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ORIGINAL RESEARCH article

Front. Mol. Biosci.
Sec. Molecular Diagnostics and Therapeutics
Volume 11 - 2024 | doi: 10.3389/fmolb.2024.1443917
This article is part of the Research Topic In Vitro Transcription (IVT) Reaction – The Gateway to New Therapeutic Modalities View all 7 articles

HPLC for at-line reaction monitoring and purification improves yield and purity of tRNA

Provisionally accepted
Polona Megušar Polona Megušar 1Ewen Calder Ewen Calder 2,3Tina Vodopivec Seravalli Tina Vodopivec Seravalli 1Sergeja Lebar Sergeja Lebar 1Louise J. Walport Louise J. Walport 2,3*Rok Sekirnik Rok Sekirnik 4*
  • 1 Sartorius, Goettingen, Germany
  • 2 The Francis Crick Institute, London, England, United Kingdom
  • 3 Imperial College London, London, England, United Kingdom
  • 4 Sartorius (Germany), Göttingen, Germany

The final, formatted version of the article will be published soon.

    Engineered transfer RNA is an emerging therapeutic modality, particularly suited to treatment of diseases caused by genetic disorders based on premature termination codons, frameshifts, or missense mutations. It is also extensively used in reprogramming of in vitro translation systems to generate non-canonical amino acid-containing proteins and peptides, such as in mRNA display. Due to its length, chemical synthesis of tRNA is challenging and production of engineered tRNA at scale is currently limited to in vitro transcription from a DNA template. Previously, the highest reported in vitro transcription yield was 2.5 g/L, significantly below the industry standard for mRNA production of 7–10 g/L. To improve this process, we implemented monitoring of nucleoside triphosphate consumption and tRNA production during in vitro transcription, using at-line high-performance liquid chromatography, with a monolithic solid phase. This allowed for optimization of nucleoside triphosphate concentration, reduction of the in vitro transcription time to <4 h, and improvement of yield up to 4.7 g/L. A step-elution purification on a DEAE chromatographic monolith with >90% step yield was then developed. These improvements in the production and purification of tRNA represent an important step in facilitating production of tRNA for research purposes, and provide a method for purification of therapeutic tRNAs that is scalable and compatible with Good Manufacturing Practice requirements for clinical production

    Keywords: tRNA, In vitro transcription, HPLC, Chromatography, anion exchange

    Received: 04 Jun 2024; Accepted: 10 Sep 2024.

    Copyright: © 2024 Megušar, Calder, Vodopivec Seravalli, Lebar, Walport and Sekirnik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Louise J. Walport, The Francis Crick Institute, London, NW1 1AT, England, United Kingdom
    Rok Sekirnik, Sartorius (Germany), Göttingen, Germany

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