{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T19:46:51Z","timestamp":1791316011879,"version":"4.3.1"},"reference-count":24,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2024,12,5]],"date-time":"2024-12-05T00:00:00Z","timestamp":1733356800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Merck Sharp and Dohme, Corp."},{"name":"a subsidiary of Merck and Co., Inc., Rahway, NJ, USA"},{"name":"Biomedical Advanced Research and Development Authority (BARDA) of the US Department of Health"},{"name":"Human Services and the Defense Threat Reduction Agency (DTRA) of the US Department of Defense"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western\u2122 is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western\u2122 is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western\u2122, as they are both run under denatured and reduced conditions. Methods: Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE. Results\/Conclusions: Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.<\/jats:p>","DOI":"10.3390\/vaccines12121373","type":"journal-article","created":{"date-parts":[[2024,12,5]],"date-time":"2024-12-05T06:24:44Z","timestamp":1733379884000},"page":"1373","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Automated, Quantitative Capillary Western Blots to Analyze Host Cell Proteins in COVID-19 Vaccine Produced in Vero Cell Line"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2156-9963","authenticated-orcid":false,"given":"Paul F.","family":"Gillespie","sequence":"first","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanjie","family":"Wang","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kuo","family":"Yin","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Groegler","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"Cunningham","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9334-1212","authenticated-orcid":false,"given":"Alyssa Q.","family":"Stiving","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7405-703X","authenticated-orcid":false,"given":"Jessica","family":"Raffaele","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalia","family":"Marusa","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher M.","family":"Tubbs","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John W.","family":"Loughney","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael A.","family":"Winters","sequence":"additional","affiliation":[{"name":"Process Research & Development, Merck & Co., Inc., West Point, PA 19486, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard R.","family":"Rustandi","sequence":"additional","affiliation":[{"name":"Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2088","DOI":"10.1002\/bit.28112","article-title":"Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts","volume":"119","author":"Wang","year":"2022","journal-title":"Biotechnol. 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