Systematic analysis of COVID-19 mRNA vaccines using four orthogonal approaches demonstrates no excessive DNA impurities

Systematic analysis of COVID-19 mRNA vaccines using four orthogonal approaches demonstrates no excessive DNA impurities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Systematic analysis of COVID-19 mRNA vaccines using four orthogonal approaches demonstrates no excessive DNA impurities Adam Achs, Tatiana Sedlackova, Lukas Predajna, Jaroslav Budis, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7340318/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Dec, 2025 Read the published version in npj Vaccines → Version 1 posted 18 You are reading this latest preprint version Abstract Robust epidemiologic data confirm that vaccination saved millions of lives and protected recipients from severe COVID-19 disease caused by SARS-CoV-2 infection. Despite this evidence, COVID-19 mRNA vaccines have become a target of largely unfounded skepticism built on coincidental health events occurring after vaccination without proven causality. An example of claims undermining trust in mRNA vaccine safety relates to excessive amounts of residual DNA from the vaccine manufacturing process. These claims are based on improperly accomplished quantification methods and/or incorrectly interpreted data. In this study, we aimed to thoroughly assess quantity, quality and identity of residual DNA in mRNA vaccines based on objective, technically correctly performed and interpreted methods using orthogonal approaches, including qPCR, fluorometry, capillary electrophoresis and short-read DNA sequencing. We analysed 15 batches of Comirnaty and Spikevax vaccines available in Slovakia, obtained from official repositories under the supervision of the State Institute for Drug Control. These included 11 batches post-expiration date that were previously claimed by others to contain “significant amounts of DNA” using data from reverse transcription-based multiplex qPCR performed with technical shortcomings. Results of our analyses utilising four orthogonal approaches clearly demonstrate that the quantity of residual DNA in all analysed vaccine batches is below approved limits, in very low quantities relative to mRNA, and degraded into small fragments, which originate from the DNA template used to transcribe mRNA during vaccine production. Our results show that reliable vaccine analysis requires rigorous application of multiple validated methods that comply with molecular characteristics of mRNA vaccine components and minimise their mutual interference. Biological sciences/Biological techniques Biological sciences/Biotechnology Health sciences/Diseases Biological sciences/Genetics Biological sciences/Immunology Biological sciences/Microbiology Biological sciences/Molecular biology mRNA vaccine residual DNA qPCR fluorometry capillary electrophoresis fragment analysis short-read DNA sequencing Full Text Supplementary Files MSAchsetalSUPPLEMENTARYINFORMATION.pdf Cite Share Download PDF Status: Published Journal Publication published 13 Dec, 2025 Read the published version in npj Vaccines → Version 1 posted Editorial decision: Revision requested 28 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviews received at journal 21 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviews received at journal 05 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers invited by journal 24 Aug, 2025 Editor assigned by journal 21 Aug, 2025 Submission checks completed at journal 14 Aug, 2025 First submitted to journal 10 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7340318","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":507655602,"identity":"7d1c3565-7b73-4a00-a4be-170c29983803","order_by":0,"name":"Adam Achs","email":"","orcid":"","institution":"Biomedical Research Center, Slovak Academy of Sciences, Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Achs","suffix":""},{"id":507655603,"identity":"291bad0b-3279-4dc9-a39f-82b9bf8477e8","order_by":1,"name":"Tatiana Sedlackova","email":"","orcid":"","institution":"Comenius University 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