(W1130-11-60) Using Cryo-TEM to Characterize Structural Details of LNPs During Manufacturing

Purpose: Optimal lipid nanoparticle (LNP) stability, encapsulation efficiency, and efficacy can be achieved via careful consideration of formulation components, and the choice and optimization of the manufacturing processes as they can directly influence the LNPs’ physicochemical characteristics. Although quality attributes such as LNP size and payload can potentially be assessed by orthogonal techniques, cryo-TEM provides direct visualization of a combination of multiple quality attributes simultaneously, including size, payload distribution, morphology, and aggregation, using the same dataset and on discreet particles preserved in near-native condition. We present the use of cryo-TEM to characterize LNPs generated using different mixing platforms, each with a unique design and operating principle, and compare variations seen in samples in response to deviations during formulation or storage.

Methods: Grids were prepared undiluted using a Vitrobot (TFS). Electron microscopy was performed using a Thermo Fisher Scientific Glacios Cryo Transmission Electron Microscope (Cryo-TEM) operated at 200kV and equipped with a Falcon 3 direct electron detector. The images were acquired at nominal magnifications of 73,000x (0.197 nm/pixel) and 28,000x (0.510 nm/pixel) using Leginon software, at a nominal underfocus of -5.5μm to -2.0μm, and electron doses of ~10-25 e-/Å2. Image analyses were carried out either manually or using a custom AI-based segmentation algorithm. The LNP formulation was a standard four lipid component system (MC3, Cholesterol, DSPC, DMG-PEG-2000), prepared by Phosphorex using a T-mixer, the Ignite+ (Precision NanoSystems), an impingement jet mixer, and the Sunshine (Unchained Labs). The mixing flow rates on each of the four mixing platforms were optimized to achieve LNPs that had a similar size, uniformity, and mRNA encapsulation efficiency according to DLS and Ribogreen measurements.

Results: LNP formulations prepared using the four different mixing systems were examined using cryo-TEM. Surprisingly, all formulations contained 40-46% of particles with bleb formations, except for the LNPs generated by the T-mixer, which showed only 27% particles with blebs. The size of the LNPs generated using the Ignite +, Jetmixer, and Sunshine, were all very similar (60-63 nm). However, particles generated using the T-mixer were slightly smaller with an average size of 52 nm and some fairly large outliers. Interestingly, DLS data (generated by Phosphorex) and cryo-TEM data showed a very similar trend; however, the overall particle size as measured from the cryo-TEM images was smaller than when measured by DLS. This mismatch is not surprising as DLS measures the hydrodynamic size and in cryoTEM measurements are taken directly from the densities in the images. We present examples where cryo-TEM is used to show that changes in buffer ionic strength not only greatly influences the size, but especially the particle morphology: particles in low ionic strength buffer contained 46% particles with bleb formations compared to 1% in high ionic strength buffer! We show that TFF produces LNPs that are 84.9±23.3 nm in size, whereas dialysis produces LNPs that are 60.0±13.9 nm in size (same formulation). TFF also resulted in a significantly higher number of particles with bleb formations than dialysis (60.1% compared to 46.0%). When changing the payload size from ~6,000 to 858 nucleotides, the particle size did not significantly change, but the number of particles displaying bleb formations was significantly less when LNPs were loaded with the smaller payload. Cryo-TEM is also immensely powerful when it comes to unanticipated changes. In this study, we looked at what happened to LNPs when inadvertently concentrating them too long, LNPs generated on a faulty device, as well as LNPs that were stored for 10 weeks at 4°C. For the latter, particles that were expected to be ~60 nm in size, now showed an average area equivalent diameter (AED) of 82 nm and exhibited a very wide size range (866 nm). In addition, many particles now showed more than two compartments, with no shortage in large aqueous compartments (bleb-structures). Changes like these can potentially be crucial when assessing differences in functional activity.

Conclusion: Although size distribution, lamellarity, and presence of payload can potentially be assessed via other individual orthogonal techniques, cryo-TEM has the unique advantage of requiring only parsimonious amounts of sample, directly observing individual particles in their near-native state, and providing information on multiple LNP features simultaneously (including structural details of the particles). In addition, structural differences, including bleb formation, cannot be observed by DLS or other rapid in-line characterization methods. And although bleb formation in LNPs is still a topic of discussion in the field, they can make all the difference when accelerating screening studies during initial LNP development or when explaining potential variations in activity or potency observed during the scale-up process or later during manufacturing. All together this makes cryo-TEM a remarkably useful characterization tool for ensuring optimization of formulation parameters, product consistency, and quality control throughout the entire LNP manufacturing process.

References: 1. Suloway C, Pulokas J, Fellmann D, et al. Automated molecular microscopy: The new Leginon system. J Struct Biol. 2005; 151: 41–60, https://doi.org/10.1016/j.jsb.2005.03.010.
2. Cheng MHY, Leung J, Zhang Y, et al., Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Advanced Materials, 2023, 35, 2303370. https://doi.org/10.1002/adma.202303370.
3. Pratsinis A, Fan Y, Portmann M, et al., Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations. International Journal of Pharmaceutics, 2023, 637, 122874, https://doi.org/10.1016/j.ijpharm.2023.122874.

Acknowledgements: NIS thanks Nicholas Boylan and Sydney Marchando at Phosphorex for their generous provision of samples for this study and assistance
in planning these experiments.

Figure 1. Cryo-TEM images of LNP formulations prepared using a T-mixer, the Ignite+, an impingement jet mixer, and the Sunshine.

Figure 2. Size measurements of LNPs following image analyses of cryo-TEM images. Left: plots showing the probability distribution function (top) and cumulative distribution function (bottom. Right: Mean Area Equivalent Diameter (AED), range, and circularity calculated for each of the four LNP preparations (top); The average diameters calculated from cryo-TEM data compared DLS measurements (bottom).

Figure 3. Size and morphology analysis data for LNPs encapsulating polyA and dialyzed into 20 mM Tris buffer, LNPs encapsulating polyA and buffer exchanged into 20 mM Tris buffer using tangential flow filtration, LNPs encapsulating polyA and dialyzed into TBS, and LNPs encapsulating hEPO mRNA and dialyzed into 20 mM Tris buffer. Representative images of LNPs in low ionic strength buffer versus high ionic strength buffer are shown in the lower right panel.