Lipid nanoparticles (LNPs) encapsulating genetic material can be produced on a large scale using the bulk-mixing method. However, this approach often lacks precise control over particle size and cargo loading, limiting its efficiency in gene delivery. We have developed a membrane extrusion process that enables large-scale production of LNPs with a narrow size distribution. Initially, an ethanolic lipid solution is mixed with an aqueous buffer containing nucleic acids, forming a pre-mix of swollen LNPs. These soft, swollen LNPs are then extruded through a polycarbonate filter membrane, producing uniform LNPs, in which the ethanol concentration and extrusion pH are adjusted for LNP fluidity. Subsequent addition of citrate buffer (pH 4) enhances encapsulation efficiency by reassembling the dissociated mRNA and lipids during the extrusion process. Finally, the LNP solution is adjusted to physiological pH through buffer exchange. Optimizing the extrusion parameters allowed us to achieve highly uniform 100 nm LNPs with over 80 % encapsulation efficiency for mRNA, siRNA, and DNA. This work provides valuable insights into LNP formation, highlights critical formulation parameters, and demonstrates the potential for large-scale, controlled LNP production.