CASE: New biomanufacturing technology for mRNA

University of Registration: University of Birmingham

Non-academic partner: Dr Tingting Cui, AstraZeneca

Project Outline

In vitro transcribed messenger RNA (IVT mRNA) technology was a remarkable success story during the COVID-19 pandemic and is largely responsible for kick-starting a revolution in RNA-based vaccines and therapeutics.[1] Part of the excitement behind RNA medicines is that they extend the domain of druggable targets beyond what can currently be achieved with small molecules and biologics, meaning that their potential to treat a wide range of conditions, including previously untreatable diseases and curing genetic diseases, is vast.[2] However, while there is much cause for optimism, looming threats of the next pandemic ‘Disease X’[3] and overzealous goals for tackling it[4] are substantially at odds with the problems facing manufacturing at world-scale.[5]

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 reactions.[1,2] 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, principal among these uncapped, shortened and lengthened RNA species in single- and double-stranded forms. The removal of residual critical impurities from single-stranded poly(A)-tailed mRNA products requires innovation in bioseparation and process analytical technology (PAT) consistent with cGMP manufacture.

It is tempting to adopt practices matured from the manufacture of biotherapeutic proteins. It is prudent, however, to recognize, and, where possible, exploit salient differences between nucleic acid and protein polymers to direct how best to purify single-stranded mRNA. One such difference is in their thermal behaviour. While some proteins show unusually high tolerance to heat, most exhibit a small operating window of temperature before they unfold irreversibly.

Nucleic acids show contrary behaviour. Past, though largely ignored, studies on RNA purification extol the benefits of temperature variation for fractionating single- from double-stranded species and small from large nucleic acid polymers under otherwise mild conditions, and for delivering the highest purities and yields. However, the wholesale adoption of ambient temperature operation in large-scale chromatography of proteins, combined with perceived difficulties in scaling up thermal chromatography, means that the use of temperature as a driving force in mRNA purification remains untapped.

In this project ‘green’ thermally-driven bioseparation systems[6,7] and supporting PAT tools[8,9] conceived at UoB will be applied to the processing of IVT mRNAs. This will entail exploiting the differential melting properties of nucleic acids in their adsorbed state for continuous separation and purification of mRNA from IVT reaction cocktails. Novel chromatography systems mounted with travelling heating and cooling reactors and employing affinity, ion exchange, hydrophobic interaction and mixed mode principles will be used. Optical measurement systems will also be advanced for high-throughput screening in mRNA purification development, and as ‘real-time’ in-line monitoring tools for use in manufacturing.

References

1. Barbier, A. et al. (2022) Nat. Biotechnol. 40:840−854.

2. Damase, T. et al. (2021) Front. Bioeng. Biotechnol. 9:628133.

3. Neumann, G. & Kawaoka, Y. (2023) Viruses 15(1):199.

4. Saville, M. et al. (2022) N. Engl. J. Med. 387(2):e3.

5. Schmidt, A. et al. (2021) Processes, 9(5):748.

6. Ketterer, B. et al. (2020) J. Chromatogr. A, 1609:460429.

7. Brean, A. et al. (2024) J. Chromatogr. A, 1731:465212.

8. Moore-Kelly, C. et al. (2019) Anal. Chem. 91:13794−13802.

9. Bruque, M. et al. (2024) Anal. Chem. 96:15151–15159.

Candidates are encouraged to contact Professor Owen Thomas to discuss the project before applying if they wish to.