IP Library › Granted Patent US 12,636,252
Granted Patent B2
US 12,636,252 · App. 18/762,242 · Granted May 26, 2026

Process of preparing mRNA-loaded lipid nano particles

Inventors: Shrirang Karve (Waltham, MA); Zarna Patel (Waltham, MA); Yi Zhang (Waltham, MA); Ashish Sarode (Waltham, MA); Rebecca L. Goldman (Waltham, MA); Frank DeRosa (Waltham, MA); Michael Heartlein (Waltham, MA)
Assignee: TRANSLATE BIO, INC.
A61K9/1277A61K9/141A61K9/51A61K31/7105
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Quick Facts
Patent No.
US 12,636,252
App. No.
18/762,242
Granted
May 26, 2026
Kind
B2
Abstract

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 solution of pre-formed lipid nanoparticles and mRNA at a low concentration.

Claims (39)

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

mixing a solution comprising pre-formed lipid nanoparticles and mRNA such that lipid nanoparticles encapsulating mRNA are formed,

wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.4 mg/ml and/or the mRNA are present in the solution at a concentration of no greater than 0.5 mg/ml.

2 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.3 mg/ml.

3 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.4 mg/ml.

4 . The process of claim 1 , wherein each of the pre-formed lipid nanoparticles and the mRNA are present in the solution at a concentration of no greater than 0.1 mg/ml.

5 . The process of claim 1 , further comprising a step of diluting the solution to achieve the concentration of no greater than 0.5 mg/ml.

6 . The process of claim 1 , wherein the pre-formed lipid nanoparticles comprise a PEG-modified lipid.

7 . The process of claim 1 , wherein the solution comprising pre-formed lipid nanoparticles and mRNA comprises less than 10 mM citrate.

8 . The process of claim 1 , wherein the solution comprising pre-formed lipid nanoparticles and mRNA comprises less than 25% non-aqueous solvent.

9 . The process of claim 1 , further comprising heating the lipid nanoparticles and mRNA to a temperature greater than ambient temperature after the mixing.

10 . The process of claim 1 , wherein the mRNA and/or the pre-formed lipid nanoparticles are heated to a temperature greater than ambient temperature prior to the mixing.

11 . The process of claim 9 , wherein the temperature is or is greater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.

12 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are formed by mixing lipids dissolved in ethanol with an aqueous solution.

13 . The process of claim 12 , wherein the pre-formed lipid nanoparticles further comprise one or more cationic lipids, and one or more non-cationic lipids.

14 . The process of claim 13 , wherein the one or more cationic lipids are selected from the group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (Imidazol-based), HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLinSSDMA, KLin-K-DMA, DLin-K-XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24), ccBene, ML7 and combinations thereof.

15 . The process of claim 6 , wherein the PEG-modified lipid comprises a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length.

16 . The process of claim 1 , wherein greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the pre-formed lipid nanoparticles have a size ranging from 75-150 nm, or wherein substantially all of the pre-formed lipid nanoparticles have a size ranging from 75-150 nm.

17 . The process of claim 1 , wherein greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the pre-formed lipid nanoparticles have a size ranging from 50-80 nm.

18 . The process of claim 1 , wherein the process results in an encapsulation rate of greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

19 . The process of claim 1 , wherein the process results in greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

20 . The process of claim 1 , wherein the process results in no substantial aggregation of lipid nanoparticles.

21 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.25 mg/ml.

22 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.15 mg/ml.

23 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.1 mg/ml.

24 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.05 mg/ml.

25 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.01 mg/ml.

26 . The process of claim 1 , wherein each of the pre-formed lipid nanoparticles and the mRNA are present in the solution at a concentration of no greater than 0.05 mg/ml.

27 . The process of claim 6 , wherein the PEG-modified lipid constitutes less than 3%, less than 2.5%, less than 2%, less than 1.5%, or less than 1% of total lipids in the lipid nanoparticles, or wherein the PEG-modified lipid constitutes between 0.1% and 3%, or between 0.75% and 2.5%, or between 0.5% and 2% of total lipids in the lipid nanoparticles, or wherein the PEG-modified lipid constitutes about 1% of total lipids in the lipid nanoparticles.

28 . The process of claim 9 , wherein the temperature ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C., or wherein the temperature is about 65° C.

29 . The process of claim 13 , wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).

30 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.3 mg/ml.

31 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.25 mg/ml.

32 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.2 mg/ml.

33 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.15 mg/ml.

34 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.1 mg/ml.

35 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.05 mg/ml.

36 . The process of claim 1 , wherein the mRNA is present in the solution at a concentration of no greater than 0.01 mg/ml.

37 . The process of claim 1 , wherein the pre-formed lipid nanoparticles are present at a concentration no greater than 0.2 mg/ml.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 17, 2026
From: KARVE, SHRIRANG; PATEL, ZARNA; SARODE, ASHISH; BALL, REBECCA L.; DEROSA, FRANK; HEARTLEIN, MICHAEL
To: TRANSLATE BIO MA, INC.
Reel/Frame 074399/0201 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 17, 2026
From: TRANSLATE BIO MA, INC.
To: TRANSLATE BIO, INC.
Reel/Frame 074399/0299 →
Continuity (5)
Continuation 17737712 · May 5, 2022
Continuation 16553747 · Aug 28, 2019
Provisional Application 62725765 · Aug 31, 2018
Provisional Application 62724582 · Aug 29, 2018
Related Publication 20250025419A1 · Jan 23, 2025
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