, and reduced therapeutic efficacy. Therefore, HCP levels are seen as a critical quality attribute which requires monitoring. Characterization of residual HCPs is essential during process development and after process changes in late project phases including post\u2010submission changes. Mass spectrometry\u2010based analysis of HCPs is increasingly employed to overcome the limitations of ELISAs, particularly in identifying individual low\u2010abundance HCPs. In this study, we systematically compared three enrichment strategies: hexapeptide enrichment, immunoaffinity chromatography (IAC), and native digest (ND), to enhance LC\u2013MS\/MS\u2010based detection of low\u2010abundance HCPs across five recombinant products expressed in CHO cells. Our results show that both ND and IAC substantially expand HCP detection beyond hexapeptide enrichment and in\u2010solution digest in total number of HCP identification. While IAC supported the detection of several potentially critical HCPs, ND uniquely identified additional low\u2010abundance HCPs not captured by antibody\u2010based methods. Furthermore, pull\u2010down assays revealed product\u2010specific HCPs likely interacting with the active pharmaceutical ingredient, suggesting that standard purification steps may be insufficient for their removal. Together, our findings emphasize the complementary nature of ND and IAC for in\u2010depth HCP profiling. We propose the combined application of both methods to ensure comprehensive detection of potentially critical HCPs in biopharmaceutical products, particularly when facing issues with low product or polysorbate stability caused by HCPs with enzymatic activities. This integrative approach supports enhanced product safety and control of consistent HCP depletion in the process.<\/jats:p>","DOI":"10.1002\/btpr.88540","type":"journal-article","created":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T05:34:26Z","timestamp":1783488866000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Deep dive into drug substance\n                    <scp>HCP<\/scp>\n                    patterns for comprehensive safety assessment in recombinant\n                    <scp>mAb<\/scp>\n                    formats"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-1058-904X","authenticated-orcid":false,"given":"Daniel M.","family":"Waldera\u2010Lupa","sequence":"first","affiliation":[{"name":"Analytical Development ProtaGene GmbH  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne","family":"Gehrke\u2010Oltmanns","sequence":"additional","affiliation":[{"name":"Analytical Development ProtaGene GmbH  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alireza","family":"Dehghani","sequence":"additional","affiliation":[{"name":"Analytical Development Biologicals Boehringer Ingelheim Pharma GmbH &amp; Co. KG  Biberach Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa","family":"Morgenroth","sequence":"additional","affiliation":[{"name":"Analytical Development ProtaGene GmbH  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antje\u2010Alke","family":"Betz","sequence":"additional","affiliation":[{"name":"Analytical Development Biologicals Boehringer Ingelheim Pharma GmbH &amp; Co. KG  Biberach Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heiner","family":"Falkenberg","sequence":"additional","affiliation":[{"name":"Analytical Development ProtaGene GmbH  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Flad","sequence":"additional","affiliation":[{"name":"Analytical Development ProtaGene GmbH  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulli","family":"Backofen","sequence":"additional","affiliation":[{"name":"Analytical Development Biologicals Boehringer Ingelheim Pharma GmbH &amp; Co. KG  Biberach Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4298-1292","authenticated-orcid":false,"given":"Thomas","family":"Waerner","sequence":"additional","affiliation":[{"name":"Analytical Development Biologicals Boehringer Ingelheim Pharma GmbH &amp; Co. KG  Biberach Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,7]]},"reference":[{"issue":"4","key":"e_1_2_12_2_1","doi-asserted-by":"crossref","DOI":"10.1002\/btpr.3448","article-title":"Host cell proteins in monoclonal antibody processing: control, detection, and removal","volume":"40","author":"Ito T","year":"2024","journal-title":"Biotechnol Prog"},{"issue":"1","key":"e_1_2_12_3_1","doi-asserted-by":"crossref","DOI":"10.1080\/19420862.2021.1955811","article-title":"Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody\u2010based therapeutic protein development","volume":"13","author":"Molden R","year":"2021","journal-title":"MAbs"},{"issue":"8","key":"e_1_2_12_4_1","doi-asserted-by":"crossref","first-page":"2870","DOI":"10.1002\/bit.27808","article-title":"\u2018High\u2010risk\u2019 host cell proteins (HCPs): a multi\u2010company collaborative 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