(T0930-03-17) Effect of Process Parameters and Buffer Exchange on the Critical Quality Attributes of siRNA Loaded Lipid Nanoparticles

Purpose: With six FDA-approved drugs available, the significance of small interfering RNA (siRNA) therapeutics in treating various diseases is evident. Lipid nanoparticle (LNP) technology, exemplified by Onpattro^®, has emerged as a promising delivery system for siRNA. Due to its scalability and robust process, microfluidic mixing is a popular manufacturing method to produce siRNA-loaded LNPs (siRNA-LNPs). Understanding the effect of process parameters, including total flow rate (TFR), flow rate ratio (FRR), and temperature, as well as the effects of buffer exchange step on the critical quality attributes (CQAs) of siRNA-LNP formulations is essential for optimizing their production and ensuring consistent quality, which is the aim of present study.

Methods: A two-level full factorial design was employed to assess the impact of three variables: TFR(1, 3 mL/min), FRR (1, 5), and temperature (8, 45 ºC) on the CQAs of siRNA-LNP formulations. Eight siRNA-LNPs were synthesized with different process parameters utilizing the NanoGenerator Flex-M microfluidic system. In brief, lipid constituents, maintaining the same molar ratio and composition as Onpattro^® [DLinMC3DMA (49.3):Cholesterol (39.04):DSPC (10.16):PEG-C-DMG (1.49)], were dissolved in anhydrous ethanol. Similarly, siRNA with a sequence and modifications similar to Onpattro^® was dissolved in 25 mM sodium acetate buffer (pH 4). These two phases were mixed in the Flex-M micromixer, and the resulting siRNA-LNPs were collected and dialyzed (utilizing a 10 kDa cutoff dialysis cassette) against phosphate buffered saline (PBS) to remove organic solvent residues and exchange buffers. Size, polydispersity index (PDI), and zeta potential of the siRNA-LNPs pre- and post-dialysis were determined using Zetasizer. The morphology of the formulations was assessed via transmission electron microscopy (TEM) imaging. RNA content and encapsulation efficiency (EE%) were evaluated before and after dialysis using the Ribogreen fluorometric assay. Lipid component quantification was conducted before and after dialysis using ultra-performance liquid chromatography (UPLC) coupled with charged aerosol detector (CAD). Subsequently, the effect of process parameters on the CQAs of the prepared siRNA-LNP formulations was analyzed using JMP software.

Results: Size, PDI, zeta potential, and EE% of formulations both pre- and post-dialysis were measured and the effect of process parameters on them was determined. Pre-dialysis, the particles exhibit a positive surface charge because the pH of 4 is lower than the apparent pKa of the ionizable lipid DLin-MC3-DMA (approximately 6.4). Post-dialysis, the pH increases to 7, resulting in the particles exhibiting near-neutral surface charge values. Analysis of the data, illustrated in Figure 1, indicates that FRR and temperature significantly affect particle size pre-dialysis, with FRR exhibiting the most significant impact, as evidenced by the lowest P value (and highest Logworth) as well as the largest size effect as evidenced by the largest absolute value of parameter estimate, also reflected in the slope steepness in the prediction profiler graph. The effect of temperature on size was less significant post-dialysis. Notably, the parameters show no significant effect on PDI pre- or post-dialysis. Higher FRR correlates with increased EE% both pre- and post-dialysis, as shown in Figure 1. Overall, the effect of parameters on responses is more pronounced pre-dialysis due to the additional variation introduced by the dialysis process. Figure 2 illustrates that at low FRR, there is a greater loss of lipid content during dialysis, possibly attributed to the removal of unincorporated lipids in LNPs. This effect leads to a significant alteration in the lipid molar ratios of particles produced at lower temperatures.

Conclusion: This study underscores the effect of process parameters, particularly FRR, on the CQAs of siRNA-LNP systems. Given the significance of siRNA-LNP CQAs—including particle size, size distribution, EE%, and lipid composition—on their safety and efficacy, the findings presented here offer valuable insights for optimizing the microfluidic manufacturing process of these systems.

Acknowledgements: The authors would like to acknowledge the U.S. Food and Drug Administration for financial support of this research (contract number: 75F40123C00118).
Conflict of Interest: Authors declare no conflict of interest.
Disclaimer: The views expressed in this abstract do not reflect the official policies of the U.S. Food and Drug Administration or the U.S. Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.


Effect summary and prediction profile of size and EE% pre- and post-dialysis. Created with BioRender.


Lipid concentrations measured by UPLC-CAD for each sample and the significant effect of FRR on the lipid loss post-dialysis. Sample labels guide: (TFR, FRR, Temperature, before(b)/after (a) dialysis). Created with BioRender.