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`US 20070042031Al
`
`(19) United States
`(12) Patent Application Publication
`MacLachlan et al.
`
`(10) Pub. No.: US 2007/0042031 Al
`Feb. 22, 2007
`(43) Pub. Date:
`
`(54) SYSTEMS AND METHODS FOR
`MANUFACTURING LIPOSOMES
`
`(75)
`
`Inventors: Ian MacLachlan, Mission (CA); Lloyd
`B. Jeffs, Delta (CA); Edward
`Yaworski, Maple Ridge (CA); Kieu
`Lam, Surrey (CA)
`
`Correspondence Address:
`TOWNSEND AND TOWNSEND AND CREW,
`LLP
`TWO EMBARCADERO CENTER
`EIGHTH FLOOR
`SAN FRANCISCO, CA 94111-3834 (US)
`
`(73) Assignee: Protiva Biotherapeutics, Inc., Burnaby
`(CA)
`
`(21) Appl. No.:
`
`111495,150
`
`(22) Filed:
`
`Jul. 27, 2006
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/703,380, filed on Jul.
`27, 2005.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 48100
`(2007.01)
`BOlD 11104
`(2006.01)
`A61K 91127
`(2006.01)
`C12N 15188
`(2006.01)
`(52) U.S. Cl ............................. 424/450; 435/458; 514/44;
`422/255
`
`(57)
`
`ABSTRACT
`
`The present invention provides apparatus and processes for
`producing liposomes. By providing a buffer solution in a
`first reservoir, and a lipid solution in a second reservoir,
`continuously diluting the lipid solution with the buffer
`solution in a mixing chamber produces a liposome. A
`therapeutic agent, such as nucleic acid, is included in one of
`the buffer solution or the lipid solution. Upon mixing a
`liposome encapsulating the therapeutic product is substan(cid:173)
`tially instantaneously formed. Thereafter the liposome solu(cid:173)
`tion formed is immediately diluted with buffer solution to
`enhance homogeneity aud maintain small particle size.
`
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`

`Patent Application Publication Feb. 22, 2007 Sheet 1 of 6
`
`US 2007/0042031 A1
`
`11 ·~
`
`Lipid
`
`Nucleic Acid Synthesis
`
`100
`¥'V
`~ 2
`
`12 ~ Lipid Solubilization in
`alkanol
`
`r
`
`Nucleic Acid Dissolution in ~ 5
`Buffer
`•
`
`13&
`
`14&
`
`15&
`
`16&
`
`Mixing of Lipid & Plasma Solutions to
`Form Liposom es
`
`Dilution
`
`Removal of Free Plasmid DNA
`(Charged Membrane Rltration)_
`
`Sample Concentration
`{Ultrafiltration)
`
`17~
`
`Ethanol Removal
`(Ultrafiltration)
`
`18~
`
`Buffer Replacement
`{Ultrafiltration)
`
`19&
`
`Sterile Rltration
`
`19(r
`
`Sterile Rll
`
`FIG. 1
`
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`

`Patent Application Publication Feb. 22, 2007 Sheet 2 of 6
`
`US 2007/0042031 Al
`
`Mixing
`Step
`
`Dilution
`Step
`I
`I
`I
`I
`
`High
`
`[Ethanol]
`
`Low
`
`Li~dl1
`
`Monomers
`
`!
`B ~~
`Meta-Stable
`SPLP
`< 200 nm
`
`C
`I
`1 Stable
`: SPLP
`1 < 200 nm
`I
`I
`
`I
`
`FIG. 2
`
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`

`

`SNALP2 Process
`(Spontaneous Vesicle Formation(cid:173)
`Stirred Tank Direct Dilution)
`
`324
`
`300
`~
`
`Key:
`
`1. Dual Heat Peristaltic Pump
`2. T-connector "mixing chamber" (1.6 or 32 mm ID)
`
`Notes:
`1. Single Stop Vesicle formation process
`2. Ethanol concentration in the Diluted samples
`Can easily be adjusted
`3. 1.6, 2.4 or 3.2 mm 10 silicon tubing used
`(depends on scale)
`
`Nucleic Acid
`Solution
`
`310
`
`326
`
`Nascent SNALP
`Collected Directly
`in Stirred Tank Containing
`Equal volume of
`Dilution Buffer
`(e.g., yielding smaller particles
`in 22.5% Ethanol)
`
`360
`
`FIG. 3A
`
`~
`
`""d a ("!> = -> :a -......
`== -......
`Q = ""d = e::
`== --· Q =
`
`......
`
`~
`
`> ........
`
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`

`

`SNALP3 Process
`(Spontaneous Vesicle Formation(cid:173)
`In-line Direct Dilutio"n)
`
`324
`
`302
`~
`
`Nucleic Acid
`Solution
`
`310
`
`326
`
`Key:
`
`1. Dual Heat Peristaltic Pump
`2. T-connector "mixing chamber" (1.6 or 32 mm ID)
`3. Peristaltic Pump calibrated to deliver at the same rate as
`combined flow rate for dual-head peristaltic pump
`
`Notes:
`1. Single Step Vesicle formation with better control of SNALP
`Dilution with Buffer
`2. Ethanol concentration in diluted SNALP can easily be adjusted
`3. 1.6, 2.4 or 3.2 mm ID silicon tubing used
`(depends on scale)
`
`Dilution
`Buffer
`
`345
`
`Diluted SNALP
`In Aqueous Ethanol
`
`350
`
`FIG. 38
`
`""d
`
`~
`
`~ -("!> = -> "C
`"C -.....
`~ --· Q = ""d = e::
`~ --· Q =
`
`.....
`
`~
`
`i"!"j
`("!>
`?'
`N
`j'J
`N
`0
`0
`--l
`
`i7l =-("!>
`
`("!> -.&;;...
`
`Q
`
`"""' 0\
`
`> .....
`
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`HBS BUFFER REAGENTS
`
`420
`HBS BUFFER STORAGE
`TANKS
`
`LIPID
`
`421
`
`ETHANOL
`STORAGE
`TANK
`
`LIPIDS
`PREPERA TION
`TANK
`
`CITRATE
`BUFFER
`REAGENTS
`
`LIPIDS STOCK STORAGE
`TANK
`
`400
`~
`
`Ultrafi It ration
`System
`COLLECTION ANLON EXCHANGE 10,000
`VESSEl
`COLUMN
`MWCO
`
`ROTARY LOBE
`COLLECTION
`PUMP
`PORT
`
`......
`CITRATE:'-;B::;:;:UFFE===::R~STORAGE
`TANK
`
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`

`

`Diagram ofT-Connector Flow Dynamics
`
`lipid
`ethanol
`
`l
`
`Plasmid
`In Buffer
`
`D =Orifice diameter (em)
`
`l Q =Volume Flow Rate (Urn in)
`
`FIG. 5
`
`SPLP
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`> .....
`
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`

`US 2007/0042031 AI
`
`Feb.22,2007
`
`1
`
`SYSTEMS AND METHODS FOR
`MANUFACTURING LIPOSOMES
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`[0001] This application is a non-provisional application
`claiming the benefit of U.S. Application No. 60/703,380,
`filed Jul. 27, 2005, entitled "SYSTEMS AND METHODS
`FOR MANUFACTURING LIPOSOMES," which is hereby
`incorporated by reference in its entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Many systems for administering active substances
`into cells are already known, such as liposomes, nanopar(cid:173)
`ticles, polymer particles, immuno- and ligand-complexes
`and cyclodextrins (see, Drug Transport in antimicrobial and
`anticancer chemotherapy. G. Papadakou Ed., CRC Press,
`1995 ). Liposomes are typically prepared in the laboratory by
`sonication, detergent dialysis, ethanol injection or dilution,
`French press extrusion, ether infusion, and reverse phase
`evaporation. Liposomes with multiple bilayers are known as
`multilamellar lipid vesicles (MLVs). MLVs are candidates
`for time release drugs because the fluids entrapped between
`layers are only released as each membrane degrades. Lipo(cid:173)
`somes with a single bilayer are known as unilamellar lipid
`vesicles (UV). UVs may be made small (SUVs) or large
`(LUVs).
`
`[0003] Some of the methods above for liposome produc(cid:173)
`tion impose harsh or extreme conditions which can result in
`the denaturation of the phospholipid raw material and encap(cid:173)
`sulated drugs. In addition, these methods are not readily
`scalable for mass production of large volumes ofliposomes.
`Further, lipid vesicle formation by conventional ethanol
`dilution. involves the injection or dropwise addition of lipid
`in an aqueous bufler. The resulting vesicles are typically
`heterogenous in size and contain a mixture of unilamellar
`and multilamellar vesicles.
`
`[0004] Conventional liposomes are fonnulated to carry
`therapeutic agents either contained within the aqueous inte(cid:173)
`rior space (water-soluble drugs) or partitioned into the lipid
`bilayer(s) (water-insoluble drugs). Active agents which have
`short half-lives in the bloodstream are particularly suited to
`delivery via liposomes. Many anti-neoplastic agents, for
`example, are known to have a short half-life in the blood(cid:173)
`stream such that their parenteral use is not feasible. How(cid:173)
`ever, the use of liposomes for site-specific delivery of active
`agents via the bloodstream is severely limited by the rapid
`clearance of liposomes from the blood by cells of the
`reticuloendothelial system (RES).
`
`[0005] U.S. Pat. No. 5,478,860, which issued to Wheeler
`eta!., on Dec. 26, 1995, and which is incorporated herein by
`reference, discloses microemulsion compositions for the
`delivery of hydrophobic compounds. Such compositions
`have a variety of uses. In one embodiment, the hydrophobic
`compounds are therapeutic agents including drugs. The
`patent also discloses methods for in vitro and in vivo
`delivery of hydrophobic compounds to cells.
`
`[0006] PCT Publication WOOl/05373 to Knopov et a!.,
`which is incorporated by reference herein, discloses tech(cid:173)
`niques for preparing lipid vesicles using an ethanol injec(cid:173)
`tion-type process with a static mixer that provides a turbu-
`
`numbers>2000).
`(e.g., Reynolds
`environment
`lent
`Therapeutic agents may then be loaded after vesicle fonna(cid:173)
`tion.
`
`[0007] Published U.S. Application 2004/0142025, which
`is incorporated by reference herein, discloses techniques for
`forming lipid particles using a sequential stepwise dilution
`process. The process disclosed produces lipid particles hav(cid:173)
`ing sizes below 200 llll1 in a non-turbulent mixing environ(cid:173)
`ment. However, the disclosed processes tend to result in less
`optimal vesicle sizes and less than optimal homogeneity,
`especially for liposomes encapsulating siRNA. Also, for
`encapsulated plasmids, an acidic buffer solution is required.
`
`[0008] Despite the advances disclosed in U.S. Pat. No.
`5,478,860, US20040142025 and WO 05373, there exists a
`need for improved processes and apparatus for formulating
`and producing lipid vesicles, and in particular lipid vesicles
`encapsulating a therapeutic agent such as nucleic acid. The
`present invention fulfills these and other needs.
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0009] The present invention provides processes and
`apparatus for making lipid vesicles that optionally contain a
`therapeutic agent. The therapeutic agent can include, for
`example, a protein, a nucleic acid, an antisense nucleic acid,
`a drug, or the like. The present invention can be used to fonn
`lipid vesicles that contain encapsulated nucleic acid or small
`molecule dmgs.ln one aspect, the lipid vesicles are prepared
`rapidly at low pressure and the approach is fully scalable. In
`certain preferred embodiments, the process does not involve
`a static mixer or specialized extrusion equipment.
`
`[0010] According to one embodiment, the present inven(cid:173)
`tion provides a method for producing a liposome. The
`process typically includes providing an aqueous solution in
`a first reservoir, the first reservoir in fluid connnunication
`with an organic lipid solution in a second reservoir, and
`mixing said aqueous solution with said organic lipid solution
`in a first mixing region to produce a liposome solution. The
`organic lipid solution mixes with said aqueous solution so as
`to substantially instantaneously produce a liposome encap(cid:173)
`sulating the therapeutic product. lrnn1ediately thereafter the
`liposome solution is mixed with a buffer solution to produce
`a diluted liposome solution. The liposome solution may be
`introduced to a buffer solution reservoir, or the liposome
`solution may be mixed with bufier in a second mixing
`region.
`
`[0011]
`In certain aspects, the aqueous solution such as a
`buffer, comprises a therapeutic product, such that the thera(cid:173)
`peutic product is encapsulated in the liposome. In other
`aspects, the organic lipid solution includes a therapeutic
`product. Suitable therapeutic products include, but are not
`limited to, a protein, a nucleic acid, an antisense nucleic
`acid, a ribozyme, tRNA, snRN.A.., siRNA (small interfering
`RNA), shRNA, ncRNA, pre-condensed DNA, an aptamer
`and an antigen. In certain preferred aspects, the therapeutic
`product is nucleic acid.
`
`[0012]
`In another embodiment, the present invention pro(cid:173)
`vides a system for producing a liposome encapsulating a
`therapeutic product. The system typically includes a first
`reservoir for holding an aqueous solution, and a second
`reservoir for holding an organic lipid solution, wherein one
`of the aqueous solution and the organic lipid solution
`
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`

`US 2007/0042031 AI
`
`Feb.22,2007
`
`2
`
`includes a therapeutic product. The system also typically
`includes a pump mechanism configured to pump the aque(cid:173)
`ous solution and the organic lipid solution into a mixing
`region at substantially equal flow rates, wherein the organic
`lipid solution mixes with the aqueous solution in the mixing
`region to substantially instantaneously fom1 a therapeutic
`product encapsulated liposome solution. The system further
`typically includes a collection reservoir, comprising a buffer
`solution, in fluid co11l11lunication with the mixing region,
`wherein the liposome solution is introduced to the collection
`reservoir substantially immediately after formation, thereby
`forming a diluted liposome solution.
`
`[0013]
`In yet another embodiment, the present invention
`provides a system for producing a liposome encapsulating a
`therapeutic product. The system typically includes a first
`reservoir for holding an aqueous solution, and a second
`reservoir for holding an organic lipid solution, wherein one
`of the aqueous solution and the organic lipid solution
`includes a therapeutic product. The system also typically
`includes a first pump mechanism configured to pump the
`aqueous solution and the organic lipid solution into a first
`mixing region at substantially equal flow rates, wherein the
`organic lipid solution mixes with the aqueous solution in the
`first mixing region to substantially instantaneously form a
`therapeutic product encapsulated liposome solution. The
`system also typically includes a buffer reservoir holding a
`buffer solution, and a second pump mechanism configured to
`pump the buffer solution into a second mixing region at a
`controlled flow rate, wherein the liposome solution is intro(cid:173)
`duced to the second mixing region substantially immediately
`after formation in the first mixing region, thereby forming a
`diluted liposome solution in the second mixing region.
`[0014] Reference to the remaining portions of the speci(cid:173)
`fication, including the drawings and claims, will realize
`other features and advantages of the present invention.
`Further features and advantages of the present invention, as
`well as the structure and operation of various embodiments
`of the present invention, are described in detail below with
`respect to the accompanying drawings. In the drawings, like
`reference numbers indicate identical or functionally similar
`elements.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`[0015] FIG. 1 illustrates a schematic for the process used
`to prepare SNALP
`[0016] FIG. 2 provides a schematic of a process of making
`liposomes according to one embodiment of the present
`invention
`[0017] FIGS. 3a and 3b show examples of an apparatus
`300 and apparatus 302, respectively, according to two
`embodiments of the present invention.
`[0018] FIG. 4 is an example of a representative schematic
`of an apparatus 400 according to one embodiment of the
`present invention.
`[0019] FIG. 5 shows aT-shaped connector and associated
`flow dynamics according to one embodiment.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`I. Definitions
`[0020] The term "nucleic acid" refers to a polymer con(cid:173)
`taining at least two nucleotides. "Nucleotides" contain a
`
`sugar deoxyribose (DNA) or ribose (RNA), a base, and a
`phosphate group. Nucleotides are linked together through
`the phosphate groups (although synthetic nucleic acids may
`be prepared using nucleotide linkers other than phosphate
`groups). "Bases" include purines and pyrimidines, which
`further include natural compounds adenine, thymine, gua(cid:173)
`nine, cytosine, uracil, inosine, and natural analogs, and
`synthetic derivatives of purines and pyrimidines, which
`include, but are not limited to, modifications which place
`new reactive groups such as, but not limited to, amines,
`alcohols, thiols, carboxylates, and alkylhalides.
`
`[0021] DNA may be in the form of antisense, plasmid
`DNA, parts of a plasmid DNA, pre-condensed DNA, prod(cid:173)
`uct of a polymerase chain reaction (PCR), vectors (Pl, PAC,
`BAC, YAC, artificial chromosomes), expression cassettes,
`chimeric sequences, chromosomal DNA, or derivatives of
`these groups. RNA may be in the form of oligonucleotide
`RNA, tRNA (transfer RNA), snRNA (small nuclear RNA),
`rRNA (ribosomal RNA), mRNA (messenger RNA), anti(cid:173)
`sense RNA, siRNA (small interfering RNA), shRNA (short(cid:173)
`hairpin RNA), ncRNA (non-coding RNA), aptamers,
`ribozymes, chimeric sequences, or derivatives of these
`groups.
`
`[0022]
`"Antisense" is a polynucleotide that interferes with
`the function of DNA and/or RNA. This may result in
`suppression of expression. Natural nucleic acids have a
`phosphate backbone, artificial nucleic acids may contain
`other types of backbones and bases. These include PNAs
`(peptide nucleic acids), phosphothioates, and other variants
`of the phosphate backbone of native nucleic acids. In
`addition. DNA and RNA may be single, double, triple, or
`quadruple stranded.
`
`[0023] The term "gene" refers to a nucleic acid (e.g.,
`DNA) sequence that comprises coding sequences necessary
`for the production of a polypeptide or precursor (e.g., herpes
`simplex virus). The polypeptide can be encoded by a full
`length coding sequence or by any portion of the coding
`sequence so long as the desired activity or fnnctional prop(cid:173)
`erties (e.g., enzymatic activity, ligand binding, signal trans(cid:173)
`duction, and the like) of the full-length or fragment are
`retained.
`
`[0024] As used herein, the term "aqueous solution" refers
`to a composition comprising in whole, or in part, water.
`
`[0025] As used herein, the tem1 "organic lipid solution"
`refers to a composition comprising in whole, or in part, an
`organic solvent having a lipid.
`
`[0026] The tenn "lipid" refers to a group of organic
`compounds that are esters of fatty acids and are character(cid:173)
`ized by being insoluble in water but soluble in many organic
`solvents. They are usually divided in at least three classes:
`(1) "simple lipids" which include fats and oils as well as
`waxes; (2) "componnd lipids" which include phospholipids
`and glycolipids; (3) "derived lipids" such as steroids.
`
`[0027] The term "amphipathic lipid" refers, in part, to any
`suitable material wherein the hydrophobic portion of the
`lipid material orients into a hydrophobic phase, while a
`hydrophilic portion orients toward the aqueous phase.
`Amphipathic lipids are usually the major component of a
`lipid vesicle. Hydrophilic characteristics derive from the
`presence of polar or charged groups such as carbohydrates,
`phosphate, carboxylic, sulfato. amino, sulfhydryL nitro,
`
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`

`US 2007/0042031 AI
`
`Feb.22,2007
`
`3
`
`hydroxy and other like groups. Hydrophobicity can be
`conferred by the inclusion of apolar groups that include, but
`are not limited to, long chain saturated and unsaturated
`aliphatic hydrocarbon groups and such groups substituted by
`one or more aromatic, cycloaliphatic or heterocyclic
`group( s ). Examples of amphipathic compounds include, but
`are not limited to, phospholipids, aminolipids and sphin(cid:173)
`golipids. Representative examples of phospholipids include,
`but are not limited to, phosphatidylcholine, phosphatidyle(cid:173)
`thanolamine,
`phosphatidylserine,
`phosphatidylinositol,
`phosphatidic acid, palmitoyloleoyl phosphatidylcholine,
`lysophosphatidylcholine,
`lysophosphatidylethanolamine,
`dipalmitoylphosphatidylcholine, dioleoylphosphatidylcho(cid:173)
`line, distearoylphosphatidylcholine or dilinoleoylphosphati(cid:173)
`dylcholine. Other compounds lacking in phosphoms, such
`as sphingolipid, glycosphingolipid families, diacylglycerols
`and ~-acyloxyacids, are also within the group designated as
`amphipathic lipids. Additionally, the amphipathic lipid
`described above can be mixed with other lipids including
`triglycerides and sterols.
`
`[0028] The term "neutral lipid" refers to any of a munber
`of lipid species that exist either in an uncharged or neutral
`zwitterionic form at a selected pH. At physiological pH,
`such lipids include, for example, diacylphosphatidylcholine,
`diacylphosphatidylethanolamine. ceramide, sphingomyelin,
`cephalin, cholesterol, cerebrosides and diacylglycerols.
`
`[0029] The tem1 "noncationic lipid" refers to any neutral
`lipid as described above as well as anionic lipids. Useful
`noncationic lipids include, for example, distearoylphos(cid:173)
`phatidylcholine
`(DSPC),
`dioleoylphosphatidylcholine
`(DOPC), dipalmitoylphosphatidylcholine
`(DPPC), dio(cid:173)
`leoylphosphatidylglycerol (DOPG), dipalmitoylphosphati(cid:173)
`dylglycerol
`(DPPG), dioleoyl-phosphatidylethanolamine
`(DOPE),
`palmitoyloleoylphosphatidylcholine
`(POPC),
`palmitoyloleoyl-phosphatidylethanolamine (POPE) and dio(cid:173)
`leoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)(cid:173)
`cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phos(cid:173)
`phatidyl
`ethanolamine
`(DPPE),
`dimyristoylphosphoethanolamine (DMPE ), distearoyl-phos(cid:173)
`phatidylethanolamine
`(DSPE), 16-0-monomethyl PE,
`16-0-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phos(cid:173)
`phatidyethanolamine (SOPE), and 1 ,2-dielaidoyl-sn-glyc(cid:173)
`ero-3-phophoethanolamine (transDOPE).
`
`[0030] The term "anionic lipid" refers to any lipid that is
`negatively charged at physiological pH. These lipids
`include, but are not limited to, phosphatidylglycerol, car(cid:173)
`diolipin, diacylphosphatidylserine, diacylphosphatidic acid,
`N-dodecanoyl
`phosphatidylethanolamines, N-succinyl
`phosphatidylethanolamines, N-glutarylphosphatidylethano(cid:173)
`lamines, lysylphosphatidylglycerols, palmitoyloleyolphos(cid:173)
`phatidylglycerol (POPG), and other anionic modifying
`groups joined to neutral lipids.
`
`yprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bro(cid:173)
`mide ("DMRIE"). Additionally, a munber of connnercial
`preparations of cationic lipids are available which can be
`used in the present invention. These include, for example,
`LIPOFECTIN® (connnercially available cationic liposomes
`comprising DOTMA and 1 ,2-dioleoyl-sn-3-phosphoethano(cid:173)
`lamine ("DOPE"), from GIBCO/BRL, Grand Island, N.Y.,
`USA); LIPOFECTAMINE® (co111111ercially available cat(cid:173)
`ionic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)(cid:173)
`N -( 2-( sperminecarboxamido )ethy I)-N,N -dime thy lal11111o(cid:173)
`nium trifluoroacetate ("DOSPA") and ("DOPE"), from
`(co111111ercially
`GIBCO/BRL); and TRANSFECTAM®
`available cationic lipids comprising dioctadecylamidoglycyl
`carboxyspermine ("DOGS") in ethanol from Promega
`Corp., Madison, Wis., USA). The following lipids are cat(cid:173)
`ionic and have a positive charge at below physiological pH:
`DODAP, DODMA, DMDMA, 1,2-DiLinoleyloxy-N,N(cid:173)
`dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,
`N-dimethylaminopropane (DLenDMA.), and the like.
`
`In addition to cationic and non-cationic lipids, the
`[0032]
`SNALP of the present invention may comprise bilayer
`stabilizing component (BSC) such as an ATTA-lipid or a
`PEG-lipid, such as PEG coupled to dialkyloxypropyls
`(PEG-DA,t\) as described in, e.g., WO 05/026372, PEG
`coupled to diacylglycerol (PEG-DA.G) as described in, e.g.,
`U.S.
`Patent Publication Nos.
`20030077829
`and
`2005008689, PEG coupled to phosphatidylethanolamine
`(PE) (PEG-PE), or PEG conjugated to ceramides, or a
`mixture thereof (see, U.S. Pat. No. 5,885,613). In one
`preferred embodiment, the BSC is a conjugated lipid that
`inhibits aggregation of the SNALP.
`
`In certain aspects, the cationic lipid typically com(cid:173)
`[0033]
`prises from about 2% to about 70%, from about 5% to about
`50%, from about 10% to about 45%, from about 20% to
`about 40%, or from about 30% to about 40% of the total
`lipid present in said particle. The non-cationic lipid typically
`comprises from about 5% to about 90%, from about 10% to
`about 85%, from about 20% to about 80%, from about 30%
`to about 70%, from about 40% to about 60% or about 48%
`of the total lipid present in said particle. The PEG-lipid
`conjugate typically comprises from about 0.5% to about
`20%, from about 1.5% to about 18%, from about 4% to
`about 15%, from about 5% to a bout 12%, or about 2% of the
`total lipid present in said particle. The nucleic acid-lipid
`particles of the present invention may further comprise
`cholesterol. If present, the cholesterol typically comprises
`from about 0% to about 10%, about 2% to about 10%, about
`10% to about 60%, from about 12% to about 58%, from
`about 20% to about 55%, or about 48% of the total lipid
`present in said particle. It will be readily apparent to one of
`skill in the art that the proportions of the components of the
`nucleic acid-lipid particles may be varied.
`
`[ 0031] The term "cationic lipid" refers to any of a nUlllber
`of lipid species which carry a net positive charge at a
`selective pH, such as physiological pH. Such lipids include,
`but are not limited to, N,N-dioleyl-N,N-dimethylammonium
`chloride ("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N(cid:173)
`trimethylal11111onium chloride ("DOTMA"); N,N-distearyl(cid:173)
`N,N-dimethylammonitun bromide ("DDAB"); N-(2,3-dio(cid:173)
`leoyloxy)propyl)-N,N,N-trimethylammonium
`chloride
`("DOTAP"); 3-(N-(N' ,N'-dimethylan1inoethane )-carbam(cid:173)
`oyl)cholesterol
`("DC-Chol") and N-(1,2-dimyristylox-
`
`In some embodiments, the nucleic acid to lipid
`[0034]
`ratios (mass/mass ratios) in a formed nucleic acid-lipid
`particle will range from about 0.01 to about 0.2, from about
`0.03 to about 0.01 or about 0.01 to about 0.08. The ratio of
`the starting materials also falls within this range. In another
`embodiment, the nucleic acid-lipid particle preparation uses
`about 400 ~tg nucleic acid per 10 mg total lipid or a nucleic
`acid to lipid ratio of about 0.01 to about 0.08, or about 0.04,
`which corresponds to 1.25 mg of total lipid per 50 ~g of
`nucleic acid.
`
`Moderna Ex 1014-p. 10
`Moderna v Arbutus
`
`

`

`US 2007/0042031 AI
`
`Feb.22,2007
`
`4
`
`[0035] "Lipid vesicle" refers to any lipid composition that
`can be used to deliver a compound including, but not limited
`to, liposomes, wherein an aqueous volume is encapsulated
`by an amphipathic lipid bilayer; or wherein the lipids coat an
`interior comprising a large molecular component, such as a
`plasmid, with a reduced aqueous interior; or lipid aggregates
`or micelles, wherein the encapsulated component is con(cid:173)
`tained within a relatively disordered lipid mixture.
`
`[0036] As used herein, "lipid encapsulated" can refer to a
`lipid formulation which provides a compound with full
`encapsulation, partial encapsulation, or both.
`
`[0037] As used herein, the term "SNALP" refers to a
`stable nucleic acid lipid particle. A SNALP represents a
`vesicle of lipids coating an interior comprising a nucleic acid
`such as a plasmid with a reduced aqueous interior.
`
`II. General
`
`[0038] The present invention provides processes and
`apparatus for making lipid vesicles. The processes can be
`used to make lipid vesicles 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 actives can be incorporated into the
`organic solvent (e.g., ethanol) with the lipid, and nucleic
`acid and hydrophilic actives can be added to an aqueous
`component. In certain aspects, the processes of the present
`invention can be used in preparing microemulsions where a
`lipid monolayer surrounds an oil-based core. In certain
`aspects, the processes and apparatus are used in preparing
`lipid vesicles, or liposomes, wherein a therapeutic agent is
`encapsulated within a liposome coincident with liposome
`formation.
`
`III. Processes of Making
`
`[0039] FIG. 1 is an example of a representative flow chart
`100 of a method of the present invention. This flow chart is
`merely an illustration and should not limit the scope of the
`claims herein. One of ordinary skill in the art will recognize
`other variations, modifications, and alternatives.
`
`In one aspect, the present method provides a lipid
`[0040]
`solution 110 such as a clinical grade lipid synthesized under
`Good Manufacturing Practice (GMP), which is thereafter
`solubilized in an organic solution 120 (e.g., ethanol). Simi(cid:173)
`larly, a therapeutic product. e.g., a therapeutic active agent
`such as nucleic acid 112 or other agent, is prepared under
`GMP. Thereafter, a therapeutic agent solution (e.g., nucleic
`acids) 115 containing a buffer (e.g., citrate) is mixed with a
`lipid solution 120 solubilized in a lower alkanol to form a
`liposomal formulation 130 (also referred to herein as "lipo(cid:173)
`some suspension" or "liposome solution"). The therapeutic
`agent is entrapped in the liposome substantially coincident
`with fonnation of the liposome. Typically, an electrostatic
`interaction between the negatively charged nucleic acid and
`positively charged cationic lipid brings about encapsulation.
`If a titratable cationic lipid is used, for example, poor NA
`encapsulation efficiencies may be achieved at higher pH
`approaching or exceeding the cationic lipids pKa. Those of
`skill in the art will realize, however, that the processes and
`apparatus of the present invention are equally applicable to
`active entrapment or loading of the liposomes a±ter fomla(cid:173)
`tion of the vesicle. In certain aspects, the liposome solution
`is substantially immediately mixed with a buffer solution
`140 to dilute the liposome solution (e.g., suspension of
`liposomes ).
`
`[0041] According to the processes and systems of the
`present invention, the action of continuously introducing
`lipid and buffer solutions into a mixing enviro11111ent, such as
`in a mixing chamber, causes a continuous dilution of the
`lipid solution with the buffer solution, thereby producing a
`liposome substantially instantaneously upon mixing. Innne(cid:173)
`diately diluting the liposome suspension, e.g., mixing the
`liposome suspension with buffer, helps prevent liposome
`particle sizes from increasing as would typically be the case
`if the liposome suspension is allowed to sit for an extended
`period of time, e.g., minutes or hours. Also, innnediate
`dilution further enhances liposome homogeneity especially
`where siRNA is the encapsulated therapeutic agent. As used
`herein, the phrase "continuously diluting a lipid solution
`with a buffer solution" (and variations) generally means that
`the lipid solution is diluted sufficiently rapidly in an hydra(cid:173)
`tion process with sufficient force to effectuate vesicle gen(cid:173)
`eration.
`
`In the processes of the present invention, the
`[0042]
`organic lipid solution typically includes an organic solvent,
`such as a lower alkanol. As mentioned above, in one aspect,
`the liposomes are immediately diluted 140 with a buffer
`(e.g., citrate) to increase nucleic acid (e.g., plasmid) entrap(cid:173)
`ment and maintain particle size. Such dilution may be by
`way of innnediate introduction of the liposome solution into
`a controlled amount of buffer solution, or by mixing the
`liposome solution with a controlled flow rate of buffer in a
`second mixing region. Before sample concentration 160,
`free therapeutic agent (e.g., nucleic acid) is removed by
`using, for example, an anion exchange cartridge 150. Fur(cid:173)
`ther, by using an ultrafiltration step 170 to remove the
`alkanol, the sample is concentrated (e.g., to about 0.9 mg/mL
`plasmid DNA), the alkanol is removed, and the buffer is
`replaced with a substitute buffer (e.g., with a saline buffer)
`180. Thereafter, the sample is filtered 190 and filled in vials
`195. The process will now be discussed in more detail herein
`below using the steps as set forth in FIG. 1.
`
`[0043] 1. Lipid Solubilization and Therapeutic Agent Dis(cid:173)
`solution
`
`In one embodiment, the liposome vesicles pro(cid:173)
`[0044]
`duced according to the processes of the present invention
`include stable nucleic acid lipid particle (i.e., SNALP)
`formulations. Those of skill in the art will appreciate that the
`following description is for illustration purposes only. The
`processes of the present invention are applicable to a wide
`range of lipid vesicle types and sizes. These lipid vesicles
`include, but are not limited to, single bilayer lipid vesicles
`known as unilamellar lipid vesicles which can be made
`small (SUV s) or large (L UV s ), as well as multilamellar lipid
`vesicles (MLVs). Further vesicles include, micelles, lipid(cid:173)
`nucleic acid particles, virosomes, and the like. Those of skill
`in the art will know of other lipid vesicles for which the
`processes and apparatus of the present invention will be
`suitable.
`
`[0045] The preferred size for liposomes made in accor(cid:173)
`dance with the present pro