Supercoiled DNA percentage: A key in-process control of linear DNA template for mRNA drug substance manufacturing

Jul 1, 2024Molecular therapy. Nucleic acids

Supercoiled DNA percentage as an important quality control in making linear DNA templates for mRNA drugs

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Abstract

Supercoiled DNA percentage significantly influences mRNA purity and transcription yield.

Higher percentages of supercoiled DNA are associated with increased mRNA purity.
Transcription yield of mRNA is positively impacted by the supercoiled DNA percentage.
Oligo-dT chromatography may reduce the negative effects of varying supercoiled DNA percentages on mRNA purity.
Key quality attributes of mRNA, such as capping efficiency and double-stranded RNA content, are also affected by supercoiled DNA levels.

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The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity andtranscription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance. in vitro in vitro

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