Method for measuring nucleic acid content in lipid nanoparticles using ultraviolet spectrometry
An ultraviolet (UV) absorbance assay for measuring the concentration of large RNA molecules such as mRNA in suspensions comprising RNA-lipid nanoparticles (RNA-LNPs) is described.
1 . A method for measuring the ribonucleic acid (RNA) concentration of a suspension of RNA-lipid nanoparticle (RNA-LNPs) wherein the RNA-LNPs comprise ionizable cationic lipids and the RNA is at least 100 nucleotides in length, the method comprising:
(a) mixing a suspension of RNA-LNPs comprising ionizable cationic lipids and RNA at least 100 nucleotides in length with an assay diluent comprising (i) a surfactant that disrupts the RNA-LNPs into their RNA and LNP components and is optically transparent at λ max of the RNA and (ii) an alkylamine that denatures the RNA by providing a pH of at least pH 11 or about pH 12, displaces the ionizable cationic lipids of the LNP that are associated with or complexed to the denatured RNA, and is optically transparent at λ max of the RNA, to provide a diluted sample solution;
(b) measuring absorbance of the diluted sample solution at lambda maximum of the RNA (λ max ) to provide a Net Absorbance or measuring absorbance of the diluted sample solution at λ max and at 400 nm and subtracting the absorbance at 400 nm from the absorbance at λ max to provide an adjusted Net Absorbance; and
(c) using the Net Absorbance or the adjusted Net Absorbance to determine the RNA concentration in the suspension of the RNA-LNPs.
2 . The method of claim 1 , wherein the alkylamine comprises a tertiary amine of the formula NRR′R″ wherein R, R′, and R″ are each independently a C1 to C18 alkyl.
3 . The method of claim 1 , wherein the alkylamine is selected from the group consisting of N,N-dimethylbutylamine (DMBA; CAS 927-62-8), N,N-diethylethanamine (TEA; CAS 121-44-8), N,N-diisopropylethylamine (DIPEA; CAS 7087-68-5), and hexan-1-amine (1-HA; CAS 111-26-2).
4 . The method of claim 1 , wherein the surfactant is selected from the group consisting of sodium dodecyl sulfate (SDS; CAS 151-21-3), cetyltrimethylammonium bromide (C-TAB; CAS 57-09-0), and polyethylene glycol alkyl ether (BRIJ).
5 . The method of claim 1 , wherein the surfactant is sodium dodecyl sulfate (SDS) and the alkylamine is N,N-dimethylbutylamine (DMBA) or the surfactant is a polyethylene glycol alkyl ether and the alkylamine is hexan-1-amine (1-HA).
6 . The method of claim 1 , wherein the ionizable cationic lipids comprise a tertiary amine.
7 . The method of claim 6 , wherein the ionizable cationic lipid comprises a tertiary amine and at least one saturated or unsaturated hydrocarbon chain comprising at least nine carbon atoms.
8 . The method of claim 1 , wherein the ionizable cationic lipid is dilinoleylmethyl-4-dimethylaminobutyrate (D-Lin-MC3-DMA; CAS 1224606-06-7).
9 . The method of claim 1 , wherein the assay diluent further includes a metal chelator.
10 . The method of claim 1 , wherein the assay diluent comprises about 750 mM N,N-dimethylbutylamine, about 10% (w/v) SDS, and about 1 mM EDTA.
11 . The method of claim 1 , wherein the RNA concentration is determined using the formula
mRNA
(
mg
mL
)
=
(
Net
Absorbance
or
adjusted
Net
Absorbance
path
length
(
cm
)
)
×
(
40
μg
·
cm
mL
)
×
(
1
mg
mL
1000
μg
mL
)
×
Dilution
Factor
wherein
Dilution
Factor
=
Diluted
Sample
Solution
volume
(
μL
)
Initial
Sample
Suspension
volume
(
μL
)
.
12 . A method for measuring the ribonucleic acid (RNA) concentration of a suspension of RNA-lipid nanoparticle (RNA-LNPs), the method comprising:
(a) providing a predetermined volume of a sample suspension comprising RNA-LNPs wherein the RNA-LNPs comprise ionizable cationic lipids and the RNA is at least 100 nucleotides in length;
(b) mixing the sample suspension of RNA-LNPs with a predetermined volume of an assay diluent comprising (i) a surfactant that disrupts the RNA-LNPs into their RNA and LNP components and is optically transparent at λ max of the RNA and (ii) an alkylamine that denatures the RNA at a pH of at least pH 11 or about pH 12, displaces the ionizable cationic lipids of the LNP that are associated with or complexed to the denatured RNA, and is optically transparent at λ max of the RNA, to provide a diluted sample solution in which the RNA is denatured and dissociated from the ionizable cationic lipids;
(c) measuring absorbance of the diluted sample solution at lambda maximum of the RNA (λ max ) in the diluted sample solution to provide a Net Absorbance or measuring absorbance of the diluted sample solution at λ max and at 400 nm and subtracting the absorbance at 400 nm from the absorbance at λ max to provide an adjusted Net Absorbance; and
(d) using the Net Absorbance or the adjusted Net Absorbance and the formula
mRNA
(
mg
mL
)
=
(
Net
Absorbance
or
adjusted
Net
Absorbance
path
length
(
cm
)
)
×
(
40
μg
·
cm
mL
)
×
(
1
mg
mL
1000
μg
mL
)
×
Dilution
Factor
wherein
Dilution
Factor
=
Diluted
Sample
Solution
volume
(
μL
)
Initial
Sample
Suspension
volume
(
μL
)
to determine the RNA concentration in the suspension of the RNA-LNPs.
13 . The method of claim 12 , wherein the alkylamine comprises a tertiary amine of the formula NRR′R″ wherein R, R′, and R″ are each independently a C1 to C18 alkyl.
14 . The method of claim 12 , wherein the surfactant is SDS and the alkylamine is N,N-dimethylbutylamine (DMBA) or the surfactant is a polyethylene glycol alkyl ether and the alkylamine is hexan-1-amine (1-HA).
15 . The method of claim 12 , wherein the ionizable cationic lipids comprise a tertiary amine.
16 . The method of claim 15 , wherein the ionizable cationic lipid comprises a tertiary amine and at least one saturated or unsaturated hydrocarbon chain comprising at least nine carbon atoms.
17 . The method of claim 12 , wherein the ionizable cationic lipid is dilinoleylmethyl-4-dimethylaminobutyrate (D-Lin-MC3-DMA; CAS 1224606-06-7).
18 . The method of claim 12 , wherein the assay diluent further includes a metal chelator.
19 . The method of claim 18 , wherein the metal chelator is ethylenediaminetetraacetic acid (EDTA).
20 . The method of claim 12 , wherein the assay diluent comprises about 750 mM N,N-dimethylbutylamine, about 10% (w/v) SDS, and about 1 mM EDTA.