Lipid nanoparticles (LNPs) are crucial in delivering mRNA vaccines and therapeutics. The properties of LNPs can be influenced by the choice of lipids and the manufacturing conditions, such as mixing parameters, lipid concentration, and the type and concentration of the aqueous buffer used. In this study, we investigated the impact of the citrate buffer molarity, the buffer commonly used to dissolve mRNA in the preparation of mRNA-LNPs. We prepared SM-102 LNPs containing firefly luciferase mRNA using citrate buffers at molarities of 50 mM, 100 mM, or 300 mM. Our findings revealed that varying the molarity of the citrate buffer did not significantly affect the particle size when considering the average diameter (z-average or Mode). All formulations exhibited low polydispersity index (PDI) and high encapsulation efficiency. Detailed analysis of particle size sub-populations (D10, D50, and D90) and morphology indicated that citrate buffer concentration might influence lipid packing during LNP production, though these differences were subtle. However, using higher citrate molarity (300 mM) to produce LNPs notably reduced cellular internalisation and in vitro transfection efficiency. This trend was also observed in vivo, where similar expression levels were noted in mice for the 50 mM and 100 mM LNP formulations, but lower expression was seen for the 300 mM formulation. Our study highlights the importance of buffer molarity in the aqueous phase during mRNA-based LNP preparation and that generally reported critical quality attributes (CQAs) for LNPs may not detect subtle formulation differences.