(T1330-12-67) Fourier Transform Infrared Spectroscopy (FT-IR) of Lyophilized RNA

Purpose: With the onset of the COVID-19 pandemic, mRNA vaccines have become a significant part of the biopharmaceutical industry. mRNA is highly unstable and currently requires ultracold temperatures (-80 ^oC) to minimise degradation, making storage and transportation difficult. Chemical degradation occurs through cleavage of the phosphodiester bonds of the mRNA backbone. The 2’ OH group of ribose initiates a nucleophilic attack on the phosphodiester bond, causing a transesterification reaction and breakage of the P-O bond. Oxidation of the nucleobases and ribose groups can also cause the cleavage of bonds and change the chemical structure of mRNA, affecting its chemical stability.^1,2 There are ongoing attempts to improve the stability of mRNA by converting to solid state. One such method that is being explored is lyophilization. Lyophilization is a drying method that involves three steps – (i) freezing of the solution (ii) removal of free water by sublimation (primary drying) (iii) removal of bound water by desorption (secondary drying). However, some reports suggest that the stresses exerted during the freezing and drying processes may cause damage to mRNA.^3,4 There are also studies showing that ‘though mRNA-LNPs retain their integrity after lyophilization, their in-vivo transfection efficacy is reduced’.^3,5 Despite these challenges, an optimised lyophilization cycle has the potential to improve the stability of mRNA. There are few studies that describe interactions of lyophilized mRNA with excipients (e.g., sugars, salts, cationic lipids) in the solid state.⁵ Here, these interactions are analysed through Fourier transform infrared spectroscopy with attenuated total reflectance (ATR-FTIR). This study provides a library of FT-IR spectra of RNA lyophilized with different excipients and at different ratios. RNA from the yeast Saccharomyces cerevisiae (average of 100 nt) is used as a simple and affordable model system for mRNA.

Methods: Solutions of yeast RNA (1mg/mL) were co-lyophilized with mannitol and sodium chloride at different ratios (RNA: Excipient = 1:2, 1:1, 2:1). Cationic lipid formulations were prepared by adding DOTAP chloride (1 mg/mL in 1% EtOH) to these solutions at two different ratios (DOTAP: RNA: Excipient = 1:2:2 and 1:1:1) before lyophilization. ATR-FTIR was carried out on each sample with 64 scans and at a resolution of 2 cm^-1. The baselines of the spectra were corrected, and spectra were normalized to account for the deviations in sample amount. Denaturing gel electrophoresis (agarose 1 % in MOPS-formaldehyde buffer) was performed on each sample to confirm the integrity of RNA after lyophilization.

Results: The FT-IR spectrum of lyophilized RNA shows three peaks of interest - (i) the band near 1683 cm^-1 is assigned to stretching vibrations of carbonyl groups, (ii) the band near 1215 cm^-1 represents antisymmetric stretching of the phosphate group and is attributed to the phosphodiester bond of the nucleic acid backbone, and (iii) the band near 1048 cm ^-1 represents C-O stretching of the ribose sugar.^6,7 The position of the bands shifted when RNA was co-lyophilized  with excipients. (i) Mannitol formulations show a slight shift of the carbonyl band in some formulations. In NaCl formulations, the position of the band is not affected by ratio. (ii) Mannitol formulations showed shifting of the phosphate band to higher wavenumber with increase in amount of RNA. The extent and direction of shift observed in NaCl formulations does not trend with ratio. (iii) The C-O band shifted to a higher wavenumber (1053cm^-1) in mannitol formulations suggesting interaction between mannitol and ribose. However, there could be interference from overlapping signals from hydroxyl groups of mannitol. In NaCl samples, a smaller shift is observed that does not trend with ratio. The effects of the cationic lipid DOTAP was studied for RNA co-lyophilized  with NaCl. For all the formulations studied, the position of the C=O and C-O band did not change from that of RNA without excipient. The phosphate band however, shifted with the change in ratio. As the DOTAP: RNA: NaCl ratio is increased to 1:2:2, an increase in wavenumber from 1218 cm^-1 to 1220 cm^-1 is observed. This suggests that DOTAP interacts with the phosphate groups of RNA even in the presence of an ionizable excipient (NaCl).

Conclusion: This study used ATR-FTIR to probe the interactions of lyophilized RNA with different excipients at varying ratios. The IR spectra of the formulations studied showed that the negatively charged phosphate group of RNA interacts with ionizable excipients. This acts as a first step in understanding the role of excipients in stability of the phosphodiester bond of RNA and its impact on developing stable solid-state mRNA formulations.

References: 1. Mikkola, S., Lönnberg, T. and Lönnberg, H. (2018) ‘Phosphodiester models for cleavage of nucleic acids’, Beilstein Journal of Organic Chemistry, 14, pp. 803–837. doi: 10.3762/bjoc.14.68.
2. Schoenmaker, L. et al. (2021) ‘mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability’, International journal of pharmaceutics. 2021/04/09. The Author(s). Published by Elsevier B.V., 601, p. 120586. doi: 10.1016/j.ijpharm.2021.120586
3. Ai, L. et al. (2022) ‘Lyophilized mRNA-lipid nanoparticle vaccine s with long-term stability and high antigenicity against SARS-CoV-2’, pp. 1–17.
4. KETTERER, T. et al. (2016) ‘Lyophilization of RNA’.
5. Zhao, P. et al. (2020) ‘Long-term storage of lipid-like nanoparticles for mRNA delivery’, Bioactive Materials. Elsevier, 5(2), pp. 358–363. doi: 10.1016/j.bioactmat.2020.03.001.
6. Geinguenaud, F., Militello, V. and Arluison, V. (2020) ‘Application of FTIR Spectroscopy to Analyze RNA Structure’, in Arluison, V. and Wien, F. (eds) RNA Spectroscopy: Methods and Protocols. New York, NY: Springer US, pp. 119–133. doi: 10.1007/978-1-0716-0278-2_10.
7. Banyay, M., Sarkar, M. and Gräslund, A. (2003) ‘A library of IR bands of nucleic acids in solution’, Biophysical Chemistry, 104(2), pp. 477–488. doi: 10.1016/S0301-4622(03)00035-8

Acknowledgments: Funding for this research is provided by EPSRC-SFI Centre for Doctoral Training in Transformative Pharmaceutical Technologies.

FT-IR spectrum of lyophilized yeast RNA



FT-IR spectra of yeast RNA co-lyophilized with a) Mannitol (RNA:Excipient = 1:0, 1:2, 1:1, 2:1) and b) NaCl (RNA:Excipient = 1:2, 1:1, 2:1)



FT-IR spectra of yeast RNA co-lyophilized with DOTAP Chloride and NaCl at ratios DOTAP:RNA:NaCl= 1:1:1 and 1:2:2