Encapsulation of messenger RNA
View Patent ↗The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.
1. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising steps of:
generating an mRNA solution by mixing a citrate buffer at a flow rate of about 220 ml/minute to about 6000 ml/minute with an mRNA stock solution; and
mixing the mRNA solution and a lipid solution by a pulse-less flow pump, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.
2. The process of claim 1 , wherein the pre-determined temperature is about 65° C.
3. The process of claim 1 , wherein the mRNA solution is mixed at a flow rate of about 200 ml/minute.
4. The process of claim 1 , wherein the lipid solution is mixed at a flow rate of about 50 ml/minute.
5. The process of claim 1 , wherein the mRNA stock solution comprises the mRNA at a concentration at or greater than 1 mg/ml.
6. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising steps of:
generating an mRNA solution by mixing an mRNA stock solution at a flow rate of about 20 ml/minute to about 600 ml/minute with a citrate buffer;
mixing the mRNA solution and a lipid solution by a pulse-less flow pump, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.
7. The process of claim 1 , wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol.
8. The process of claim 1 , wherein the mRNA solution and the lipid solution are mixed into a 20% ethanol, resulting in a suspension of lipid nanoparticles.
9. The process of claim 8 , wherein the lipid nanoparticles are further purified by Tangential Flow Filtration.
10. The process of claim 6 , wherein the pre-determined temperature is about 65° C.
11. The process of claim 6 , wherein the mRNA solution is mixed at a flow rate of about 200 ml/minute.
12. The process of claim 6 , wherein the lipid solution is mixed at a flow rate of about 50 ml/minute.
13. The process of claim 6 , wherein the mRNA stock solution comprises the mRNA at a concentration at or greater than 1 mg/ml.
14. The process of claim 6 , wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol.
15. The process of claim 6 , wherein the mRNA solution and the lipid solution are mixed into a 20% ethanol, resulting in a suspension of lipid nanoparticles.
16. The process of claim 15 , wherein the lipid nanoparticles are further purified by Tangential Flow Filtration.