(T1230-12-77) Analytical Characterization for Nucleic Acid Therapy: A Case Study

Purpose: Nucleic acid therapeutics are considered the third major drug discovery platform in addition to small molecules and antibodies – they enable control of gene expression by gene inhibition, addition, replacement or editing and provide long-lasting effects. Therefore, the development of robust tools for the analysis of nucleic acids is of high relevance. (m)RNA therapeutic efficiency is closely linked to its structure and the maintenance of its integrity. Most reported methods to assess mRNA integrity are based on liquid chromatography and electrophoresis. However, these methods rely on considerable amounts of material and cannot be easily expedited. Herein, we propose a novel method to monitor (m)RNA integrity, using a differential scanning fluorimetry (DSF). The data gathered through the complementary analytical techniques – liquid chromatography, electrophoresis and DSF – allowed to understand the impact on mRNA integrity, when submitted to stress-inducing conditions. Moreover, with this case study we demonstrated that DSF can be used as an expedite and low-consumption technique to assess mRNA integrity.

Methods: Samples composed of different proportions of mRNA, excipients and stressing agent were evaluated using ion pair reversed-phase chromatography, agarose gel electrophoresis and differential scanning fluorimetry. Ion pair reversed-phase chromatography (IP-RP HPLC) was performed in a ACQUITY UPLC H-Class PLUS (Waters, USA). mRNA sample separation was accomplished with a DNAPac RP column with 4-µm particles and dimensions of 2.1 × 100 mm (Thermo Fisher Scientific) at a flow rate of 0.45 mL/minute and column temperature of 80 °C. Mobile phase A consisted of 0.1 M Triethylamine Acetate (TEAA) buffer and mobile phase B consisted of 50:50 %v/v acetonitrile (ACN) (Sigma-Aldrich) and 0.1 M TEAA buffer. Agarose gel electrophoresis was performed in Wide Mini-Sub Cell GT (BioRad) and mRNA samples were incubated with sample buffer and Sybr Green II (Invitrogen™, 10,000X stock solution)) and heated 5 minutes at 70ºC, before being loaded in the gel. An RNA Ladder (RiboRuler High Range RNA Ladder, Thermo Scientific™) was used as control. Gels were imaged on a Chemidoc MP imaging system (BioRad). Fluorescence profiles of mRNA samples were obtained in CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

Results: The integrity profile of mRNA control sample was monitored by IP-RP HPLC and gel electrophoresis and was aligned with literature and supplier information. When mRNA was incubated with the stress agent, a controlled hydrolysis of mRNA molecule was noticeable: with IP-RP HPLC, a decrease in peak area of mRNA main peak was observed (Fig.1,a), and, also, a smear-like band was evident in gel electrophoresis (Fig.1,b). Moreover, at specific conditions of stress agent, a possible aggregation into insoluble complexes prevented migration of samples in the gel (Fig.1,b). With this preliminary work, we were able to establish a protocol for controlled mRNA hydrolysis, with a lower limit of quantification and higher repeatability when using IP-RP HPLC (Fig.2). Then, mRNA was incubated with the stress agent and its fluorescence profile monitored by DSF. A correlation between stress agent and fluorescence was found, being aligned with the data gathered by IP-RP HPLC and electrophoresis. mRNA was further incubated with stress agent and excipients at different conditions and its integrity evaluated through the three different analytical. Similarly, the results demonstrated DSF was aligned with classic methods (Fig.3).

Conclusion: In this work, mRNA was submitted to stress-inducing conditions and a protocol for mRNA-controlled hydrolysis was established through three different analytical techniques: IP-RP HPLC, agarose gel electrophoresis and DSF. The kinetic fluorescence profile results correlated with classic IP-RP HPLC and agarose gel electrophoresis, sustaining that we can leverage DSF to assess mRNA integrity.

References: [1] J. Currie, J. R. Dahlberg, J. Eriksson, F. Schweikart, G. A. Nilsson, and E. Örnskov, “Stability Indicating Ion-Pair Reversed-Phase Liquid Chromatography Method for Modified mRNA.” ChemRxiv, 2021, doi: 10.26434/chemrxiv-2021-mgx9q.
[2] G. J. Guimaraes, J. Kim, and M. G. Bartlett, “Characterization of mRNA therapeutics,” Mass Spectrom. Rev., no. February, pp. 1–25, Jul. 2023, doi: 10.1002/mas.21856.
[3] R. Silvers, H. Keller, H. Schwalbe, and M. Hengesbach, “Differential Scanning Fluorimetry for Monitoring RNA Stability,” ChemBioChem, vol. 16, no. 7, pp. 1109–1114, May 2015, doi: 10.1002/cbic.201500046.

Acknowledgements:This work is supported by Fundação para a Ciência e Tecnologia (FCT) through the PhD scholarship SFRH/BD/151219/2021 and Hovione Farmaciência S.A.

mRNA integrity and degradation profile assessed by IP-RP HPLC and agarose gel electrophoresis.

Correlation between % degradation and stress agent.

Agreement between results for mRNA integrity assessed through the complementary analytical techniques.