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Determination of linearized pDNA template in mRNA production process using HPLC

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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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References

  1. Rosa SS, Prazeres DMF, Azevedo AM, Marques MPC. mRNA vaccines manufacturing: challenges and bottlenecks. Vaccine. 2021;39(16):2190–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Baiersdörfer M, Boros G, Muramatsu H, Mahiny A, Vlatkovic I, Sahin U, et al. A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA. Mol Ther Nucleic Acids. 2019;15:26–35.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Korenč M, Mencin N, Puc J, Skok J, Nemec KŠ, Celjar AM, et al. Chromatographic purification with CIMmultusTM Oligo dT increases mRNA stability. Cell Gene Therapy Insights. 2021;7(9):1207–16.

    Article  Google Scholar 

  4. Kramps T, Elbers K. Introduction to RNA vaccines. Methods Mol Biol. 2017;1499:1–11.

    Article  CAS  PubMed  Google Scholar 

  5. Anindyajati R, Artarini AA, Riani C, Retnoningrum DS. Plasmid copy number determination by quantitative polymerase chain reaction. Sci Pharm. 2016;84(1):89–101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ahlemeyer B, Colasante C, Baumgart-Vogt E. Analysis of the level of plasmid-derived mRNA in the presence of residual plasmid DNA by two-step quantitative RT-PCR. Methods Protoc. 2020;3(2):40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pavlin N, Černigoj U, Bavčar M, Plesničar T, Mavri J, Zidar M, et al. Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels. ELECTROPHORESIS [Internet]. [cited 2023 Nov 14];n/a(n/a). Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/elps.202300031

  8. Smith CR, DePrince RB, Dackor J, Weigl D, Griffith J, Persmark M. Separation of topological forms of plasmid DNA by anion-exchange HPLC: shifts in elution order of linear DNA. J Chromatograph B. 2007;854(1–2):121–7.

    Article  CAS  Google Scholar 

  9. Mota É, Sousa Â, Černigoj U, Queiroz JA, Tomaz CT, Sousa F. Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger. J Chromatograph A. 2013;1291:114–21.

    Article  CAS  Google Scholar 

  10. Molloy MJ. Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed. Nucl Acids Res. 2004;32(16):e129–e129.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Colote S, Ferraz C, Liautard JP. Analysis and purification of plasmid DNA by reversed-phase high-performance liquid chromatography. Anal Biochem. 1986;154(1):15–20.

    Article  CAS  PubMed  Google Scholar 

  12. Diogo MM, Queiroz JA, Prazeres DMF. Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography. J Chromatograph A. 2003;998(1):109–17.

    Article  CAS  Google Scholar 

  13. Černigoj U, Vidič J, Ferjančič A, Sinur U, Božič K, Mencin N, et al. Guanidine improves DEAE anion exchange-based analytical separation of plasmid DNA. Electrophoresis. 2021;42(24):2619–25.

    Article  PubMed  Google Scholar 

  14. Schmid A. Considerations for producing mRNA vaccines for clinical trials. In: Kramps T, Elbers K, editors. RNA vaccines [Internet]. New York, NY: Springer New York; 2017 [cited 2023 Oct 8]. p. 237–51. (Methods in Molecular Biology; vol. 1499). Available from: http://link.springer.com/10.1007/978-1-4939-6481-9_15

  15. Yang H. Establishing acceptable limits of residual DNA. PDA J Pharm Sci Technol. 2013;67(2):155–63.

    Article  CAS  PubMed  Google Scholar 

  16. The Moderna COVID-19 (mRNA-1273) vaccine: what you need to know [Internet]. [cited 2023 Aug 16]. Available from: https://www.who.int/news-room/feature-stories/detail/the-moderna-covid-19-mrna-1273-vaccine-what-you-need-to-know

  17. The Pfizer BioNTech (BNT162b2) COVID-19 vaccine: what you need to know [Internet]. [cited 2023 Aug 16]. Available from: https://www.who.int/news-room/feature-stories/detail/who-can-take-the-pfizer-biontech-covid-19--vaccine-what-you-need-to-know

  18. Pregeljc D, Skok J, Vodopivec T, Mencin N, Krušič A, Ličen J, et al. Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring. Biotech Bioeng. 2023;120(3):737–47.

    Article  CAS  Google Scholar 

  19. Lee C, Kim J, Shin SG, Hwang S. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol. 2006;123(3):273–80.

    Article  CAS  PubMed  Google Scholar 

  20. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–22.

    Article  CAS  PubMed  Google Scholar 

  21. EMA. European Medicines Agency. 2018 [cited 2023 Dec 4]. ICH Q2(R2) Validation of analytical procedures - scientific guideline. Available from: https://www.ema.europa.eu/en/ich-q2r2-validation-analytical-procedures-scientific-guideline

  22. Bloom K, van den Berg F, Arbuthnot P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021;28(3):117–29.

    Article  CAS  PubMed  Google Scholar 

  23. Oda Y, Kumagai Y, Kanai M, Iwama Y, Okura I, Minamida T, et al. Persistence of immune responses of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2. The Lancet Infectious Diseases [Internet]. 2024 Feb 1 [cited 2024 Feb 10];0(0). Available from: https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(24)00060-4/fulltext

  24. Miklavčič R, Megušar P, Kodermac Š, Bakalar B, Dolenc D, Sekirnik R, et al. High recovery chromatographic purification of mRNA at room temperature and neutral pH. Int J Mol Sci. 2023;24:14267.

    Article  PubMed  PubMed Central  Google Scholar 

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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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Authors and Affiliations

  1. Sartorius BIA Separations d.o.o., Mirce 21, 5270, Ajdovščina, Slovenia

    Marta Leban, Tina Vodopivec Seravalli, Nina Mencin, Sandra Potušek, Andrej Thompson, Aleš Štrancar & Rok Sekirnik

  2. Biomay AG, Ada-Lovelace-Strasse 2, 1220, Vienna, Austria

    Martina Hauer & Ernst Böhm

  3. Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000, Ljubljana, Slovenia

    Jurij Trontelj

Authors
  1. Marta Leban
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  2. Tina Vodopivec Seravalli
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  3. Martina Hauer
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  4. Ernst Böhm
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  5. Nina Mencin
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  6. Sandra Potušek
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  7. Andrej Thompson
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  8. Jurij Trontelj
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  9. Aleš Štrancar
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  10. Rok Sekirnik
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Contributions

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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Correspondence to Rok Sekirnik.

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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

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