Abstract
The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.
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Acknowledgements
The authors would like to thank Polona Megušar and Andreja Krušič for providing downstream process samples of saRNA, and Mario Perković and Tim Beissert (TrON, Mainz, Germany) for providing pDNA construct encoding saRNA. Biomay is acknowledged for providing cell pastes containing plasmid encoding eGFP and Cas9 mRNA. The authors also thank Andreja Gramc Livk, Tomas Kostelec, Rok Miklavčič, and Urh Černigoj for constructive feedback during manuscript preparation.
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Marta Leban: methodology, investigation, writing — original draft, writing — review and editing. Tina Vodopivec Seravalli: methodology, investigation, writing — review and editing. Martina Hauer — methodology, writing — review and editing. Ernst Böhm — methodology, writing — review and editing. Andrej Thompson: methodology, writing — original draft. Aleš Štrancar: supervision, resources, writing — review and editing. Jurij Trontelj: writing — review and editing. Rok Sekirnik: conceptualization, supervision, project administration, writing — original draft, writing — review and editing.
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Leban, M., Vodopivec Seravalli, T., Hauer, M. et al. Determination of linearized pDNA template in mRNA production process using HPLC. Anal Bioanal Chem 416, 2389–2398 (2024). https://doi.org/10.1007/s00216-024-05204-0
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DOI: https://doi.org/10.1007/s00216-024-05204-0