Standardization of pilot scale microfluidic manufacturing of mRNA-lipid nanoparticles

Introduction: RNA delivery technology using lipid nanoparticles (LNPs) has emerged as a new category of therapeutic agents and plays an essential role in the application of mRNA vaccines and RNA-based therapies.LNPs are considered a safe, effective and stable delivery system that protects nucleic acid from degradation and allows cellular uptake and release of RNA.They offer some advantages such as greater stability and low cost, enabling large-scale production.In this study, the microfluidic technique was used to encapsulate mRNA in LNPs, as it ensures greater precision in the control of nanoparticle size and high reproducibility.Objectives: Standardize the pilot scale production of mRNA-LNPs, using as reference the process parameters obtained at lab scale.Methodology: A specific equipment was used for the encapsulation (E).The lipid formulation was composed of a neutral structural lipid (1), cholesterol (2), PEG-lipid (3), ionizable lipid (4) and polyadenylic acid (Poly(A)/Sigma) was used as mRNA.Results: To optimize the mRNA-LNP encapsulation, data (under patent secrecy) obtained on a lab scale were used: nitrogen/phosphate (N/P) molar ratio: 9:1; lipid mix (%): 1:10, 2:48, 3:2 and 4:40; Encapsulation flow ratio (mRNA:lipids): 3:1.Different Encapsulation flow rates (mL/min) were analyzed: 115, 200, 250, 300 and 350.200mL/min was the one that obtained the best results (LNP size: ~66nm, polydispersity index (PDI): 0.145, Zeta potential (Z): 5mV and E efficiency (EE): ~83%).These conditions were repeated twice with reproducibility.The test with N/P ratio 6:1 and flow ratio 2:1 was analyzed, but the result was unsatisfactory (size: ~151nm and PDI: 0.407).At pilot scale, volume produced was ~50x greater than at lab scale. Conclusion:Criteria such as stability, EE, drug release profile, biodistribution and cellular uptake are among the parameters most influenced by size and PDI.Smaller LNPs (<100nm) tend to prolong circulation times and to accumulate in target tissues.In view of this, the results of the preliminary tests were satisfactory.Capacity to manufacture LNPs on a large scale is essential for the action policy of the Ministry of Health for nationalization of strategic products and in strengthening the industrial complex.