(T1430-01-03) Importance of Early-Stage Process Design Space Assessment for Lipid Nanoparticles (LNP) Technology Transfer

Purpose: Lipid nanoparticles (LNPs) represent the most advanced platform for the systemic delivery of nucleic acid and the success of mRNA vaccine and siRNA products have accelerated the development of nucleic acid delivery. LNP manufacturing is a complex process. Technology is developed in R&D laboratories and transferred to a GMP facility for clinical and commercial production. Difference in R&D and GMP equipment influences process reproducibility during technology transfer and scale-up. Often, process design assessment is overlooked during early-stage process development. This assessment is critical to predict impact of formulation and process changes on product quality and develop strategies to ensure product quality and minimize product loss during technology transfer. In this study, we aimed to explore process design space assessment on a scaled-down basis for LNP manufacturing for siRNA delivery, focusing on the mixing phase of LNP-siRNA fabrication.

Methods: The fabrication of siRNA-loaded LNP was done via T-junction microfluidic mixing technology. Concisely, the lipids at the proper ratio were dissolved in ethyl alcohol and the siRNA was dissolved in citrate buffer. . Our study investigated the relationship between four key process parameters and how they affected the resultant particles: concentration of total lipids (ration was kept constant), concentration of siRNA, flow rate of lipid solution, and flow rate of siRNA solution. Nitrogen-to-phosphate (N:P) ratio of lipid and RNA was calculated for each test condition. Product mixing time was 6 seconds for each test condition. Generated LNPs were diluted in-line in PBS (phosphate buffered saline), with PBS solution flow rate was kept constant. The particle size (z-average), polydispersity index (PDI), and aggregation percentages were measured using dynamic light scattering (DLS).

Results: A batch of LNPs was considered successful if the measured Z-average was less than 100nm and PDI was less than 0.1. The product was also expected to be free from particle aggregation. When data was analyzed, it was found that all test conditions met criteria for Z-average and PDI, but particle aggregation was observed for few conditions as highlighted below. Solution flow rate: The results showed lipid solution flow rate was critical for particle aggregation. Changes in siRNA solution flow rate did not lead to particle aggregation. Lipid solution concentration: Particle aggregation was seen when lipid solution concentration was lowered. No aggregation was observed when concentration was increased. siRNA solution concentration: Changes in siRNA solution concentration led to particle aggregation when lipid solution concentration was at the target or lower. Change in siRNA solution concentration did not cause particle aggregation only at higher lipid solution concentrations than target. Nitrogen-to-phosphate (N:P) ratio of lipid and RNA: The N:P ratio was overall good predictor of particle aggregation.

Conclusion: This study signifies the importance of  process design space assessment to find critical process parameters (CPPs) that must be precisely controlled to keep product quality and overall project timelines for technology transfer.

Figure 1: Effect of Solution Concentration on LNP Aggregation

Figure 2: Effect of Solution Flow Rate on LNP Aggregation