(T1430-01-07) Importance of Quality Control of In Vitro Transcribed RNA

Purpose: The recent Covid-19 pandemic has raised awareness of RNA-based vaccines and the speed at which they can be produced in case of disease outbreaks. Currently the only approved RNA therapeutics are the Covid-19 vaccines, however RNA is being investigated for other prophylactic vaccines, therapeutics, and therapeutic vaccines. One way to produce the RNAs required for these new vaccines and therapeutics is in vitro transcription (IVT). When producing any IVT RNA, quality control (QC) of the RNA is essential to ensure that the product is the correct length, does not contain contaminants or degradation products, and verify the absence of the DNA template used for IVT RNA production.

Methods: The Fragment Analyzer and TapeStation systems can be used for QC of DNA and RNA samples. Concerning the IVT RNA workflow, each system offers solutions for the analysis of both the DNA templates and the final IVT RNA products for the assessment of size, purity, and integrity. To assess the performance of the 5200 Fragment Analyzer and 4200 TapeStation system for QC in the IVT RNA workflow, DNA templates were prepared by PCR amplification and analyzed with the dsDNA 930 reagent kit (75-20000 bp) and D5000 ScreenTape assay, respectively. IVT RNA was generated from PCR-amplified DNA templates using a standard protocol and analyzed with the RNA kit (15 nt) and RNA ScreenTape assay, respectively.

Results: Using the 4200 TapeStation system, the DNA and IVT RNA samples were analyzed for sizing accuracy and precision. All tested DNA and IVT RNA samples showed a low percent error below 10% and percent coefficient of variation (CV) below 1% for DNA and below 5% for IVT RNA, respectively. Using the 5200 Fragment Analyzer system, the IVT RNA samples were analyzed for sizing accuracy and precision. All tested IVT RNA samples showed a low percent error below 10% and percent coefficient of variation (CV) below 1%. Moreover, with the 5200 Fragment Analyzer system unspecific amplification of the DNA template and the presence of DNA template in the IVT RNA product can be detected within the specifications of the kits.

Conclusion: The Fragment Analyzer and TapeStation systems have been successfully deployed by major pharmaceutical companies for automated QC and size analysis during the IVT RNA production workflow. With both systems it can be verified that the appropriate templates are used in the IVT reaction and that the final IVT RNA is suitable for the intended downstream application ensuring a quick, safe, and effective distribution of vaccines.