A recent publicationi investigates the characterization of viral and non-viral vectors using multiple Malvern Panalytical technologies. This application note takes a close look at how researchers used the MicroCal PEAQ DSC to analyze the thermal stability of mRNA-LNP formulations.
What are lipid nanoparticles?
Lipid nanoparticles present a promising alternative to viral delivery vectors for vaccines and therapeutic drugs, with the benefits of cell-free production and the potential for rapid scale-up. However, a better understanding of their complex structure and behavior in solution is required to control production and ensure the final product functions as designed, remains stable in storage and conforms to release specifications.
Figure 1 The basic structure and compositions of the mRNA-LNP characterized in this study.
What is differential scanning calorimetry (DSC)?
DSC measures the heat change associated with a molecule’s thermal denaturation when heated at a constant rate. The technique is used in the development and manufacturing of several commercial vaccines and is a well-established technique to monitor a number of different events:
- phase transitions of lipids
- structural transitions of RNA and DNA molecules
- their complexes with cationic lipids
- thermal stability of lipid-based delivery vectors
How is MicroCal PEAQ DSC applied in LNP research & manufacture?
LNP developers use the MicroCal PEAQ DSC to characterize the stability of LNPs by measuring the tiny heat change associated with the breakdown of the structure. This is known as thermal denaturation. By measuring this thermal unfolding, the MicroCal PEAQ DSC provides a fingerprint of the higher order structure of the particles AND of the RNA inside these particles. This data is highly valuable in product characterization, formulation development, stress and comparability studies.
Developers are increasingly augmenting the characterization insights they get from light scattering techniquesiii with DSC (and SEC–SLS) data to build a deeper, more reliable picture of LNP attributes.
What does thermal stability tell you about mRNA-LNP formulations?
Two mRNA-LNP formulations, LNP1 and LNP2, and their change in thermal transitions during storage were measured using the MicroCal PEAQ DSC. The results of the measurements on mRNA-LNP1 and mRNA-LNP2 are presented in Figure 1 and Table 1.
Figure 1 Overlays of the thermograms obtained for (a) mRNA-LNP1 and (b) mRNA-LNP2 batch 1
The LNP formulations used in this study, mRNA-LNP1 (Figure 1a) and mRNA-LNP2 (Figure 1b), show at least two transitions of significantly different peak amplitudes identified in a lower temperature range and in a higher temperature range. The overall amplitudes of the transitions are consistently higher for the mRNA-LNP1 samples than for mRNA-LNP2. Possible reasons are due to differences in the composition and concentration of the samples (ca. factor 2 difference) and/or different structural arrangements within mRNA-LNP particles.
The thermograms represent a qualitative fingerprint specific to each sample, showing well-reproduced DSC profiles. The corresponding numerical parameters of the thermal transitions, such as integrated heat effect and the temperature of both thermal transitions, Tm1 and Tm2 show only minor changes over the storage condition after 9 days (Table 1).
|Sample||Run #||Tm1 (C)||Tm2 (C)||Total Area (mJ)|
Table 1. Summary of the results of DSC measurements of mRNA-LNP samples. Repeat measurements were made with an interval of nine days.
What can reversibility of transitions reveal about LNP formulations?
Reversibility is an important aspect of the structural transitions observed in DSC. Therefore, the free mRNA and all mRNA-LNP formulations were tested for reversibility of the thermal transitions by performing a re-scan following each DSC scan. Figure 2 shows overlays of the DSC data obtained for the scans (blue) and re-scans (red) of mRNA-LNP1 and free mRNA samples.
Irreversibility of the thermal transition of mRNA-LNP1 (Figure 2a) is a prominent feature of all the mRNA-LNP samples tested in this study (data not shown). It is likely due to the complexity of structure and interaction networks within the mRNA-lipid complexes and lipid phases resulting in kinetically controlled states and slow (if any) relaxation to the original structuresiv.
Figure 2. Overlays of the raw DSC data as traces of differential power, DP over temperature for a scan (blue) and a re-scan (red) of (a) mRNA-LNP1 and (b) free mRNA samples.
The free mRNA sample used in this study has at least three transitions (Figure 2b): The 1st thermal transition around 65°C appears to show about 40% reversibility), whilst the thermal transitions observed above 100°C are not reversible upon the re-scan.
RNA molecules are known to be structurally flexible and can be organized in diversity of secondary and tertiary structuresv and/or built of several domains which can undergo cooperative and non-cooperative transitionsvi. Structural transitions can be triggered by factors, such as temperature, pH, the addition of salts or ligand bindingvii. Encapsulation of mRNA molecules into structurally complex LNPs results in a complex network of interactions, and reversibility of some structural transitions of mRNA, if present, can be expected to be kinetically controlled and may depend on the direction of the change.
DSC and high order structure (HOS)
In addition to multiple stability metrics, DSC provides a fingerprint of higher order structure (HOS) defined by a range of intra- and inter-molecular interactions in a sample.
Figure 3 shows an overlay of DSC thermograms obtained for the free mRNA (red) and the LNP formulations mRNA-LNP1 (blue) and mRNA-LNP2 (green). The main peak observed for the free mRNA (68 °C) is shifted to higher temperatures in the mRNA-LNP1 and mRNA-LNP2 samples (Figure 3a).
If one compares the heat effects of this transition for all three samples and normalizes it to the mRNA concentration per mole (Figure 3b), the free mRNA sample shows the smallest normalized heat effect (red). The mRNA concentration in the LNP samples is about an order of magnitude lower but the heat effects observed for the main transitions in both LNP formulations are significantly higher and cannot be accounted for by the heat of transition of the free mRNA only (Figure 3b).
Figure 3. Overlays of the DSC traces of mRNA-LNP1, mRNA-LNP2 and free mRNA samples corrected for the instrumental blank and baseline and presented as (a) not normalized differential power and as (b) normalized per mole of mRNA for each sample and expressed as apparent excess heat capacity.
This is likely due to the dynamics of mRNA-cationic lipid complex and the likely linkage between the mRNA structural transitions and the disruption of interactions within the complex, with potential effects on the overall lipid assembly and phases, such as hexagonal and lamellar phases described elsewhereviii-x. The MicroCal PEAQ DSC results suggest that the contribution of the mRNA and lipid transitions to the DSC thermogram profile of the mRNA-LNP samples are different from an additive combination of transitions of individual components.
Change in temperature is a common stress factor for biologics. Measuring the impact of thermal stress on complex structures, such as viral and lipid-based vectors, helps to understand the behavior and structure of these biomolecular assemblies in solution. It also helps to inform on rational approaches to the development and design of stable liquid formulation.
MicroCal PEAQ DSC thermograms provide several parameters which serve as sample-specific fingerprints, enabling detailed characterization and comparison of thermal stability and higher order structures, helping scientists to reveal the structure, stability and function of the delivery vector.
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i N. Markova, et al. Vaccines 10, 49 (2022).
ii Horowitz, E.D.; Rahman, K.S.; Bower, B.D.; Dismuke, D.J.; Falvo, M.R.; Griffith, J.D.; Harvey, S.C.; Asokan, A. Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release. J. Virol. 2013, 87, 2994–3002. [CrossRef] [PubMed].
iii Specifically: dynamic light scattering (DLS), multi-angle dynamic light scattering, (MADLS), nanoparticle tracking analysis (NTA) and electrophoretic light scattering (ELS).
iv Larson, N.R.; Hu, G.; Wei, Y.; Tuesesca, A.; Forrest, M.L.; Middaugh, C.R. pH-Dependent Phase Behavior and Stability of Cationic Lipid–mRNA Nanoparticles. J. Pharm. Sci. 2021. [CrossRef]
v Draper, D.E. Strategies for RNA folding. TIBS 1996, 21, 145–149. [CrossRef]; Blake, R.D.; Delcourt, S.G.; Breslauer, K.J. High-resolution calorimetric and optical melting profiles of DNA plasmids: Resolving contributions from intrinsic melting domains and specifically designed inserts. Biopolymer 1999, 50, 303–318. [CrossRef]; Spink, C.H. Differential Scanning Calorimetry. Methods Cell Biol. 2008, 84, 115–141. [PubMed]
vi Koltover, I.; Salditt, T.; Raedler, J.O.; Sanya, C.R. An Inverted Hexagonal Phase of Cationic Liposome-DNA Complexes Related to DNA Release and Delivery. Science 1998, 281, 78–81. [CrossRef]; Li, S.J.; Marshall, A.G. Multistage unfolding of wheat germ ribosomal 5S RNA analyzed by Differential Scanning Calorimetry. Biochemistry 1985, 24, 4047–4052. [CrossRef]
vii Draper, D.E. Strategies for RNA folding. TIBS 1996, 21, 145–149. [CrossRef]
viii Koltover, I.; Salditt, T.; Raedler, J.O.; Sanya, C.R. An Inverted Hexagonal Phase of Cationic Liposome-DNA Complexes Related to DNA Release and Delivery. Science 1998, 281, 78–81. [CrossRef]
ix 74. Middaugh, C.R.; Ramsey, J.D. Analysis of cationic lipid—Plasmid DNA complexes. Anal. Chem. 2007, 79, 7240–7248.
x Larson, N.R.; Hu, G.; Wei, Y.; Tuesesca, A.; Forrest, M.L.; Middaugh, C.R. pH-Dependent Phase Behavior and Stability of Cationic Lipid–mRNA Nanoparticles. J. Pharm. Sci. 2021. [CrossRef]