Process of preparing mRNA-loaded lipid nanoparticles

A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising:

• (a) providing a lipid mixture comprising a cationic lipid, a non-cationic lipid, a polyethylene glycol (PEG)-modified lipid, and a cholesterol-based lipid dissolved in ethanol, at a total lipid concentration of up to 100 mg/ml;
• (b) mixing the lipid mixture with citrate buffer using a pump system to obtain a solution comprising pre-formed, empty lipid nanoparticles in citrate buffer containing ethanol, wherein the pre-formed, empty lipid nanoparticles are of a homogenous particle size of less than 150 nm;
• (c) buffer-exchanging the solution comprising the pre-formed, empty lipid nanoparticles into an aqueous solution comprising one or more of trehalose, sucrose, lactose, and mannitol to remove the citrate buffer and ethanol;
• (d) mixing the aqueous solution comprising the pre-formed, empty lipid nanoparticles with an aqueous solution comprising mRNA at a concentration of up to 5.0 mg/ml and a pH of about 3.5-6.5;
• wherein said mixing takes place in the presence of less than 1% ethanol and less than 1 mM citrate;
• and
• (e) heating the lipid nanoparticle and mRNA solution obtained in step (d) at a temperature of or greater than 50° C.