METHOD OF PREPARING LIPID VESICLES

Abstract

There is described a method of preparing lipid vesicles, said method comprising dispersing a first liquid phase in a second liquid phase; wherein said first liquid phase comprises a lipid phase and said second liquid phase comprises an aqueous phase; or said first liquid phase comprises an aqueous phase and said second liquid phase comprises a lipid phase; said method comprising controlling provision of the first liquid phase in a first flow direction to a membrane, said membrane defining a plurality of pores; and controlling provision of the second liquid phase to the membrane in a crossflow to the first flow direction, via the plurality of pores, to form a lipid vesicle suspension.

Claims

1. A method of preparing lipid vesicles, said method comprising dispersing a first liquid phase in a second liquid phase; wherein said first liquid phase comprises a lipid phase and said second liquid phase comprises an aqueous phase; or said first liquid phase comprises an aqueous phase and said second liquid phase comprises a lipid phase; said method comprising controlling provision of the first liquid phase in a first flow direction to a membrane, said membrane defining a plurality of pores; and controlling provision of the second liquid phase to the membrane in a crossflow to the first flow direction, via the plurality of pores, to form a lipid vesicle suspension.

2. The method according to claim 1 wherein the first liquid phase comprises a lipid phase and the second liquid phase comprises an aqueous phase.

3. The method according to claim 1 wherein the first liquid phase comprises an aqueous phase and the second liquid phase comprises a lipid phase.

4. The method according to claim 1 wherein the lipid vesicles are liposomes or lipid nanoparticles (LNPs).

5. (canceled)

6. (canceled)

7. A method of preparing lipid vesicles, said method comprising dispersing a first liquid phase in a second liquid phase, wherein said first liquid phase comprises a lipid phase; wherein said method uses a crossflow emulsification apparatus; said crossflow emulsification apparatus (AXF) comprising: an outer tubular sleeve provided with a first inlet at a first end; a lipid vesicle suspension outlet; and a second inlet, distal from and inclined relative to the first inlet; a tubular membrane provided with a plurality of pores and adapted to be positioned inside the tubular sleeve; and optionally an insert adapted to be located inside the tubular membrane, said insert comprising an inlet end and an outlet end, each of the inlet end and an outlet end being provided with chamfered region; the chamfered region is provided with a plurality of orifices and a furcation plate; and controlling provision of the first liquid phase to the tubular membrane; and controlling provision of a second liquid phase to the tubular membrane via the plurality of pores to form a lipid vesicle suspension.

8. (canceled)

9. (canceled)

10. The method according to claim 7 wherein the lipid vesicles are liposomes or lipid nanoparticles (LNPs).

11. (canceled)

12. (canceled)

13. The method according to claim 1 wherein the aqueous phase includes one or more active agents.

14. The method according to claim 13 wherein the aqueous phase comprises a buffered solution.

15. The method according to claim 14 wherein the aqueous phase buffers include, but shall not be limited to, MES (2-N-morpholino)ethanesulfonic acid), citrate, phosphate, acetate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS (tris(hydroxymethyl)aminomethane) and PBS (phosphate-buffered saline); and combinations thereof.

16. The method according to claim 7 wherein when the one or more active agents is hydrophobic, then the one or more active agents may be included in the lipid phase.

17. (canceled)

18. The method according to claim 1 wherein the lipid vesicles are produced unloaded and loaded afterwards (active loading).

19. The method according to claim 1 wherein the lipid vesicles are produced loaded (passive loading).

20. (canceled)

21. The method according to claim 13 wherein the one or more active agents is a bioactive agents, such as a therapeutic agent (drug), vaccine and the like.

22. The method according to claim 21 wherein the bioactive agent is a therapeutic nucleic acid such as one encoding for an antigen.

23. The method according to claim 22 wherein therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir and aptamers.

24. (canceled)

25. (canceled)

26. The method according to claim 1 wherein the lipid vesicles are LNPs and the LNPs are ionisable or cationic LNPs.

27. The method according to claim 1 wherein the lipid vesicles comprise cationic lipid vesicles, such as, DDA (dimethyl dioctadecyl ammonium bromide) or DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) may suitably be used.

28. (canceled)

29. The method according to claim 1 wherein the lipid vesicles comprise neutral lipid vesicles and the neutral lipid vesicles comprise sphingosylphosphorylcholine (SPC), L-α-hydrogenated phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC) and 1-palmitoyl-2-oleoylphosphatidylcholine, (POPC), and the like; and combinations thereof.

30. (canceled)

31. The method according to claim 1 wherein the lipid vesicles comprise a lipid having a pKa in the range of 5.0 to 7.6 and the lipids includes a tertiary amine.

32. The method according to claim 31 wherein the lipid vesicles comprise 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane. Another suitable lipid having a tertiary amine is 1,2-dioleyloxy-N,Ndimethyl-3-aminopropane.

33.-35. (canceled)

36. The method according to claim 1 wherein the lipid vesicles comprise a pegylated lipid.

37. The method according to claim 36 wherein the pegylated lipids include, but shall not be limited to, 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated diacylglycerol (PEG-DAG), e.g. 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG), e.g. 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-({acute over (ω)}-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate, e.g. {acute over (ω)}-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, 2,3-di(tetradecanoxy)propyl-N-({acute over (ω)}-methoxy(polyethoxy)ethyl)carbamate or distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) 2000]; and combinations thereof.

38. (canceled)

39. (canceled)

40. A method according to claim 7 wherein the apparatus includes an insert.

41.-51. (canceled)

52. A method according to claim 7 wherein the crossflow apparatus comprises a plurality of tubular membranes.

53.-79. (canceled)

80. A method according to claim 7 wherein the apparatus is suitable for preparing lipid vesicles with a PDI of from about 0.02 to about 0.3.

81. Lipid vesicles prepared by the method according to claim 1.

82.-99. (canceled)

Description

[0123] The present invention will now be described by way of example only, with reference to the accompanying Examples and Figures in which:

[0124] FIG. 1(a) illustrates the effect of Total Flow Rate (TFR) on particle size for unloaded liposomes;

[0125] FIGS. 1(b)-(d) illustrate the size distribution by intensity for unloaded liposomes at flow rates of 120 mL/min, 267 mL/min and 567 mL/min, respectively;

[0126] FIG. 2(a) illustrates the effect of Membrane Pore Diameter on particle size for unloaded liposomes;

[0127] FIGS. 2(b)-(d) illustrate the size distribution by intensity for unloaded liposomes at membrane pore diameters of 10 μm, 20 μm and 40 μm respectively;

[0128] FIGS. 3(a)-(d) illustrate the size distribution by intensity for unloaded liposomes at insert diameters of 7 mm, 9 mm and 9.5 mm respectively;

[0129] FIG. 4(a) illustrates the effect of Total Flow Rate (TFR) on particle size for pegylated liposomes;

[0130] FIGS. 4(b)-(d) illustrate the size distribution by intensity for pegylated liposomes at flow rates of 120 mL/min, 200 mL/min and 1,000 mL/min, respectively;

[0131] FIGS. 5(a) and (b) illustrate the particle size verses the polydispersity index (PDI) for LNPs loaded with an RNA analogue flow rate on particle size verses the polydispersity index (PDI) for LNPs loaded with an RNA analogue at a lipid phase flow rate of 125 mL/min; and

[0132] FIGS. 6(a) and (b) illustrate the effect of flow rate on particle size verses the polydispersity index (PDI) for LNPs loaded with an RNA analogue at flow rates of 200 mL/min, 300 mL/min and 500 mL/min respectively.

EXAMPLES

Example 1

[0133] Effect of Flow Rate on Particle Size of Unloaded Liposomes

[0134] A vessel containing an aqueous PBS buffer was prepared, alongside another vessel containing a solution of lipids and cholesterol in ethanol at a total concentration of 20 mg/mL.

[0135] The AXF micromixing equipment consisted of the device housing, a membrane with 10 μm pores and a spacing of 200 μm in a square grid, and an insert 9.5 mm in diameter.

[0136] Peristaltic pumps were used to pump both phases, using tygon tubing. The aqueous buffer phase was pumped through the centre of the AXF membrane micromixing equipment, at rates of 96, 214 and 454 mL/min. The lipid phase was pumped at rates of 24, 53 and 113 mL/min, into the top port of the device, through the membrane, and into the aqueous phase flow, maintaining the 4:1 aqueous:organic phase ratios across all the experiments.

[0137] The resultant 20% ethanol solution was further diluted by the addition of aqueous PBS buffer, and the resulting dilute solution was concentrated via ultrafiltration.

[0138] The resulting suspension was analysed via Dynamic Light Scattering/Quasi-Elastic Light Scattering (DLS/QELS), and intensity Z.sub.average particle size and PDI values were recorded and reported in Table 1 and FIGS. 1(a)-(d).

TABLE-US-00001 TABLE 1 Total flow rate (TFR) Z average (nm) PDI 120 99.35 0.13 267 76.37 0.134 567 65.32 0.115

Example 2

[0139] Effect of Membrane Pore Size on Particle Size and Distribution of Unloaded Liposomes

[0140] A vessel containing an aqueous PBS buffer was prepared, alongside another vessel containing a solution of lipids and cholesterol in ethanol at a total concentration of 20 mg/mL.

[0141] The AXF micromixing equipment consisted of the device housing, a membrane, and an insert 9.5 mm in diameter. 3 membranes were used, with pore diameters of 10, 20 and 40 μm. All had a pore spacing (pitch) of 200 μm in a square grid.

[0142] Peristaltic pumps were used to pump both phases, using tygon tubing. The aqueous buffer phase was pumped through the centre of the AXF membrane micromixing equipment, at a rate of 240 mL/min. The lipid phase was pumped at rates of 40 mL/min, into the top port of the device, through the membrane, and into the aqueous phase flow, giving a 6:1 aqueous:organic phase ratio.

[0143] The resultant ˜14% ethanol solution was further diluted by the addition of aqueous PBS buffer, and the resulting dilute solution was concentrated via ultrafiltration.

[0144] The resulting suspension was analysed via DLS/QELS, and intensity Z.sub.average particle size and PDI values were recorded and reported in Table 2 and FIGS. 2(a)-(d).

TABLE-US-00002 TABLE 2 Membrane Pore Size (μm) Z average (nm) PDI 10 89.68 ± 0.44 0.156 ± 0.006 20 91.24 ± 0.14 0.153 ± 0.006 40 99.63 ± 0.24 0.178 ± 0.013

Example 3

[0145] Effect of Insert Diameter on Particle Size and Distribution of Unloaded Liposomes A vessel containing an aqueous PBS buffer was prepared, alongside another vessel containing a solution of lipids and cholesterol in ethanol at a total concentration of 20 mg/mL.

[0146] The AXF micromixing equipment consisted of the device housing, a membrane with a pore diameter of 10 μm and a pore spacing (pitch) of 200 μm in a square grid, and an insert. 3 inserts were used, with diameters of 7.0 mm, 9.0 mm and 9.5 mm.

[0147] Peristaltic pumps were used to pump both phases, using tygon tubing. The aqueous buffer phase was pumped through the centre of the AXF membrane micromixing equipment, at a rate of 240 mL/min. The lipid phase was pumped at rates of 40 mL/min, into the top port of the device, through the membrane, and into the aqueous phase flow, giving a 6:1 aqueous:organic phase ratio.

[0148] The resultant ˜14% ethanol solution was further diluted by the addition of aqueous PBS buffer, and the resulting dilute solution was concentrated via ultrafiltration.

[0149] The resulting suspension was analysed via DLS/QELS, and intensity Z.sub.average particle size and PDI values were recorded and reported in Table 3 and FIGS. 3(a)-(d).

TABLE-US-00003 TABLE 3 Insert Diameter (mm) Z average (nm) PDI 7.0 114.10 ± 0.85  0.121 ± 0.003 9.0 95.97 ± 1.13 0.142 ± 0.011 9.5 92.92 ± 0.30 0.144 ± 0.007

Example 4

[0150] Production of Pegylated Liposomes

[0151] A vessel containing an aqueous HEPEs buffer (10 mM, pH 7.4) was prepared, alongside another vessel containing a solution of lipids and cholesterol in ethanol. The lipids were HSPC (S PC-3, Lipoid GmBH), DSPC-mPEG2000 (Lipoid GmBH), and cholesterol (Sigma Aldrich) at a molar ratio of HSPC/Cholesterol/Pegylated Lipid of 56.2/38.5/5.3, at a total concentration of 10 mg/mL.

[0152] The AXF micromixing equipment consisted of the device housing, a membrane with 10 μm pores and a spacing of 200 μm in a square grid, and a 9.5 mm insert.

[0153] Peristaltic pumps were used to pump both phases, using tygon tubing. Both phases were held above the T.sub.c of the lipids. The aqueous buffer phase was pumped through the centre of the AXF membrane micromixing equipment, at rates of 90 mL/min, 150 mL/min and 750 mL/min. The lipid phase was pumped at rates of 30 mL/min, 50 mL/min and 250 mL/min, into the top port of the device, through the membrane, and into the aqueous phase flow, giving a 3:1 aqueous:organic phase ratio.

[0154] The resultant 25% ethanol solution was further diluted by the immediate addition of aqueous HEPEs buffer, and the resulting dilute solution was concentrated via ultrafiltration.

[0155] The resulting suspension was analysed via DLS/QELS, and intensity Z.sub.average particle size and PDI values were recorded and reported in Table 3 and FIGS. 4(a)-(d).

TABLE-US-00004 TABLE 3 Total Flow Rate (mL/min) Z average (nm) PDI 120 62.40 ± 0.76 0.153 ± 0.014 200 63.30 ± 0.18 0.142 ± 0.008 1000 45.64 ± 0.33 0.132 ± 0.003

Example 5

[0156] Reproducibility in Production of LNPs Loaded with an RNA Analogue

[0157] A vessel containing an aqueous 100 mM citrate buffer system (pH 6) and the RNA analogue polyA was prepared, alongside another vessel containing a solution of lipids in ethanol. The lipids were the cationic lipid DDAB, the structural lipid DSPC, the pegylated lipid DMG-PEG2000 and cholesterol, at a total lipid concentration of 3 mM and a molar ratio of DDAB/DSPC/Chol/DMG-PEG2000 of 40/10/48/2. The nitrogen-to-phosphate ratio (N/P; nitrogen from the cationic lipid and phosphate from the nucleic acid) was 6.

[0158] The AXF micromixing equipment consisted of the device housing, a membrane with 10 μm pores and a spacing of 200 μm in a square grid, and an insert 9.0 mm in diameter.

[0159] Gear pumps were used to pump both phases, using PFA tubing. The aqueous buffer phase was pumped through the centre of the AXF membrane micromixing equipment, at a rate of 375 mL/min. The lipid phase was pumped at a rate of 125 mL/min, into the top port of the device, through the membrane, and into the aqueous phase flow, giving a 3:1 aqueous:organic phase ratio.

[0160] The resultant 25% ethanol solution was further diluted by the addition of aqueous buffer, and the resulting dilute solution was concentrated via ultrafiltration.

[0161] The experiment was run 3 times, and the resulting suspensions were analysed via DLS/QELS, and intensity Z.sub.average particle size and PDI values were recorded. Nucleic acid loading was quantified by Ribogreen assay. These values are reported in Table 4 and FIGS. 5(a)-(b).

TABLE-US-00005 TABLE 4 Run Z average (nm) PDI EE (%) n1 105.03 ± 1.01 0.21 ± 0.010 96.45 ± 0.33 n2  94.01 ± 3.83 0.21 ± 0.010 97.81 ± 0.29 n3 109.57 ± 0.25 0.22 ± 0.018 97.09 ± 0.39

Example 6

[0162] Effect of Flow Rate in the Production of LNPs Loaded with an RNA Analogue

[0163] A vessel containing an aqueous 100 mM citrate buffer system (pH 6) and the RNA analogue polyA was prepared, alongside another vessel containing a solution of lipids in ethanol. The lipids were the cationic lipid DDAB, the structural lipid DSPC, the pegylated lipid DMG-PEG2000 and cholesterol, at a total lipid concentration of 3 mM and a molar ratio of DDAB/DSPC/Chol/DMG-PEG2000 of 40/10/48/2. The nitrogen-to-phosphate ratio (N/P; nitrogen from the cationic lipid and phosphate from the nucleic acid) was 6.

[0164] The AXF micromixing equipment consisted of the device housing, a membrane with 10 μm pores and a spacing of 200 μm in a square grid, and an insert 9.0 mm in diameter.

[0165] Gear pumps were used to pump both phases, using PFA tubing. The aqueous buffer phase was pumped through the centre of the AXF membrane micromixing equipment. The lipid phase was pumped into the top port of the device, through the membrane, and into the aqueous phase flow. The total flow rates were 100 mL/min, 200 mL/min, 300 mL/min and 500 mL/min. A 3:1 aqueous:organic phase ratio was maintained for all runs.

[0166] The resultant 25% ethanol solution was further diluted by the addition of aqueous buffer, and the resulting dilute solution was concentrated via ultrafiltration.

[0167] The experiment was run 3 times, and the resulting suspensions were analysed via DLS/QELS, and intensity Z.sub.average particle size and PDI values were recorded. Nucleic acid loading and encapsulation efficiency (EE) was quantified by Ribogreen assay. All values are reported in Table 5 and FIGS. 6(a) and (b).

TABLE-US-00006 TABLE 5 Total Flow Rate (mL/min) Z average (nm) PDI EE (%) 200  113.7 ± 1.65 0.160 ± 0.019 93.80 300 110.55 ± 1.62 0.183 ± 0.002 97.10 500 105.23 ± 1.00 0.214 ± 0.008 97.45