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Lipid-nanoparticle (LNP), micropump, stability, processing

Abstract

The use of lipid-nanoparticles (LNPs) for delivering nucleic acids has become widely popular in clinical studies, including vaccine development and other innovative therapies (Jung et al. 2022). LNPs typically consist of four lipid types, each playing a critical role in stability, delivery, and cellular uptake:  cationic or ionizable amino lipids, helper phospholipids, cholesterol, and PEGylated lipids. The production of LNPs involves rapid mixing of a lipid-containing ethanol phase with an aqueous nucleic acid-containing phase. The increasing polarity of the surrounding media during the mixing process  induces the electrostatic interaction of the phosphate backbone of the nucleic acid and the amino lipid. Further increasing polarity yields lipid particles with an electron-dense core containing the nucleic acids. To eliminate residual ethanol and neutralize the pH, cross-flow filtration (CFF) is employed. All  processing steps, particularly CFF applications, necessitate transportation through pumping. While the effects of pumping and shear stress on proteins have excessively been studied (Fanthom et al. 2023), LNP stability has only been evaluated for storage and filtration so far (Mehta et al. 2023).

This study presents a systematic approach to evaluate the influence of pump-induced stress on LNP quality attributes in a lab-scale setup. The combination of online Z-average determination using dynamic light scattering (DLS) during pump-induced transportation and offline analytics to evaluate LNP  quality attributes yields a comprehensive picture of LNP quality and stability during processing. To reduce the required volumes and minimize the scale, a piezoelectric titanium-based microfluidic pump with passive spring valves (Bußmann et al. 2021) is used. For a full set of analytics, the in-process  sampling took place and was monitored in terms of stability. Besides size monitoring via DLS, the further LNP quality attributes surface charge, lipid composition, and encapsulation efficiency of the nucleic acids are assessed by electrophoretic light scattering, reversed-phase high-performance liquid  chromatography coupled with a charged aerosol detector, and fluorescence-based assays.

In the second part of the study, LNPs loaded with nucleic acid molecules of different sizes were tested using the same pumping process. The LNP formulation parameters, such as nitrogen-to-phosphate ratio, lipid concentration, and lipid ratio, as well as process-related parameters, such as flow rate  ratio and total flow rate, were kept constant. This allowed us to evaluate the impact of nucleic acid molecular weight and length on the stability of LNP in isolation.

In conclusion, a combination of online and offline monitoring of LNP size, along with offline analysis of LNP quality attributes can help to accurately identify pumping-induced quality issues. Moreover, this analytical panel and lab-scale setup can be utilized in the future to assess different pump types or  tubing types helping to increase process knowledge and understanding for LNP large-scale processes.

Acknowledgement

This research project is part of Moore4Medical which received funding from the ECSEL JU, under grant agreement H2020-ECSEL-2019-IA-876190. See also https://moore4medical.eu (accessed on 22nd February 2024).

References

  • Bußmann, A. B., Durasiewicz, C. P., Kibler, S. H. A., Wald, C. K. (2021). Piezoelectric titanium-based microfluidic pump and valves for implantable medical applications. Sensors and Actuators A: Physical, 323, 112649, DI: 10.1016/j.sna.2021.112649.
  • Fanthom, T. B., Wilson, C., Gruber, D., & Bracewell, D. G. (2023). Solid-Solid Interfacial Contact of Tubing Walls Drives Therapeutic Protein Aggregation During Peristaltic Pumping. Journal of pharmaceutical sciences, 112(12), 3022–3034. DOI: 10.1016/j.xphs.2023.08.012
  • Mehta, M., Bui, T. A., Yang, X., Aksoy, Y., Goldys, E. M., and Deng, M. (2023). Lipid-Based Nanoparticles for Drug/Gene Delivery: An Overview of the Production Techniques and Difficulties Encountered in Their Industrial Development. ACS Materials Au 2023 3 (6), 600-619 DOI: 10.1021/acsmaterialsau.3c00032
  • Jung, H. N., Lee, S. Y., Lee, S., Youn, H., & Im, H. J. (2022). Lipid nanoparticles for delivery of RNA therapeutics: Current status and the role of in vivo imaging. Theranostics, 12(17), 7509–7531. DOI: 10.7150/thno.77259