Process and formulation of lipid nanoparticles
The present invention provides an improved process for preparing a stable lyophilized composition comprising lipid nanoparticles encapsulating mRNA, which results in high mRNA integrity suitable for both short- and long-term storage for therapeutic use. In some embodiments, the present invention provides a method of preparing a stable lyophilized composition comprising the steps of adding ascorbic acid to an aqueous solution comprising one or more lipid nanoparticles, freezing the aqueous solution to obtain a frozen solution, drying the frozen solution to obtain a lyophilized composition, and increasing and holding the temperature of the lyophilized composition to a temperature of between 15° C. to 30° C.
1 . A method of preparing a stable lyophilized composition comprising a lipid nanoparticle encapsulating mRNA, the method comprising the steps of:
adding between 5 mM and 200 mM of ascorbic acid to an aqueous solution comprising the lipid nanoparticle encapsulating mRNA having an initial mRNA integrity,
freezing the aqueous solution to obtain a frozen solution, and
drying the frozen solution for a period of greater than 10 hours to obtain the stable lyophilized composition, wherein:
the step of drying the frozen solution comprises a primary drying phase and a secondary drying phase;
the primary drying phase is performed at a first temperature between 0° C. and −100° C.;
the secondary drying phase is performed at a second temperature between 5° C. and 40° C.;
the primary drying phase and/or the secondary drying phase is performed at a pressure of between 10 mTorr and 80 mTorr;
the stable lyophilized composition has a moisture content of less than 1% after the secondary drying phase; and
upon reconstitution of the stable lyophilized composition following storage of the stable lyophilized composition for four weeks at a temperature of 15° C. to 25° C., an mRNA integrity following reconstitution is at least 50% of the initial mRNA integrity.
2 . The method of claim 1 , wherein the method further comprises a step of removing citric acid from the aqueous solution prior to the step of adding between 5 mM and 200 mM of the ascorbic acid to the aqueous solution.
3 . The method of claim 2 , wherein the step of removing the citric acid from the aqueous solution comprises washing the aqueous solution with a citrate buffer comprising EDTA in which the buffer has a pH of 6 to 8.
4 . The method of claim 3 , wherein the citrate buffer is between 1 mM to 10 mM.
5 . The method of claim 1 , wherein the second temperature is between 15° C. to 30° C.
6 . The method of claim 1 , wherein the aqueous solution comprises about 10 mM to 50 mM of the ascorbic acid.
7 . The method of claim 1 , wherein the initial mRNA integrity is higher than 50%.
8 . The method of claim 1 , wherein the primary drying phase and the secondary drying phase are each performed at a pressure of between 10 mTorr and 80 mTorr.
9 . The method of claim 8 , wherein the primary drying phase and the secondary drying phase are each performed at a pressure of between 20 mTorr and 60 mTorr.
10 . The method of claim 1 , wherein the aqueous solution further comprises trehalose or sucrose.
11 . A stable lyophilized composition comprising a lipid nanoparticle encapsulating mRNA and between 10 mM and 200 mM of ascorbic acid, wherein:
the stable lyophilized composition has a moisture content of less than 1%; and
upon reconstitution of the stable lyophilized composition following storage of the stable lyophilized composition for four weeks at a temperature of 15° C. to 25° C., an integrity of the mRNA following reconstitution is at least 50% of an initial mRNA integrity.
12 . The stable lyophilized composition of claim 11 , wherein the stable lyophilized composition has less than 0.1 mM citrate.
13 . The stable lyophilized composition of claim 11 , wherein the stable lyophilized composition comprises between 10 mM and 50 mM of the ascorbic acid.
14 . The stable lyophilized composition of claim 11 , wherein the initial mRNA integrity is higher than 50%.
15 . The stable lyophilized composition of claim 11 , wherein the stable lyophilized composition comprises trehalose or sucrose.
16 . The stable lyophilized composition of claim 11 , wherein the stable lyophilized composition has a pH below 7.0.
17 . The stable lyophilized composition of claim 11 , wherein the lipid nanoparticle comprises a cationic lipid, a helper lipid and a PEG-modified lipid.
18 . The stable lyophilized composition of claim 17 , wherein the lipid nanoparticle further comprises cholesterol.
19 . The stable lyophilized composition of claim 17 , wherein the helper lipid is DOPE or DEPE.
20 . The stable lyophilized composition of claim 17 , wherein the PEG-modified lipid is DMG-PEG 2000.