Encapsulation of messenger rna

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 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 - 50 .(canceled) 
     
     
         51 . A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a buffered mRNA solution and a lipid solution,
 wherein the buffered mRNA solution comprises an mRNA stock solution and a buffer having a concentration of about 10 mM or greater, and   wherein the buffered mRNA solution and the lipid solution are at a pre-determined temperature from about 50-70° C.   
     
     
         52 . The process of  claim 51 , wherein the mRNA stock solution comprises mRNA at a concentration of greater than about 1 mg/mL. 
     
     
         53 . The process of  claim 51 , wherein the buffered mRNA solution has a pH no greater than about 4.5. 
     
     
         54 . The process of  claim 51 , wherein the buffer is a citrate buffer. 
     
     
         55 . The process of  claim 51 , wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, and one or more PEG-modified lipids. 
     
     
         56 . The process of  claim 55 , wherein the one or more helper lipids comprise non-cationic lipids and cholesterol-based lipids. 
     
     
         57 . The process of  claim 55 , wherein the one or more cationic lipids, one or more helper lipids, and one or more PEG-modified lipids are dissolved in absolute ethanol. 
     
     
         58 . The process of  claim 57 , wherein the lipid solution has a total lipid concentration ranging from about 1.0-15 mg/mL. 
     
     
         59 . The process of  claim 56 , wherein the one or more helper lipids are selected from the group consisting of DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE, cholesterol, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, and combinations thereof. 
     
     
         60 . The process of  claim 56 , wherein the one or more PEG-modified lipids comprise 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. 
     
     
         61 . The process of  claim 51 , wherein the mRNA solution is mixed at a rate of at least 3× greater than the rate of the lipid solution. 
     
     
         62 . The process of  claim 51 , wherein the mRNA comprises one or more modified nucleotides. 
     
     
         63 . A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising
 a. separately heating an mRNA solution and a lipid solution to a pre-determined temperature from about 50-70° C. to generate a heated mRNA solution and a heated lipid solution;   b. mixing the heated mRNA solution and the heated lipid solution to generate a suspension of lipid nanoparticles,
 wherein the heated mRNA solution is mixed at a rate of at least 3× greater than the rate of the heated lipid solution; and 
   c. purifying the lipid nanoparticles by tangential flow filtration (TFF).   
     
     
         64 . The process of  claim 63 , wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, and PEG-modified lipids. 
     
     
         65 . The process of  claim 63 , wherein 95% of the purified lipid nanoparticles have an individual particle size of less than about 100 nm. 
     
     
         66 . A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising
 a. mixing an mRNA stock solution and a buffer solution at ambient temperature to form a buffered mRNA solution,
 wherein the mRNA stock solution has an mRNA concentration of greater than about 1 mg/mL, and 
 wherein the buffer solution has a buffer concentration of about 10 mM or greater; 
   b. separately heating the buffered mRNA solution and a lipid solution to a pre-determined temperature from about 50-70° C. to generate a heated mRNA solution and a heated lipid solution,
 wherein the lipid solution has a total lipid concentration ranging from about 1.0-15 mg/mL; 
   c. mixing the heated mRNA solution and the heated lipid solution to generate a suspension of lipid nanoparticles; and   d. purifying the lipid nanoparticles by tangential flow filtration (TFF).   
     
     
         67 . The process of  claim 66 , wherein the buffer solution has a pH of about 4.5. 
     
     
         68 . The process of  claim 66 , wherein the buffer solution is a citrate buffer. 
     
     
         69 . The process of  claim 66 , wherein the heated mRNA solution is mixed at a rate of at least 3× greater than the rate of the heated lipid solution. 
     
     
         70 . The process of  claim 66 , wherein the lipid solution is in absolute ethanol.