Quantification of objective concentrations of DNA impurities in mRNA vaccines

The COVID-19 pandemic has demonstrated the benefits and advantages of mRNA technologies in combination with lipid nanoparticle (LNP) delivery systems for vaccine development, revolutionizing the field. Vaccine mRNA synthesis by in vitro transcription (IVT) is a cell-free process to produce plasmid-encoded mRNA by recombinant RNA polymerase activity. During mRNA vaccine manufacturing, the linearized plasmid DNA serving as template for IVT of vaccine mRNA, is enzymatically hydrolysed resulting in short digestion products that are removed, and residual amounts of template DNA remain as impurities in the vaccine product. These residual plasmid DNA quantities are defined as process- and product-related impurities and were subject of several discussions on various social media platforms. A recent study claimed to provide evidence for DNA impurities in mRNA vaccines exceeding permissible DNA concentrations by up to 534-fold. Due to the potential risk exceeding amounts of foreign DNA could pose for the safety of vaccinees as a consequence of an innate immune response, we set out to investigate the claims presented in this publication by attempting to reproduce the published data.

We applied various methods including Qubit fluorimetric quantitation and liquid chromatography-mass spectrometry (LC-MS/MS) to accurately quantify RNA and DNA concentrations in several individual mRNA vaccine vials.

Our results reproducibly confirm DNA:RNA mass ratios of 1:1000 which is consistent with the specifications of the approved mRNA vaccine product. We demonstrate that earlier published claims of 534-fold higher amounts of DNA impurities in vaccine mRNA products are not correct and a consequence of extraordinary high RNA and lipid concentrations. According to our data, gathered by applying different, orthogonal methods, we provide evidence that the concentration of product-related DNA impurities in both approved mRNA vaccines, BNT162b2 (Comirnaty) and mRNA-1273 (Spikevax) coincide with the approved mRNA specifications.