"Abstract
For some of the COVID-19 vaccines, the drug substances released to market were manufactured differently than those used in clinical trials. Manufacturing nucleoside-modified mRNA (modRNA) for commercial COVID-19 vaccines relies on RNA polymerase transcription of a plasmid DNA template. Previous studies identified high levels of plasmid DNA in vials of modRNA vaccines, suggesting that the removal of residual DNA template is problematic. Therefore, we quantified the DNA load in a limited number of Pfizer-BioNTech and Moderna COVID-19 modRNA vaccine vials using two independent methods. Total DNA and specific DNA targets were quantified by Qubit fluorometry and quantitative polymerase chain reaction (qPCR), respectively on 32 vials representing 16 unique vaccine lots. RNase A treatment was used to assess the impact of RNA crosstalk in DNA fluorometry. A preliminary assessment of DNA fragment length and DNase I sensitivity were also performed. Total DNA ranged 371–1,548 ng/dose and 1,130–6,280 ng/dose in Pfizer and Moderna products, respectively... These data demonstrate the presence of 1.23 × 108 to 1.60 × 1011 plasmid DNA fragments per dose encapsulated in lipid nanoparticles. Using fluorometry, total DNA in all vials tested exceeded the regulatory limit for residual DNA set by the US Food & Drug Administration (FDA) and the World Health Authorization [sic] (WHO) by 36–153-fold for Pfizer and 112–627-fold for Moderna after accounting for nonspecific binding to modRNA. When tested by qPCR, all Moderna vials were within the regulatory limit, but 2/6 Pfizer lots (3 vials) exceeded the regulatory limit for the SV40 promoter-enhancer-ori by 2-fold. The presence of the SV40 promoter-enhancer element in Pfizer vials raises significant safety concerns...
Discussion
... These data involving vaccine vials distributed in Canada are consistent with several non-peer reviewed and peer reviewed reports of DNA contamination in modRNA vaccines...
Our exploratory analysis of the relationship between the residual DNA content and SAEs [serious adverse events] reported to VAERS is preliminary and limited in sample size but warrants confirmation by examining many more lots and vials. This preliminary analysis appears to show a correlation between the adverse events reported for each lot and the levels of DNA, especially in the context of the ori... [O]ur findings suggest that beyond plasmid DNA levels, issues related to the LNPs, toxicity, autoimmunity and/or aberrant immune responses due to ribosomal frameshifting may... contribute to AEs shortly after vaccination... Higher DNA loads might also contribute to long-term health risks, such as cancer from genomic integration events...
Conclusion
... The PCR results for the most recent XBB.1.5 Moderna and Pfizer vaccines suggest that DNA residues have not been reduced from previous vaccine versions."
© 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
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