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Increasing productivity of IVT reaction using at-line monitoring by HPLC chromatography

Increasing productivity of IVT reaction using at-line monitoring by HPLC chromatography

mRNA is produced by in vitro transcription (IVT) reaction, which is typically performed as a batch process. Considering its catalytic basis, it is possible to extend reaction time and yields by continuous addition of consumed reagents to the reaction mixture, however fed-batch strategies reported to date have only managed to achieve a 40 – 100 % increase in the production of mRNA. One of the main limitations of development has been low throughput of analytics for quantification of mRNA and NTP precursors; productivity is typically determined as an end-point measurement of mRNA concentration.

Webinar will discuss an HPLC-based analytical methodology using a multimodal ligand based on anion exchange/hydrogen-bonding ligand (PrimaS) to monitor the IVT reaction, which allows for simultaneous quantification of NTPs, capping reagent, plasmid, and mRNA within 3.5 min.

When applied to monitoring IVT reaction a batch approach with an average productivity of 3-5 mg/mL can be converted to fed-batch yielding 10-12 mg/mL. Two approaches for controlling IVT reaction will be demonstrated, one focusing on productivity of uncapped mRNA, another on productivity of capped mRNA, thereby requiring a precise control of NPT:capping reagent ratio

Attend this webinar to increase your understanding of:

  • Benefits of implementing HPLC-based analytics in mRNA production workflow
  • Principles of monolith chromatography and its use for mRNA analytics and purification
  • Principles of IVT reaction and factors affecting rate and yield
  • Principles of conversion from batch to fed-batch process

Rok Sekirnik

Head of Process Development, mRNA/pDNA, BIA Separations

Rok Sekirnik graduated in Chemistry at University of Oxford, where he also completed his PhD. He has worked in the areas of mAb, vaccine and gene therapy vector development in various roles, including Principal Scientist at Novartis and Project Manager at Batavia Biosciences. His team at BIA Separations is working on developing high yielding, cost-effective purification approaches for pDNA and mRNA production.


Rok Sekirnik
Rok Sekirnik
Head Process Development for mRNA and pDNA at BIA Separations

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