(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`(43) International Publication Date
`28 December 2017 (28.12.2017) WIPO I PCT
`
`(10) International Publication Number
`WO 2017/223135 A1
`
`(51) International Patent Classification:
`A 61K 9/127 (2006.01)
`B82 F 40/00 (2011.01)
`A61K 9/133 (2006.01)
`A61M5/20 (2006.01)
`B82Y5/00 (2011.01)
`(21) International Application Number:
`PCT/US2017/038426
`
`(22) International Filing Date:
`
`21 June 2017 (21.06.2017)
`English
`English
`
`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`24 June 2016 (24.06.2016)
`62/354,351
`US
`(71) Applicant: MODERNATX, INC. [US/US]; 200 Technol­
`ogy Square, Cambridge, MA 02139 (US).
`(72) Inventor: GELDHOF, Benjamin, Frank; 165 Woodside
`Avenue, Winthrop, MA 02152 (US).
`= (74) Agent: BELLIVEAU, Michael, J.; Clark & Elbing LLP,
`101 Federal Street, 15th Floor, Boston, MA 02110 (US).
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available)'. AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
`
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
`HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,
`KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available)'. ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`as to applicant's entitlement to apply for and be granted a
`patent (Rule 4.17(ii))
`as to the applicant's entitlement to claim the priority of the
`earlier application (Rule 4.17(Hi))
`of inventorship (Rule 4.17(iv))
`
`(57) Abstract: The invention features methods and apparatus for producing lipid
`nanoparticles. Methods of the invention include inj ecting a lipid solution into an aque­
`ous solution at an automated rate (e.g., a rate controlled by a servo pump). The in­
`vention provides methods and apparatus for making lipid nanoparticles possessing a
`wide range of lipid components and hydrophilic encapsulants, including nucleic acids
`(e.g., mRNA). Also provided are nanoparticles and compositions thereof made by
`methods and apparatus of the invention.
`
`(54) Title: LIPID NANOPARTICLES
`
`FIG. 1
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`Stepper Motor
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`mi r
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`I ead Screw with
`plunger attachment
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`Syringe Barrel
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`Pipette Tip Adaptor
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`Pipette Tip
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`'
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`IT) m
`m
`CJ
`CJ
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`o
`CJ o
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`[Continued on next page]
`
`ARBUTUS - EXHIBIT 2037
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
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`WO 2017/223135 A1 llll II II11 III I II III III I III 11 III II III
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`Published:
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`LIPID NANOPARTICLES
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`BACKGROUND OF THE INVENTION
`Nanoparticles are useful for the delivery of various therapeutic, diagnostic, or experimental
`agents to cells and tissues. Nanoparticles are hypothesized to have enhanced interfacial cellular uptake
`because of their sub-cellular size, achieving a local effect. It is also hypothesized that there is enhanced
`cellular uptake of agents encapsulated in nanoparticles compared to the corresponding agent
`administered in free form. Thus, nanoparticle-entrapped agents have enhanced and sustained
`concentrations inside cells, thereby increasing therapeutic effects. Furthermore, nanoparticle-entrapped
`agents are protected from metabolic inactivation before reaching the target site, as often happens upon
`systemic administration of free agents. Therefore, the effective local nanoparticle dose required for the
`local pharmacologic effect may be several fold lower than with systemic or oral doses. Lipid
`nanoparticles, in particular, are useful in enhancing the delivery of agents such as nucleic acids.
`Widespread utility of lipid nanoparticles is limited, in part, due to manufacturing and processing
`constraints. In particular, large-scale production of lipid nanoparticle formulations can introduce variability
`in lipid nanoparticle characteristics, such as chemical composition, surface charge, size, batch-to-batch
`concentration, and purity. Such processing limitations have generated a need in the field for new
`methods and apparatus for synthesizing lipid nanoparticles.
`
`SUMMARY OF THE INVENTION
`The invention provides a method for producing lipid nanoparticles, the method including the steps
`of providing an aqueous solution; providing a lower alkanol solution including lipids; and injecting at an
`automated rate (e.g., at a rate controlled by a servo pump) the lower alkanol solution to the aqueous
`solution to produce the lipid nanoparticles. In some embodiments, the steps of the method of the
`invention are repeated one or more (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, 10, or more) times.
`In some embodiments, the lipid nanoparticles have a mean diameter between 80 nm and 100 nm
`(e.g., between 82 nm and 98 nm, between 84 nm and 96 nm, between 86 nm and 94 nm, between 88 nm
`and 92 nm, about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86
`nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm,
`about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, or about 100 nm) and
`a polydispersity index of 0.25 or less (e.g., 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15,
`0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less).
`In some embodiments, the aqueous solution includes a nucleic acid (e.g., DNA, RNA, e.g.,
`mRNA). The nucleic acid may be at a concentration between 50 jjg per ml and 200 jjg per ml of the
`aqueous solution (e.g., about 50 jjg/ml, about 60 jjg/ml, about 70 jjg/ml, about 80 jjg/ml, about 90 jjg/ml,
`about 100 jjg/ml, about 110 jjg/ml, about 111 jjg/ml, about 111.11 jjg/ml, about 120 jjg/ml, about 130
`jjg/ml, about 140 jjg/ml, about 150 jjg/ml, about 175 jjg/ml, or about 200 jjg/ml). All of or a portion of the
`nucleic acid may be encapsulated in the lipid nanoparticles. In some embodiments, the method yields a
`nucleic acid encapsulation efficiency of at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
`88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater). In some embodiments,
`the method yields a nucleic acid encapsulation efficiency of at least 94%.
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`In some embodiments, the lower alkanol solution provides 50% or less (e.g., between 25% and
`50%, e.g., 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%,
`34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,
`16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less) of the total
`volume. In some embodiments, the lower alkanol solution provides about 33% or about 25% of the total
`volume.
`In some embodiments, the injecting is at a rate from about 1,000 to about 5,000 microliters per
`second (|jl/s; e.g., from about 1,000 to about 5,000 |jl/s, from about 1,500 to about 4,500 jjl/s, from about
`2,000 to about 4,000 jjl/s, or from about 2,500 to about 3,000 jjl/s). For example, injecting may be at a
`rate of 2,600 (Jl/s.
`In some embodiments, the aqueous solution further includes a buffer. Examples of suitable
`buffers include, but are not limited to, a citrate buffer (e.g., 100 mM citrate buffer), a phosphate buffer
`(e.g., phosphate buffered saline (PBS)), or a IRIS buffer (e.g., TRIS/Sucrose). In some embodiments,
`the aqueous solution has a pH from about 3.0 to about 8.0 (e.g., about 3.0, about 3.1, about 3.2, about
`3.3, about 3.4, about 3.5, about 3.6., about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2,
`about 4.3, about 4.4, about 4.5, about 4.6., about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about
`5.2, about 5.3, about 5.4, about 5.5, about 5.6., about 5.7, about 5.8, about 5.9, about 6.0, about 6.1,
`about 6.2, about 6.3, about 6.4, about 6.5, about 6.6., about 6.7, about 6.8, about 6.9, about 7.0, about
`7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.9, or about 8.0). The
`aqueous solution can have an osmolality from about 200 mOsm to about 400 mOsm (e.g., from about
`250 mOsm to about 350 mOsm, or about 260 mOsm, 270 mOsm, 280 mOsm, 290 mOsm, 300 mOsm,
`310 mOsm, 320 mOsm, 330 mOsm, 340 mOsm, or 350 mOsm).
`In some embodiments, the lower alkanol solution includes heptatriaconta-6,9,28,31-tetraen-19-yl-
`4-(dimethylamino)butanoate (DLin-MC3-DMA), phosphatidylcholine (1,2-distearoyl-sn-glycero-3-
`phosphocholine (DSPC), cholesterol, and polyethylene glycol-dimyristolglycerol (PEG-DMG). The ratio of
`DLin-MC3-DMA:DSPC:Cholesterol:PEG-DMG may be, for example, 50:10:38.5:1.5.
`In some embodiments, the method further includes purifying and/or concentrating the dispersion
`of lipid nanoparticles, e.g., through the use of a desalting column, dialysis, or tangential flow filtration.
`Purification and/or concentration may be performed as part of a buffer exchange procedure, e.g.,
`complete buffer exchange. The method may further include sterilizing the dispersion of lipid
`nanoparticles by filtration, e.g., microfiltration.
`In some embodiments, the invention provides a method of producing nanoparticles having an
`encapsulation efficiency of greater than 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
`99%, or 100%).
`In another aspect, the invention provides a lipid nanoparticle produced by injecting a lower
`alkanol solution into an aqueous solution having a lipid, wherein the injecting is automated at a rate from
`about 1,000 to about 5,000 microliters per second (jjl/s; e.g., from about 1,000 to about 5,000 jjl/s, from
`about 1,500 to about 4,500 jjl/s, from about 2,000 to about 4,000 jjl/s, or from about 2,500 to about 3,000
`|j|/s). In some embodiments, the injecting is at a rate of 2,600 jjl/s.
`In another aspect, the invention provides an apparatus for producing lipid nanoparticles, the
`apparatus having an injector configured to transfer a lower alkanol solution from a first reservoir to a
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`second reservoir configured to hold an aqueous solution; and a servo pump configured to operate the
`injector at a rate from 1,000 to 5,000 jjl/s (e.g., from 1,500 to 4,000 jjl/s, 2,000 to 3,500 jjl/s, or from 2,500
`to 3,000 |jl/s. In some embodiments, the servo pump is configured to operate the injector at a rate of
`2,600 MI/S.
`In some embodiments, the injector is configured to move in three dimensions relative to the
`second reservoir. In some embodiments, the injector is configured to move in three dimensions relative
`to the first reservoir and second reservoir.
`In some embodiments, the invention provides a pharmaceutical composition including the lipid
`nanoparticles and a pharmaceutically acceptable carrier.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIGURE 1 is a schematic diagram of an injector including a stepper motor, lead screw with
`plunger, syringe barrel, pipette tip adaptor, and pipette tip.
`
`DEFINITIONS
`The term "nucleic acid" refers to a molecule of two or more nucleotides or alternative nucleotides.
`The term, "nucleotide" refers to a nucleoside including a phosphate group. The term "nucleoside" refers
`to a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in
`combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to
`herein as a "nucleobase"). Examples of nucleic acids include but are not limited to DNA, RNA, tRNA
`(transfer RNA), mRNA (messenger RNA), siRNA (small interfering RNA), miRNA (micro RNA), shRNA
`(short hairpin RNA), ncRNA (non-coding RNA), aptamers, ribozymes, and shorter oligonucleotide
`sequences of any of the foregoing. Alterations of the base, sugar, and phosphate moiety of a nucleotide
`are encompassed by this definition. Herein, in a nucleotide, nucleoside or polynucleotide (such as the
`nucleic acids of the invention, e.g., mRNA molecule), the terms "alteration" or, as appropriate,
`"alternative" refer to alteration with respect to A, G, U or C ribonucleotides. Generally, herein, these
`terms are not intended to refer to the ribonucleotide alterations in naturally occurring S'-terminal mRNA
`cap moieties.
`As used herein, the terms "alteration" or "alternative" of a nucleotide, nucleoside, or
`polynucleotide (such as the polynucleotides of the invention, e.g., mRNA molecule), refer to alteration
`with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to
`the ribonucleotide alterations in naturally occurring 5'-terminal mRNA cap moieties.
`As used herein, the term "nanoparticle" refers to a particle having one or a plurality of
`components, the particle having any one structural feature on a scale of less than about 1000 nm that
`exhibits novel properties as compared to a bulk sample of the same material or component materials.
`Routinely, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than
`about 400 nm, less than about 300 nm, less than about 200 nm or less than about 100 nm. In exemplary
`embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-500
`nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the
`order of about 10-1000 nm. A spherical nanoparticle would have a diameter, for example, of between 10­
`100 nm or 10-1000 nm.
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`A nanoparticle most often behaves as a unit in terms of its physical or biophysical properties,
`e.g., transport. It is noted that novel properties that differentiate nanoparticles from the corresponding
`bulk material typically develop at a size scale of under 1000 nm, or at a size of under 500 nm, but
`nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. The
`size at which materials display different properties as compared to the bulk material is material-dependent
`and can be seen for many materials much larger in size than 100 nm and even for some materials larger
`in size than 1000 nm. Nanoparticles can be employed in a variety of drug delivery technologies (e.g.,
`nucleic acid drug delivery technologies) and can be employed for various purposes including, but not
`limited to, controlled drug delivery, protection of the drugs from degradation, and protection of the body
`from the toxic effects of the drugs.
`As used herein, the term "microparticle" refers to a small particle or particulate system, generally
`larger than about one micrometer (1 |im) in diameter and can be used to describe both microcapsules
`and microspheres.
`The term "lipid nanoparticle" refers to a nanoparticle as described above that includes lipids and
`that is stable and dispersible in aqueous media. In exemplary embodiments, lipid nanoparticles may be
`from 10 nm to 500 nm in diameter, e.g., from 70 nm to 120 nm.
`The term "lower alkanol" refers to an alcohol with 6 or fewer carbon atoms. Examples of lower
`alkanols include but are not limited to methanol, ethanol, propanol, pentanol, their isomers, and mixtures
`thereof.
`The term "injector" refers to a structure, through which a solution passes, that is configured to
`guide the flow of a solution into a reservoir.
`The term "injection rate" refers to the volume of fluid that is injected per unit time.
`The term "total volume after injecting" refers to the volume of suspension (e.g., including the
`lower alkanol solution, the aqueous solution, and any encapsulants) at a time point immediately following
`the termination of injecting, prior to any subsequent processing, such as filtration, lyophilization, etc.
`The term "particle size" or "particle diameter" refers to the mean diameter of the particles in a
`sample, as measured by dynamic light scattering (DLS), multiangle light scattering (MALS), nanoparticle
`tracking analysis, or comparable techniques. It will be understood that a dispersion of lipid nanoparticles
`as described herein will not be of uniform size but can be described by the average diameter and,
`optionally, the polydispersity index.
`In preferred embodiments, the lipid nanoparticles in the formulation of the present invention have
`a single mode particle size distribution (i.e., they are not bi- or poly-modal). The particle size distribution
`relates to the amount of particles by size within a given population. This is derived using Mie theory,
`where the assumption is that all particles are spherical, and the optical properties of the particles are
`known. A particle size distribution can be measured by dynamic light scattering (DLS) or other particle
`tracking systems (e.g., diffraction tracking and Brownian motion analysis).
`The term "encapsulation efficiency" as used herein refers to the percentage of nucleic acid in the
`lipid nanoparticles that is not degraded after exposure to serum or a nuclease assay that would
`significantly degrade free nucleic acids. Encapsulation efficiency can be measured as follows: Dilute the
`lipid nanoparticle formulation to about 1 -10 jjg/ml in 1 x TE buffer. Place 50 |il of the sample in a well in a
`polystyrene 96 well plate, and 50 |il in the well below it. Add 50 |il of 1x TE buffer to the top well, and 50
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`of 2% Triton X-100 to the bottom well. For the reference wells, replace the sample with 50 |il of 1x TE
`buffer. Allow the 96 well plate to incubate at 370C for 15 minutes. During this time, allow RiboGreen® to
`thaw. Once thawed, dilute the RiboGreen 1:100 in 1x TE buffer. After the 15 minute incubation, add 100
`|il of diluted RiboGreen reagent to each well, mixing thoroughly by pipetting. Once addition of the
`RiboGreen is complete, the plate is then read by a fluorescence plate reader (FITC settings); after
`subtracting the fluorescence values of the blanks from each sample well, the percent of free mRNA may
`be determined by dividing the fluorescence of the intact liposome sample (no Triton X-100) by the
`fluorescence value of the disrupted liposome sample (with Triton X-100).
`
`Entrapped fraction = 1 - free fraction.
`Encapsulation efficiency = 100 x entrapped fraction.
`
`The term "fully encapsulated" as used herein indicates that the nucleic acid in the particles is not
`significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free
`nucleic acids. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is
`degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than
`10% and most preferably less than 5% of the particle nucleic acid is degraded. Fully encapsulated also
`indicates that the particles are serum stable, that is, that they do not rapidly decompose into their
`component parts upon in vivo administration.
`
`DETAILED DESCRIPTION
`The invention provides methods and apparatus for producing lipid nanoparticles that involve
`injecting a lipid solution into an aqueous solution at an automated rate. The methods can be used to
`make lipid nanoparticles possessing a wide range of lipid components including, but not limited to,
`cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG) lipids, hydrophilic polymer lipids,
`fusogenic lipids, and sterols. Hydrophobic agents can be incorporated into the organic solvent (e.g.,
`ethanol) with the lipid, and nucleic acids can be added to an aqueous component. The methods and
`apparatus can be used to prepare homogeneous dispersions of lipid nanoparticles.
`
`Methods
`Lipid nanoparticles can be produced (e.g., allowed to self-assemble, e.g., spontaneously) by
`injecting a lower alkanol solution containing lipids into an aqueous solution. Various lipids can be used to
`achieve desired properties, such as size, surface charge, and capacity for encapsulants. Such properties
`can also be influenced by the composition of the aqueous solution. Lipid nanoparticles of the invention
`can encapsulate a wide range of hydrophilic molecules, e.g., nucleic acids such as DNA and RNA, or
`alternative versions of DNA or RNA. Variations in lipid nanoparticle size can be affected by controlling
`process parameters. In particular, the rate at which the lower alkanol solution is injected into the aqueous
`solution is inversely related to the resulting lipid nanoparticle size. Similarly, minimizing variance in the
`rate of injection will minimize variance in lipid nanoparticle size, yielding homogeneous suspensions of
`lipid nanoparticles, e.g., within a single batch or among multiple batches. A precise rate of injection can
`be attained, e.g., through a servo pump.
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`Typically, the lipid nanoparticles are liposomes with a lipid bilayer surrounding an aqueous
`interior. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV)
`which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers
`separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller
`than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 nm and 500 nm
`in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve
`the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to,
`endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the
`pharmaceutical formulations.
`The formation of liposomes may depend on the physicochemical characteristics such as, but not
`limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the
`medium in which the lipid vesicles are dispersed (e.g., osmolality or pH), the effective concentration of the
`entrapped substance and its potential toxicity, any additional processes involved during the application
`and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the
`intended application, and the batch-to-batch reproducibility and possibility of large-scale production of
`safe and efficient liposomal products.
`The invention further provides methods of injecting the lipid-containing lower alkanol solution into
`the aqueous solution at a precise rate. Without wishing to be bound by theory, a high rate of injection
`creates greater turbulence and provides more energy for lipid assembly, yielding small lipid nanoparticles.
`In some embodiments, the volume of the aqueous solution is at most 5.0 ml. In conditions in which the
`volume of the aqueous solution is 5.0 ml, the rate of injection of the lower alkanol solution can be
`between 1,000 |jl/s and 5,000 jjl/s (e.g., between 1,500 |jl/s and 4,000 jjl/s, between 2,000 and 3,000
`|j|/s, between 2,400 jjl/s and 2,800 jjl/s, or about 2,600 jjl/s), or between 20% and 100% (e.g., between
`30% and 80%, between 40% and 60%, or between 48% and 56%, e.g., about 52%) of the total volume
`per second. Lipid nanoparticles of the invention may have a diameter of, e.g., between 70 nm and 110
`nm (e.g., between 75 nm and 105 nm, between 80 nm and 100 nm, between 82 nm and 98 nm, between
`84 nm and 96 nm, between 86 nm and 94 nm, between 88 nm and 92 nm, about 85 nm, about 90 nm, or
`about 95 nm) with a polydispersity index of 0.25 or less (e.g., 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19,
`0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or
`less), as measured by dynamic light scattering (DLS). In some embodiments, all or a portion of the
`nucleic acid is encapsulated in the lipid nanoparticles. In some embodiments, the method yields a nucleic
`acid encapsulation efficiency of at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 92%,
`at least 94%, at least 95%, at least 96%, at least 97%, or at least 98%, e.g., 80%, 81%, 82%, 83%, 84%,
`85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater).
`The volume fraction may be modified according to parameters known in the art. For example, a
`lower alkanol solution having a greater concentration of lipid molecules may necessitate a lesser volume
`of lower alkanol solution relative to aqueous solution, e.g., to maintain the molar ratio of lipid to
`hydrophilic encapsulants.
`The lower alkanol solution can be injected automatically (e.g., at an automated rate, e.g., by an
`automatic injector, e.g., a servo-powered injector, e.g., as part of a robotic pipetting apparatus) into a
`volume of aqueous solution, e.g., contained within a reservoir, e.g., a well of a multi-well plate, test tube,
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`PCT/US2017/038426
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`or flask. Reservoirs can be part of a multi-reservoir unit, e.g., a multi-well plate or a rack of multiple tubes
`(e.g., 0.25 ml tubes, 0.5 ml tubes, 1.0 ml tubes, 1.5 ml tubes, 2.0 ml tubes, 2.5 ml tubes, 5.0 ml tubes, 10
`ml tubes, 15 ml tubes, or 50 ml tubes, e.g., included as part of a multiple tube rack, e.g., a 2-tube rack, a
`4-tube rack, a 6-tube rack, an 8-tube rack, a 10-tube rack, a 12-tube rack, a 16 tube rack, a 24-tube rack,
`a 48 tube rack, or a 96-tube rack). In some embodiments, one or more multi-reservoir units (e.g., tube
`racks) can be processed in a single run. The aqueous solution may be stationary or under mixing or
`agitation. Multiple, repeated injections, each into a corresponding reservoir of aqueous solution, can
`produce multiple suspensions of lipid nanoparticles. The injector can be moved relative to the aqueous
`solution reservoir, or, alternatively, the aqueous solution reservoir can be moved relative to the injector.
`An injector may additionally move in the Z direction, relative to the aqueous solution reservoir, e.g., such
`that ejection of lower alkanol solution from the injector occurs below the surface of the aqueous solution.
`Alternatively, ejection of the lower alkanol solution from the injector can occur above the surface of the
`aqueous solution, e.g., to prevent spillage and/or crossover of aqueous solution between reservoirs.
`The total volume of the lower alkanol solution per injection is generally equal to or less than the
`total volume of suspension upon injection completion (e.g., the mixture of lower alkanol solution and
`aqueous solution). In some embodiments, the volume of the lower alkanol solution per injection is
`between 10% and 100% (e.g., between 20% and 90%, between 30% and 80%, between 40% and 75%,
`between 50% and 70%, or between 60% and 70%, e.g., about 20%, about 30%, about 40%, about 50%,
`about 60%, about 65%, about 66.67%, about 70%, about 80%, about 90%, or about 100%) of the volume
`of the aqueous solution or between 5% and 50%, between 10% and 45%, between 15% and 40%,
`between 20% and 38%, or between 25% and 35%, e.g., about 20%, about 30%, about 33%, about 40%,
`or about 40% of the total volume of suspension upon injection completion). Other volume fractions can
`be used. In general, the greater the volume of the lower alkanol solution relative to the aqueous solution,
`the more concentrated the suspension of lipid nanoparticles will be upon completion of injection.
`Conventional downstream processing can be employed as part of the present invention. For
`example, lipid nanoparticles can be purified or concentrated, e.g., by tangential flow filtration, dialysis, or
`desalting column (e.g., a PD-10 desalting column). In methods involving dialysis, the filter membrane
`geometry, including the area of the filter and the fiber diameter can be varied to achieve an optimal rate of
`filtration according to known parameters, such as lipid nanoparticle size, encapsulants size, and liquid
`viscosity (e.g., buffer viscosity). A person of skill in the art will understand that the effect of varying each
`of these parameters can be informed by the Stokes-Einstein equation, below, where D is the diffusion
`constant of a particle, ka is Boltzmann's constant, Tis temperature, 77 is viscosity, and ris the radius of
`the particle.
`
`knT
`D =
`Gnrjr
`
`For tangential flow filtration procedures, the effect of liquid flow reduces the influence of diffusion
`on buffer exchange rate. In this case, increasing the recirculation rate will increase the shear rate and
`enhance the rate of buffer exchange. The transmembrane and permeate pressures can also be varied.
`
`7
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`WO 2017/223135
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`PCT/US2017/038426
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`In some embodiments, the nanoparticle suspension can be exchanged with a solution containing
`a cryoprotectant (e.g., for long term storage in, e.g., frozen or lyophilized form). Physiologically suitable
`cyroprotectants for lipid nanoparticles are known in the art and include, e.g., sucrose, glucose, mannitol,
`glycerol, and other carbohydrates and polyalcohols. In one non-limiting example, a lipid nanoparticle
`solution is dialyzed against a sucrose solution (e.g., a TRIS/sucrose buffer).
`A sterile filtration step may also be employed, and the membrane area, pore size and filtration
`force can be varied, as described above. The lipid nanoparticles described herein may be made in a
`sterile environment by the system and/or methods described in U.S. Publication No. 20130164400.
`
`Lower Alkanol Solution
`A lower alkanol solution of the invention provides the lipid components that assemble into lipid
`nanoparticles upon injection into the aqueous solution. Lipid components that can be included in the
`lower alkanol solution include, but are not limited to, cationic lipids, anionic lipids, neutral lipids,
`polyethyleneglycol lipids, hydrophilic polymer lipids, and fusogenic lipids.
`In one embodiment, the lower alkanol solution includes at least one lipid. The lipid may be
`selected from, but is not limited to, L604, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA,
`DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In another
`aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-
`DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid may be the
`lipids described in and/or made by the methods described in U.S. Publication No. US20130150625,
`herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid may be 2-
`amino-3-[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1 -yloxy]methyl}propan-1 -
`ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-
`yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-
`yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-
`[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol
`(Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.
`Lipid nanoparticle formulations may include, e.g., an ionizable cationic lipid, for example, 2,2-
`dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-
`dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl) 9-((4-
`(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and
`a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
`In one embodiment, the lower alkanol solution includes (i) at least one lipid selected from the
`group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-
`4-dimethylaminobutyrate (DLin-MC3-DMA), and di(