In the development of certain COVID-19 vaccines, variations occurred between the manufacturing processes for drug substances intended for market release and those employed during clinical evaluations. The production of nucleoside-modified messenger RNA (modRNA) for these commercial vaccines involves in vitro transcription using RNA polymerase on a plasmid DNA template. Prior investigations have revealed elevated concentrations of plasmid DNA remnants in modRNA vaccine vials, indicating challenges in the effective elimination of residual template DNA. Consequently, this study evaluated DNA content in a select cohort of Pfizer-BioNTech and Moderna modRNA COVID-19 vaccine vials through dual independent assays. Quantification of overall DNA and targeted DNA sequences was achieved via Qubit fluorometry and quantitative polymerase chain reaction (qPCR), respectively, across 32 vials encompassing 16 distinct lots. To mitigate RNA interference in DNA fluorometric measurements, RNase A digestion was applied. Additionally, initial evaluations of DNA fragment sizes and susceptibility to DNase I were conducted.
Total DNA concentrations varied from 371 to 1,548 ng per dose in Pfizer-BioNTech samples and from 1,130 to 6,280 ng per dose in Moderna samples. For specific plasmid-derived DNA sequences, levels ranged between 0.22 and 7.28 ng per dose for Pfizer-BioNTech, and between 0.01 and 0.78 ng per dose for Moderna. Detection of the SV40 promoter-enhancer-origin sequence (ranging from 0.25 to 23.72 ng per dose) was confined to Pfizer-BioNTech vials. Sequencing via Oxford Nanopore on a single vial yielded average and peak DNA fragment lengths of 214 base pairs and 3.5 kilobases, respectively. These findings indicate the encapsulation of 1.23 × 10^8 to 1.60 × 10^11 plasmid DNA fragments per dose within lipid nanoparticles.
Fluorometric analysis revealed that DNA levels in all examined vials surpassed the residual DNA thresholds established by the US Food and Drug Administration (FDA) and the World Health Organization (WHO) by factors of 36 to 153 for Pfizer-BioNTech and 112 to 627 for Moderna, following adjustments for non-specific modRNA interactions. In contrast, qPCR assessments showed all Moderna vials complying with these limits, whereas two of six Pfizer-BioNTech lots (encompassing three vials) exceeded the threshold for the SV40 promoter-enhancer-origin by a twofold margin. The identification of the SV40 promoter-enhancer in Pfizer-BioNTech vials poses notable safety implications. This investigation underscores the critical role of methodological precision in quantifying residual plasmid DNA within modRNA formulations, particularly given the enhanced transfection capabilities of lipid nanoparticles and the potential health risks associated with repeated administration and cumulative exposure.
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