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New downstream processing technology for in vitro transcribed messenger RNA

Primary Supervisor: Professor Owen R.T. Thomas (OT), School of Chemical Engineering

Secondary supervisor: Prof Tim R Dafforn (TD)

PhD project title: New downstream processing technology for in vitro transcribed messenger RNA

University of Registration: University of Birmingham

Project outline:

In vitro transcribed messenger RNA (IVT mRNA) is being touted as the next big thing in the biopharmaceutical sector. The idea is beautiful in its simplicity … construct an mRNA sequence, deliver it to the target cell, then sit back and allow the cell’s own machinery to spew forth the encoded protein, which elicits the desired therapeutic effect … but the implementation is infinitely more complex. Some passionate advocates of IVT mRNA based therapies claim that they will disrupt the drug industry, rendering current and new therapies obsolete, and that IVT mRNA is the veritable ‘Columbus Egg’ we’ve all been waiting for.1 While there is much cause for optimism, cold reality should temper expectation. Prior to the COVID-19 pandemic, the first mRNA therapies were some way off, i.e. either in preclinical development, Phase 1 or 2 clinical trials.2 While some smaller pioneering companies at the forefront of mRNA drug development believe molecular biology based kits and procedures for the extraction, purification and analysis of nucleic acids are appropriate for future commercial manufacture of therapeutic grade mRNA,3 larger partnering biopharmaceutical manufacturers do not. Compared with biopharmaceutical proteins such as monoclonal antibodies which are manufactured under cGMP using mature standardised platform processes developed over the past four decades, there is a definitive lack of enabling technology to support process development and manufacture of future mRNA biopharmaceuticals.

IVT mRNAs are produced in cell-free systems by in vitro transcription from DNA templates using cocktails containing a RNA polymerase, ribonucleotides and 5′ capping agents. Following mRNA synthesis, the mRNA product must be recovered from a complex soup containing spent polymerase, unincorporated ribonucleotides, DNA template, and an assortment of product-related impurities (PRI), principal among these uncapped, shortened and lengthened RNA species in single- and double-stranded forms. While biological solutions currently under development are expected to improve the yield and fidelity of in vitro transcription, reduce PRI load and diversity, and lessen purification demands, the removal of residual critical impurities from single-stranded poly(A)-tailed mRNA products requires innovation in downstream process separation technology consistent with cGMP manufacture and specifically tailored to the characteristics of nucleic acids.

Affinity chromatography, the lynchpin in commercial production of therapeutic antibodies will likely play a dominant role in the future platform manufacture of mRNA therapeutics. Continuous magnetic particle based affinity separations4-6 likewise offer attractive possibilities for mRNA products. Affinity separation of IVT mRNA via hydridization of its poly(A) tail to solid-supported oligodeoxythymine (dT) can be highly selective. Despite early studies indicating thermal elution delivers the highest purities and yield it is not practiced today, likely arising from perceived difficulties in scaling up thermoresponsive adsorption processes.7 In this project the differential melting properties of nucleic acids in their adsorbed state will be exploited for the purification of IVT mRNA using three powerful continuous separation systems being developed at U. Birmingham in association with KIT, Germany. One of these is chromatographic (travelling heating zone reactor chromatography7), the others magnetic (high-gradient magnetic fishing4,5 and continuous magnetic extraction6), and each promises substantial improvements in productivity and purity of mRNA cf. current benchmarks.

References:

  1. J Transl Med 17(1):54
  2. https://www.modernatx.com/, https://ethris.com/, https://biontech.de/
  3. 3-month manufacturing process for BioNTech COVID-19 mRNA vaccine is the same as that approved 10 years ago to supply IVT mRNA for clinical testing
  4. Biotechnol Bioeng, 110(6): 1714
  5. J Chem Technol Biotechnol 93: 1901
  6. J Chromatogr A, 1305, 7
  7. J Chromatogr A, 1609, 460429

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth:Industrial Biotechnology & Integrated Understanding of Health: Pharmaceuticals

Techniques that will be undertaken during the project:

mRNA preparation: In vitro transcription (IVT) reactions, Poly (A) tailing reactions, mRNA extraction from cells using magnetic bead kits

Analytical techniques: UV/Vis Spectrophotometry, Circular Dichroism, Linear Dichroism, Gel electrophoresis, Size exclusion chromatography

Conventional fixed bed chromatography: Oligo(dT) affinity, Anion Exchange, Size Exclusion

Avantgarde separation technology: Continuous single column travelling heating zone reactor (THZR) chromatography; Continuous high-gradient magnetic affinity separation; Continuous magnetic extraction

Contact: Professor Owen Thomas, University of Birmingham