Methods of preparing lipid nanoparticles

1 . A method of producing a lipid nanoparticle (LNP) formulation, the method comprising:

mixing a lipid solution comprising an ionizable lipid, a phospholipid, and a structural lipid with an aqueous buffer solution having a pH in a range of about 4.6 to about 5.5, thereby forming a lipid nanoparticle composition comprising a lipid nanoparticle;

storing the lipid nanoparticle composition for at least 1 day, at about 10° C. to 25° C.; and

after the storing, adding a nucleic acid solution comprising a nucleic acid to the lipid nanoparticle composition, thereby forming a lipid nanoparticle (LNP) formulation comprising the lipid nanoparticle associated with the nucleic acid.

2 . The method of claim 1 , wherein before the storing:

the lipid nanoparticle of the lipid nanoparticle composition has an average lipid nanoparticle diameter of less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, or less than about 30 nm; or

the lipid nanoparticle of the lipid nanoparticle composition has an average lipid nanoparticle diameter of about 25 nm to about 125 nm, about 30 nm to about 110 nm, about 35 nm to about 100 nm, about 40 nm to about 90 nm, about 45 nm to about 80 nm, or about 50 nm to about 70 nm.

3 . The method of claim 1 , wherein the lipid nanoparticle composition has a pH that is less than the pKa of the ionizable lipid.

4 . The method of claim 1 , further comprising processing the lipid nanoparticle formulation.

5 . The method of claim 1 , wherein the nucleic acid is a ribonucleic acid.

6 . The method of claim 1 , wherein the nucleic acid is a messenger RNA (mRNA).

7 . The method of claim 1 , wherein the lipid solution and/or the lipid nanoparticle composition comprises a first organic solvent.

8 . The method of claim 7 , wherein the first organic solvent is an alcohol.

9 . The method of claim 7 , wherein the first organic solvent is ethanol.

10 . The method of claim 4 , wherein the processing comprises at least one step selected from the group consisting of filtering, pH adjusting, buffer exchanging, dialyzing, concentrating, freezing, lyophilizing, or packing.

11 . The method of claim 1 , wherein the lipid solution, the lipid nanoparticle (LNP) composition, and/or the lipid nanoparticle (LNP) formulation further comprises an encapsulation agent, wherein the encapsulation agent is

i) a compound of Formula (EA-I):

or a salts or isomer thereof, wherein

R 201 and R 202 are each independently selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, and (C═NH)N(R 101 ) 2 wherein each R 101 is independently selected from the group consisting of H, C 1 -C 6 alkyl, and C 2 -C 6 alkenyl;

R 203 is selected from the group consisting of C 1 -C 20 alkyl and C 2 -C 20 alkenyl;

R 204 is selected from the group consisting of H, C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C(O)(OC 1 -C 20 alkyl), C(O)(OC 2 -C 20 alkenyl), C(O)(NHC 1 -C 20 alkyl), and C(O)(NHC 2 -C 20 alkenyl);

n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

ii) a compound of Formula (EA-II):

or a salt or isomer thereof, wherein

X 101 is a bond, NH, or O;

R 101 and R 102 are each independently selected from the group consisting of H, C 1 -C 6 alkyl, and C 2 -C 6 alkenyl;

R 103 and R 104 are each independently selected from the group consisting of C 1 -C 20 alkyl and C 2 -C 20 alkenyl; and

n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; or

iii)ethyl lauroyl arginate or a salt or isomer thereof.

12 . The method of claim 1 , wherein the lipid nanoparticle further comprises a PEG lipid.

13 . The method of claim 12 , wherein the PEG lipid

i) is selected from a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, and a PEG-modified dialkylglycerol;

ii) is a compound of Formula (PL-I):

or salts or isomer thereof, wherein:

R 3 is —OR O ;

R O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

r is an integer between 1 and 100, inclusive;

L 1 is optionally substituted C 1-10 alkylene, wherein at least one methylene of the optionally substituted C 1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N );

D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

each instance of L 2 is independently a bond or optionally substituted C 1-6 alkylene, wherein one methylene unit of the optionally substituted C 1-6 alkylene is optionally replaced with O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, —OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N );

each instance of R 2 is independently optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), —OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(═NR N ), C(═NR N )N(R N ), NR N C(═NR N ), NR N C(═NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, —OS(O)O, OS(O) 2 , S(O) 2 O, OS(O) 2 O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O) 2 , N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or —N(R N )S(O) 2 O;

each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

p is 1 or 2;

iii) is a compound of Formula (PL-II):

or a salt or isomer thereof, wherein:

R 3 is —OR O ;

R O is hydrogen, optionally substituted alkyl or an oxygen protecting group;

r is an integer between 1 and 100;

R 5 is optionally substituted C 10-40 alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl; and optionally one or more methylene groups of R 5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), —C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(═NR N ), C(═NR N )N(R N ), NR N C(═NR N ), NR N C(═NR N )N(R N ), C(S), —C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O) 2 , S(O) 2 O, —OS(O) 2 O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O) 2 , —N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or N(R N )S(O) 2 O; and

each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

iv) is a compound of Formula (PL-III):

or a salt or isomer thereof, wherein s is an integer between 1 and 100, or

v) is

14 . The method of claim 1 , wherein the structural lipid is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

15 . The method of claim 1 , wherein the phospholipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

16 . The method of claim 1 , wherein the ionizable lipid

i) is a compound of Formula (IL-1):

or a salt or isomers thereof, wherein:

R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR″, —YR″, and —R″M′R′;

R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;

R 4 is selected from the group consisting of hydrogen, a C 3-6 carbocycle, —(CH 2 ) n Q, (CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —N(R)R 8 , N(R)S(O) 2 R 8 , —O(CH 2 ) n OR, —N(R)C(—NR 9 )N(R) 2 , —N(R)C(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) 2 R, —N(OR)C(O)OR, —N(OR)C(O)N(R) 2 , —N(OR)C(S)N(R) 2 , —N(OR)C(═NR 9 )N(R) 2 , —N(OR)C(—CHR 9 )N(R) 2 , —C(═NR 9 )N(R) 2 , —C(═NR 9 )R, —C(O)N(R) OR, and —C(R)N(R) 2 C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5;

each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;

each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—,

—C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group, in which M″ is a bond, C 1-13 alkyl or C 2-13 alkenyl;

R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;

R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle;

R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;

each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;

each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C 3-15 alkyl and C 3-15 alkenyl;

each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;

each Y is independently a C 3-6 carbocycle;

each X is independently selected from the group consisting of F, Cl, Br, and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R 4 is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, or —CQ(R) 2 , then (i) Q is not —N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2,

or

ii) is a compound of Formula (IL-II):

or a salt or isomer thereof, wherein

1 is selected from 1, 2, 3, 4 and 5; M1 is a bond or M′; R 4 is hydrogen, unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which n is 2, 3, or 4, and Q is —OH, —NHC(S)N(R) 2 , —NHC(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)R 8 , —NHC(—NR 9 )N(R) 2 , —NHC(—CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —OC(O)-M″—C(O)O—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl; or

iii) is a compound of formula (IL-III):

or a salt or isomer thereof, wherein,

W is

ring A is

t is 1 or 2;

A 1 and A 2 are each independently selected from CH or N;

Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;

R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R″MR′, —R*YR″, —YR″, and —R*OR″;

R X1 and R X2 are each independently H or C 1-3 alkyl;

each M is independently selected from the group consisting of —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —C(O)S—, —SC(O)—, an aryl group, and a heteroaryl group;

M* is C 1 -C 6 alkyl,

W 1 and W 2 are each independently selected from the group consisting of —O— and —N(R 6 )—;

each R 6 is independently selected from the group consisting of H and C 1-5 alkyl;

X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, —CH 2 —, —(CH 2 ) 2 —, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —(CH 2 ) n —C(O)—, —C(O)—(CH 2 ) n —, —(CH 2 ) n —C(O)O—, —OC(O)—(CH 2 ) n —, —(CH 2 ) n —OC(O)—, —C(O)O—(CH 2 ) n —, —CH(OH)—, —C(S)—, and —CH(SH)—;

each Y is independently a C 3-6 carbocycle;

each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;

each R is independently selected from the group consisting of C 1-3 alkyl and a C 3-6 carbocycle;

each R′ is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;

each R″ is independently selected from the group consisting of C 3-12 alkyl, C 3-12 alkenyl and —R*MR′; and

n is an integer from 1-6;

wherein when ring A is

 then i) at least one of X 1 , X 2 , and X 3 is not —CH 2 —; and/or ii) at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is —R″MR′;

iv) is

or a salt or isomer thereof; or

v) is selected from 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), and (2S)—2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)).

17 . A lipid nanoparticle (LNP) formulation prepared by the method of claim 1 .

18 . The method of claim 1 , wherein during the storing:

i) an average lipid nanoparticle diameter of the lipid nanoparticles in the lipid nanoparticle composition increases by less than about 25% during the storing; and/or

ii) a polydispersity index (PDI) of the lipid nanoparticles in the lipid nanoparticle composition increases by less than about 0.25 during the storing.