(427e) Understanding the Interplay between Magnesium Pyrophosphate Crystallization and mRNA Production

The in vitro transcription (IVT) reaction is the primary industrial pathway for producing mRNA vaccines and therapies [1]. Pyrophosphate (PPi), one of the byproducts of the IVT reaction, forms an insoluble salt with magnesium (Mg₂PPi), whose crystallization is associated with a reduction in mRNA production yield [2]. The prevailing explanation is that Mg₂PPi crystals bind with the DNA template, thereby halting the reaction [3]. However, the characterization and methodologies supporting this conclusion are inconclusive and require further investigation.

The objective of this project is to investigate the IVT reaction from a crystallization viewpoint. This involves establishing solubility curves, measuring crystal nucleation and growth rates of Mg₂PPi in the presence and absence of DNA, and gaining a fundamental molecular-level comprehension of the DNA-Mg₂PPi interaction. The complexity of electrolyte solutions' thermodynamics regarding the speciation and sensitivity of ionic concentrations to changes in pH, temperature, and the presence or absence of other ionic species, necessitated the development of various techniques to determine supersaturation as the driving force of crystallization for this system. Additionally, we explored whether the heterogeneous nucleation of Mg₂PPi on DNA could lead to a different induction time compared to homogeneous nucleation of Mg₂PPi in the absence of DNA. Consequently, high-throughput nucleation induction time studies were utilized to gain insights into the nucleation rate of this process.

Overall, delving into the complexities of Mg₂PPi crystallization within IVT reactions deepens our understanding of mRNA production mechanisms and offers promising avenues for enhancing production efficiency and yield.

[1] Barbier, A. J., Jiang, A. Y., Zhang, P., Wooster, R., & Anderson, D. G. (2022). The Clinical Progress of mRNA Vaccines and Immunotherapies. Nature Biotechnology, 40(6), 840854. https://doi.org/10.1038/s41587-022-01294-2

[2] Stover, N. M.; Ganko, K.; Braatz, R. (2023) Mechanistic Modeling of InVitro Transcription. Authorea. DOI: 10.22541/au.169299542.25533105/v1

[3] Akama, S., Yamamura, M., & Kigawa, T. (2012). A Multiphysics Model of In Vitro Transcription Coupling Enzymatic Reaction and Precipitation Formation. Biophysical Journal, 102(2), 221–230. https://doi.org/10.1016/j.bpj.2011.12.014

Acknowledgements: This research was supported by the U.S. Food and Drug Administration under the FDA BAA-22-00123 program, Award Number 75F40122C00200.