Methods for Producing Adjuvants

Abstract

The present invention relates to a method for producing homogeneous and heterogeneous adjuvant formulations comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), including dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSP ... and (ii) at least one phospholipid selected from the group consisting of diphosphopyridine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), and further provided is an adjuvant formulation produced from the method.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 485,964, filed February 20, 2023, and U.S. Provisional Application No. 63 / 319,418, filed March 14, 2022. The entire contents of each of the above-mentioned applications are incorporated herein by reference.

[0002] Technical Field The present invention relates to a method for producing homogeneous and heterogeneous adjuvant formulations comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), including dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSP ... and (ii) at least one phospholipid selected from the group consisting of diphosphopyridine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), and further provided is an adjuvant formulation produced from the method. [Background technology]

[0003] A commercially available liposomal adjuvant formulation known as AS01, which contains both monophosphoryl lipid A (MPLA) and QS-21 saponin, was used as an adjuvant for the shingles vaccine for adults over 50 years of age in the United States in 2017. The United States Army has developed another liposome-based formulation known as ALFQ (Army Liposome Formulation Q), which contains both MPLA and QS-21 saponin. ALFQ has been shown to be efficacious as a liposomal vaccine adjuvant in rodent and non-human primate studies, and has also been shown to be non-pyrogenic and non-toxic in preclinical studies. The improved safety profile of ALFQ can be attributed to irreversible binding by liposomal cholesterol to free QS-21 forming a complex that prevents hemolysis resulting from binding of QS-21 to red blood cells. When soluble QS-21 is added to the suspension of liposome intermediates, the size of ALFQ increases from 50-100 nm to approximately 30,000 nm during the production of ALFQ. As described in the literature, ALFQ liposome intermediates are prepared by a rehydration process (Beck et al., Biochimica et Biophysica Acta 1848 (2015) 775-780; Singh et al., Biochemical and Biophysical Research Communications 529 (2020) 362-365; Matyas et al., Methods in Enzymology 373 (2003) 34-50). In the process, lipids are mixed, dissolved in organic solvents, dried under vacuum, and then liposomes are formed in PBS and downsized to 30-100 nm by a microfluidizer. In the most widely used of these methods, a thin lipid film (from an organic solvent) is deposited on the walls of a vessel, an aqueous solution of the substance to be encapsulated is added, and the vessel is agitated (Bangham et al., J. Mol. Biol. 13 (1965) 238-252). Under the correct conditions, this process leads to the formation of liposomal multilamellar vesicles.However, this method is not generally scalable for manufacturing due to limitations in the size of the equipment used, i.e., the rotary evaporator.

[0004] In another process, an organic solution of lipids is freeze-dried to obtain a freeze-dried product that has the physical property of being easily hydrated by aqueous solutions of the material to be encapsulated. Problems associated with this production method include batch-to-batch variability in drug encapsulation in liposomes. For example, Conrad et al. (Biochim. Biophys. Acta 332 (1974) 36-46) show that a standard deviation of encapsulation efficiency of 12-13% was found among independently prepared liposomal formulations using this method. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for a reproducible process that can control the size and polydispersity of liposome adjuvant formulations that contain MPLA and saponin, including but not limited to QS-21.In addition, there is a need for a process that is sufficiently robust for large-scale use, i.e., scalable for clinical and commercial manufacturing. [Means for solving the problem]

[0006] The present invention provides a novel reproducible process that can control the size and polydispersity of liposomal adjuvant formulations containing MPLA and saponin, including but not limited to QS-21.In addition, the present invention provides a process for large-scale manufacturing of liposomal adjuvant formulations containing MPLA and saponin, where large-scale manufacturing (i.e., scalable manufacturing) produces liposomal adjuvant formulations in sufficient quantities for clinical and commercial use.

[0007] The present invention relates to a first method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), and distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) concentrating the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5. thereby producing a homogenous adjuvant formulation.

[0008] In one embodiment of the first method for producing a homogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or mixtures thereof. In a preferred embodiment, the saponin is QS-21.

[0009] The present invention relates to a second method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent or mixture of organic solvents to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) filtering the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5. thereby producing a homogenous adjuvant formulation.

[0010] In one embodiment of the second method for producing a homogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or mixtures thereof. In a preferred embodiment, the saponin is QS-21.

[0011] The present invention relates to a first method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid solution as an organic phase; (ii) injecting the organic and aqueous phases of step (i) together at a specific flow rate and in a specific ratio of organic solvent phase to aqueous phase to form liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) treating the intermediate liposomes of step (iv) with a microfluidizer at about 17,500 psi for 10 passes; (vi) filtering the intermediate liposomes of step (v) using a 0.22 um membrane; (vii) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0012] In one embodiment of the first method for producing a heterogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof. In a preferred aspect, the saponin is QS-21.

[0013] The present invention relates to a second method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid formulation as an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) concentrating the intermediate liposomes of step (iii); (v) removing the organic phase of the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0014] In one embodiment of the second method for producing a heterogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof. In a preferred aspect, the saponin is QS-21.

[0015] The present invention provides a third method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) preparing a lyophilized organic phase comprising phospholipids, cholesterol and MPLA; (ii) rehydrating the lyophilized organic phase of step (i) with an aqueous phase to form intermediate liposomes; (iii) downsizing the intermediate liposomes of step (ii) using a microfluidizer; (iv) combining the intermediate liposomes of step (iii) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0016] In one embodiment of the third method for producing a heterogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof. In a preferred aspect, the saponin is QS-21.

[0017] The present invention also provides an adjuvant formulation produced by any one of the methods described herein. [Brief description of the drawings]

[0018] [Figure 1] 1 is a graph showing neutralization titers of individual animals (rats) immunized with C. difficile toxoid antigen formulated with various LiNA-2 adjuvants (homogeneous and heterogeneous). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates to a process for preparing an adjuvant formulation comprising a monophosphoryl lipid A (MPLA)-containing liposomal composition and at least one saponin (e.g., QS-21). Examples of such non-toxic adjuvant formulations are described in U.S. Pat. No. 10,434,167 and PCT International Publication WO2015148648, each of which is incorporated herein by reference in its entirety. The adjuvant formulation comprises a monophosphoryl lipid A (MPLA)-containing liposomal composition and at least one saponin, the liposomal composition comprising i) a lipid bilayer comprising phospholipids and ii) cholesterol at a molar percentage concentration of the liposomal composition greater than about 50% (mol / mol). The saponin may be selected from QS-7, QS-18, QS-21, or a mixture thereof. Preferably, the saponin is QS-21.

[0020] Thus, the adjuvant formulation comprises a monophosphoryl lipid A (MPLA)-containing liposome composition and at least one saponin, the liposome composition comprising i) a lipid bilayer comprising phospholipids and ii) cholesterol, the molar ratio of cholesterol to phospholipid being greater than about 1. The saponin may be selected from QS-7, QS-18, QS-21, or a mixture thereof. Preferably, the saponin is QS-21. Described herein are several processes for preparing heterogeneous and homogeneous liposomal adjuvant formulations that are scalable for manufacturing. The homogeneous process is a controlled and robust process that results in size-controlled liposomes. Both adjuvant formulations comprise MPLA-containing liposomes with saponin (e.g., QS-21). The non-toxic adjuvant formulation described in U.S. Pat. No. 10,434,167 is a highly heterogeneous product produced by a process that is very difficult to scale up for clinical and commercial manufacturing. Example 3 describes three novel processes for producing this heterogeneous liposomal adjuvant formulation that are highly reproducible and can be easily scaled up for clinical and commercial manufacturing.

[0021] Examples 1 and 2 describe two processes that are scalable to produce homogeneous liposomal adjuvant formulations with the same components as the heterogeneous liposomal adjuvant formulation described in U.S. Patent No. 10,434,167. The processes described by Examples 1 and 2 create homogeneous formulations with liposomes with a size range of less than 1 micrometer and a controlled polydispersity index (PDI) that ranges from 0.05 to 0.3. The processes described herein are reproducible and well controlled, allowing for the production of homogeneous liposomal adjuvant formulations with defined sizes.

[0022] Several novel solvent injection methods for preparing intermediate liposomes are also described herein. Solvent injection involves the rapid injection of a lipid solution in ethanol or other organic solvent into an aqueous medium. The process is carried out at room temperature or at higher temperatures (e.g., 60°C). Intermediate liposomes can be produced in sizes from 30 nm to 200 nm with PDI<0.25. The size and PDI of the intermediates can be controlled by adjusting the parameters of the solvent injection, such as temperature, flow rate, flow rate ratio of the organic phase to the aqueous phase, or by the microfluidizer process. The final size of the liposomal adjuvant formulation can be influenced by the size of the intermediate liposomes and the manufacturing process (e.g., number of passes through the microfluidizer, etc.), as described herein.

[0023] In addition, the preparation of heterogeneous liposomes using lyophilized lipid is also described herein.In this preparation, there is no problem of drug loading variation in different batches, since there is no drug in adjuvant formulation.This method ensures batch-to-batch uniformity in clinical and commercial scale, and facilitates quality control.

[0024] In general, the present specification provides a novel process for preparing adjuvant formulations, including MPLA-containing liposome compositions containing saponin (e.g., QS-21).These processes can control the size and polydispersity of liposome adjuvant formulations, and are easily scalable for clinical and commercial manufacturing, and are reproducible compared to conventional processes.In addition, the present specification provides a novel homogeneous MPLA-containing liposome composition containing saponin (e.g., QS-21).

[0025] Accordingly, the present invention provides a first method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) concentrating the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5. thereby producing a homogenous adjuvant formulation.

[0026] In one embodiment of the first method for producing a homogeneous adjuvant composition, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or mixtures thereof. In a preferred embodiment, the saponin is QS-21.

[0027] In another embodiment of the first method for producing a homogeneous adjuvant composition, in step (i), the phospholipid, cholesterol and MPLA are dissolved in an organic solvent by sonication, heat or a combination thereof, preferably by heating. In one aspect, the organic solvent is ethanol or isopropyl alcohol. In another aspect, the organic phase is heated to a temperature between 45°C and 65°C.

[0028] In another embodiment of the first method for producing a homogeneous adjuvant composition, the aqueous phase comprises water and, optionally, a buffer. In a preferred embodiment, the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In a preferred embodiment, the aqueous phase is 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In another embodiment, the aqueous phase is at a temperature between 20° C. and 60° C. In another embodiment, the flow rate of step (ii) is between 0.5 mL / min and 400 mL / min or is rapid addition. In another embodiment, the flow rate of step (ii) is between 0.5 mL / min and 400 mL / min. In another embodiment, the injection of step (ii) is performed by rapid injection of the organic phase of step (i).

[0029] In another embodiment of the first method for producing a homogenous adjuvant composition, the intermediate liposomes of step (iii) are stirred at a speed of between 700 rpm and 900 rpm.

[0030] In another embodiment of the first method for producing a homogeneous adjuvant composition, the ratio of organic phase to aqueous phase in step (ii) ranges from 1:4 to 1:16. In a preferred embodiment, the ratio is 1:8.

[0031] In another embodiment of the first method for producing a homogenous adjuvant composition, the size of the intermediate liposomes in step (iv) is downsized by using a high pressure extruder or a microfluidic homogenizer. In one aspect, the size of the liposomes is downsized in step (iv) by using a membrane size in the range of 50 nm to 120 nm or a homogenization pressure between 17000 PSI to 24000 PSI, or a combination thereof.

[0032] In another embodiment of the first method for producing a homogenous adjuvant composition, the organic solvent is removed prior to downsizing in step (iv) or after downsizing in step (iv). In one aspect, the removal of the organic phase of the intermediate liposomes in step (iv) is by tangential flow filtration (TFF). In another aspect, the TFF is TFF diafiltration. In another aspect, the TFF comprises a membrane having a molecular weight cut off (MWCO) in the range of 100-500 kDa.

[0033] In another embodiment of the first method for producing a homogeneous adjuvant composition, the concentration in step (v) is by ultrafiltration. In one aspect, the ultrafiltration comprises a bioburden reduction filter and a sterilizing filter.

[0034] In another embodiment of the first method for producing a homogenous adjuvant composition, the blending in step (vi) is at a mixing speed of 300 rpm. In one aspect, the blending in step (vi) is for a period ranging from 1 hour to 24 hours. In another aspect, the blending in step (vi) is performed at room temperature, or at 2-8°C.

[0035] In another embodiment of the first method for producing a homogeneous adjuvant composition, the final adjuvant formulation has a size range of about 30 nm to 400 nm.

[0036] In another embodiment of the first method for producing a homogeneous adjuvant composition, the final adjuvant formulation has a polydispersity of between 0.05 and 0.5.

[0037] In a further embodiment of the first method for producing a homogenous adjuvant composition, the injecting in step (ii) is by pump or syringe injection. In a preferred aspect, the injecting in step (ii) is by pump.

[0038] In one embodiment of the first method for producing a homogeneous adjuvant composition, the at least one phospholipid is a mixture of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG).

[0039] In one embodiment, the first method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof, and (ii) cholesterol, wherein the molar ratio of cholesterol to phospholipid in the liposomal composition is greater than 1, comprises: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) concentrating the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5. thereby producing a homogenous adjuvant formulation.

[0040] The present invention relates to a second method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent or mixture of organic solvents to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) filtering the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5. thereby producing a homogenous adjuvant formulation.

[0041] In one embodiment of the second method for producing a homogeneous adjuvant composition, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or mixtures thereof. In a preferred embodiment, the saponin is QS-21.

[0042] In another embodiment of the second method for producing a homogeneous adjuvant composition, in step (i), the phospholipid, cholesterol and MPLA are dissolved in an organic solvent by sonication, heat, stirring or a combination thereof. In one aspect, the organic solvent is ethanol or isopropyl alcohol or other organic solvent. In one aspect, the organic solvent is ethanol or isopropyl alcohol. In another aspect, the organic phase is heated to a temperature between 45°C and 65°C, preferably between 50°C and 65°C or between 45°C and 55°C.

[0043] In another embodiment of the second method for producing a homogeneous adjuvant composition, the aqueous phase comprises water and, optionally, a buffer. In one embodiment, the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In a preferred embodiment, the aqueous phase is 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In another embodiment, the aqueous phase is at a temperature between 20° C. and 65° C. In another embodiment, the flow rate of step (ii) is between 5 mL / min and 15 mL / min. In another embodiment, the flow rate of step (ii) is 12 mL / min. In another embodiment, the intermediate liposomes of step (ii) are stirred at a speed between 100 rpm and 1000 rpm. In another embodiment, the ratio of organic phase to aqueous phase of step (ii) ranges from 1:2 to 1:16. In a preferred embodiment, the ratio is 4:7.

[0044] In another embodiment of the second method for producing a homogeneous adjuvant composition, the concentration in step (iii) is by ultrafiltration.

[0045] In another embodiment of the second method for producing a homogenous adjuvant composition, the removal of the organic phase of the intermediate liposome in step (iv) is by tangential flow filtration (TFF). In one aspect, the TFF is TFF diafiltration. In another aspect, the TFF comprises a membrane having a molecular weight cut off (MWCO) in the range of 100-500 kDa.

[0046] In another embodiment of the second method for producing a homogeneous adjuvant composition, the filtration in step (v) comprises a bioburden reduction filter and a sterilizing filter.

[0047] In another embodiment of the second method for producing a homogenous adjuvant composition, the blending in step (vi) is at room temperature for 1 hour with a mixing speed of 350 rpm.

[0048] In another embodiment of the second method for producing a homogeneous adjuvant composition, the final adjuvant formulation has a size range of about 50-200 nm. Preferably, the final adjuvant formulation has a size of about 100 nm.

[0049] In another embodiment of the second method for producing a homogeneous adjuvant composition, the final adjuvant formulation has a polydispersity of 0.05 to 0.5. Preferably, the final adjuvant formulation has a PDI of <0.2.

[0050] In a further embodiment of the second method for producing a homogeneous adjuvant composition, the injecting in step (ii) is by pump or syringe injection. In a preferred aspect, the injecting in step (ii) is by pump.

[0051] In one embodiment of the second method for producing a homogeneous adjuvant composition, the at least one phospholipid is a mixture of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG).

[0052] In one embodiment, the second method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof, and (ii) cholesterol, wherein the molar ratio of cholesterol to phospholipid is greater than 1, comprises: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent or mixture of organic solvents to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) filtering the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5. thereby producing a homogeneous adjuvant formulation.

[0053] The present invention relates to a first method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid solution as an organic phase; (ii) injecting the organic and aqueous phases of step (i) together at a specific flow rate and in a specific ratio of organic solvent phase to aqueous phase to form liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) treating the intermediate liposomes of step (iv) with a microfluidizer at about 17,500 psi for 10 passes; (vi) filtering the intermediate liposomes of step (v) using a 0.22 um membrane; (vii) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0054] In one embodiment of the first method for producing a heterogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof. In a preferred aspect, the saponin is QS-21.

[0055] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, in step (i), the phospholipid, cholesterol and MPLA are dissolved in an organic solvent by sonication, heat, stirring or a combination thereof. In one aspect, the organic solvent is ethanol or isopropyl alcohol or a mixture thereof. In another aspect, the lipid formulation organic phase is heated to a temperature between 45°C and 65°C.

[0056] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the aqueous phase comprises water and, optionally, a buffer. In one aspect, the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In a preferred aspect, the aqueous phase is 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In another aspect, the aqueous phase is at a temperature between 45° C. and 60° C.

[0057] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, in step (ii), the organic phase has a flow rate of 0.5-2 ml / min and the aqueous phase has a flow rate of 5-15 ml / min. In another embodiment of the third method, in step (ii), the organic phase has a flow rate of 1.333 ml / min and the aqueous phase has a flow rate of 10.667 ml / min.

[0058] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the intermediate liposomes of step (iii) are stirred at a speed between 100 rpm and 900 rpm.

[0059] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the ratio of organic phase to aqueous phase in step (ii) ranges from 1:4 to 1:8. In a preferred aspect, the ratio is 1:8.

[0060] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the removal of the organic phase of the intermediate liposomes in step (iii) and step (iv) is by tangential flow filtration (TFF). In one aspect, the TFF is TFF ultrafiltration and diafiltration. In another aspect, the TFF comprises a membrane having a molecular weight cut-off (MWCO) in the range of 100 to 500 kDa.

[0061] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the concentration in step (iii) is by ultrafiltration.

[0062] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the size of the intermediate liposomes in step (iv) is manipulated by using a microfluidizer.

[0063] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the organic solvent is removed prior to the microfluidizing of step (v).

[0064] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, a buffer is added to the blending in step (vii). In one aspect, the blending in step (vii) is at room temperature for 1 hour to 48 hours with a mixing speed of 350 rpm, or is stirred for 1 hour. In another aspect, after blending in step (vii), the intermediate liposomes are stored at room temperature without stirring for up to 48 hours.

[0065] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the final adjuvant formulation has a size of about 300 nm to 1000 nm.

[0066] In another embodiment of the first method for producing a heterogeneous adjuvant formulation, the final adjuvant formulation has a polydispersity of between 0.4 and 1.0.

[0067] In a further embodiment of the first method for producing a heterogeneous adjuvant formulation, the injecting in step (ii) is by Nanoassembblr or pump or syringe injection.

[0068] In one embodiment of the first method for producing a heterogeneous adjuvant formulation, the at least one phospholipid is a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0069] In one embodiment, the first method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof, and (ii) cholesterol, wherein the molar ratio of cholesterol to phospholipid is greater than 1, comprises: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid solution as an organic phase; (ii) injecting the organic and aqueous phases of step (i) together at a specific flow rate and in a specific ratio of organic solvent phase to aqueous phase to form liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) treating the intermediate liposomes of step (iv) with a microfluidizer at about 17,500 psi for 10 passes; (vi) filtering the intermediate liposomes of step (v) using a 0.22 um membrane; (vii) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0070] The present invention relates to a second method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein at least one phospholipid is selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid formulation as an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic solvent phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) concentrating the intermediate liposomes of step (iii); (v) removing the organic layer of the intermediate liposome of step (iv); (vi) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0071] In one embodiment of the second method for producing a heterogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof. In a preferred aspect, the saponin is QS-21.

[0072] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, in step (i), the phospholipid, cholesterol and MPLA are dissolved in an organic solvent by sonication, heat, stirring or a combination thereof. In one aspect, the organic solvent comprises ethyl acetate and isopropyl alcohol. In another aspect, the lipid formulation organic phase is heated to a temperature of 50°C to 65°C.

[0073] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the aqueous phase comprises water and, optionally, a buffer. In one aspect, the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In a preferred aspect, the aqueous phase is 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In another aspect, the aqueous phase is at a temperature between 20° C. and 25° C. In a further aspect, the flow rate of step (ii) is between 10 and 30 mL / min. In a further aspect, the flow rate of step (ii) is 20 mL / min.

[0074] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the intermediate liposomes of step (iii) are stirred at a speed between 100 rpm and 900 rpm.

[0075] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the ratio of organic phase to aqueous phase in step (ii) ranges from 1:4 to 1:8. In a preferred aspect, the ratio is 1.8.

[0076] In another embodiment of the second process for producing a heterogeneous adjuvant formulation, the organic solvent is removed prior to the combination in step (vi).

[0077] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the removal of the organic phase of the intermediate liposome in step (iv) and step (v) is by tangential flow filtration (TFF). In one aspect, the TFF is TFF ultrafiltration and diafiltration. In another aspect, the TFF comprises a membrane having a molecular weight cut-off (MWCO) in the range of 100-500 kDa.

[0078] In another embodiment of the second process for producing a heterogeneous adjuvant formulation, the concentration in step (vi) is by ultrafiltration.

[0079] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, a buffer is added to the formulation in step (vi).

[0080] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the blending in step (vi) is at room temperature for 1 hour with a mixing speed of 300 rpm. In one aspect, after blending in step (vii), the intermediate liposomes are stored at room temperature without stirring for 24 hours.

[0081] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the final adjuvant formulation has a size range of 300 nm to 1000 nm.

[0082] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the final adjuvant formulation has a polydispersity of 0.4 to 1.0.

[0083] In another embodiment of the second method for producing a heterogeneous adjuvant formulation, the injecting in step (ii) is by pipette, pump or syringe injection.

[0084] In one embodiment of the second method for producing a heterogeneous adjuvant formulation, the at least one phospholipid is a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0085] In one embodiment, the second method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof, and (ii) cholesterol, wherein the molar ratio of cholesterol to phospholipid is greater than 1, comprises: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid formulation as an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) concentrating the intermediate liposomes of step (iii); (v) removing the organic phase of the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0086] The present invention provides a third method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from at least one phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), the at least one phospholipid being selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) preparing a lyophilized organic phase comprising phospholipids, cholesterol and MPLA; (ii) rehydrating the lyophilized organic phase of step (i) with an aqueous phase to form intermediate liposomes; (iii) downsizing the intermediate liposomes of step (ii) using a microfluidizer; (iv) combining the intermediate liposomes of step (iii) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0087] In one embodiment of the third method for producing a heterogeneous adjuvant formulation, the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof. In a preferred aspect, the saponin is QS-21.

[0088] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, in step (i), the phospholipid, cholesterol and MPLA are dissolved in an organic solvent by sonication, heat, stirring or a combination thereof. In one aspect, the organic solvent comprises tert-butyl alcohol (TBA) or a mixture thereof. In another aspect, the lipid organic phase is heated to a temperature between 25° C. and 65° C. In another aspect, the organic phase solution is lyophilized.

[0089] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the aqueous phase comprises water or a buffer. In one aspect, the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In a preferred aspect, the aqueous phase is 10 mM phosphate at pH 6.2 containing 150 mM NaCl. In another aspect, the aqueous phase is at a temperature between 20° C. and 70° C.

[0090] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the intermediate liposomes of step (ii) are stirred at a speed of 100 to 1000 rpm.

[0091] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the intermediate liposomes in step (iii) are downsized using a microfluidizer and a pressure at or about 18640 PSI.

[0092] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the organic solvent is removed prior to rehydration in step (ii). In one aspect, removal of the organic solvent is by lyophilization.

[0093] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, a buffer is added to the formulation in step (iv).

[0094] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the blending in step (iv) is at room temperature for 1 hour with a mixing speed of 300 rpm or with stirring.

[0095] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the compounded intermediate liposomes of step (iv) are stored at room temperature without stirring for 24 hours.

[0096] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the final adjuvant formulation has a size >300 nm.

[0097] In another embodiment of the third method for producing a heterogeneous adjuvant formulation, the final adjuvant formulation has a polydispersity >0.4.

[0098] In one embodiment of the third method for producing a heterogeneous adjuvant formulation, the at least one phospholipid is a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0099] In one embodiment, the third method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof, and (ii) cholesterol, wherein the molar ratio of cholesterol to phospholipid is greater than 1, comprises: (i) preparing a lyophilized organic phase comprising phospholipids, cholesterol and MPLA; (ii) rehydrating the lyophilized organic phase of step (i) with an aqueous phase to form intermediate liposomes; (iii) downsizing the intermediate liposomes of step (ii) using a microfluidizer; (iv) combining the intermediate liposomes of step (iii) with saponin; thereby producing a heterogeneous adjuvant formulation.

[0100] The present invention also provides an adjuvant formulation produced by any one of the methods described herein.

[0101] In one embodiment, the liposome composition of the adjuvant formulation may include cholesterol at a molar percent concentration of greater than 50% (mol / mol), about 55% to about 71% (mol / mol), or preferably about 55% (mol / mol). In one embodiment, the liposome composition of the adjuvant formulation may include a phosphatidylcholine phospholipid (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearyl phosphatidylcholine (DSPC). In another embodiment, the liposome composition of the adjuvant formulation may include a phosphatidylglycerol phospholipid (PG) selected from the group consisting of dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). In a further aspect, the liposomal composition of the adjuvant formulation may comprise a combination of (i) a phosphatidylcholine phospholipid (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearyl phosphatidylcholine (DSPC), and (ii) a phosphatidylglycerol phospholipid (PG) selected from the group consisting of dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). The liposome composition of the adjuvant formulation may have a PC to PG ratio (mol / mol) of about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. The liposome composition of the adjuvant formulation may comprise multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs), where small unilamellar vesicles are about 50 to about 100 nm in diameter and multilamellar vesicles are about 1 to about 4 μm in diameter.In a preferred embodiment, the liposomal composition of the adjuvant formulation comprises PC and PG, where PC is dimyristoyl phosphatidylcholine (DMPC) and PG is dimyristoyl phosphatidylglycerol (DMPG), with a molar ratio of PC to PG (mol / mol) of about 9:1.

[0102] In another embodiment, the liposomal composition of the adjuvant formulation may comprise about 5 mg or less, about 4 mg or less, about 3 mg or less, about 2 mg or less, about 1 mg or less, about 0.9 mg or less, about 0.8 mg or less, about 0.7 mg or less, about 0.6 mg or less, about 0.5 mg or less, about 0.4 mg or less, about 0.3 mg or less, about 0.2 mg or less, about 0.1 mg or less, about 0.09 mg or less, about 0.08 mg or less, about 0.07 mg or less, about 0.06 mg or less, about 0.05 mg or less, about 0.04 mg or less, about 0.03 mg or less, about 0.02 mg or less, or about 0.01 mg or less (total weight per ml of liposomal suspension) of MPLA. The liposome composition of the adjuvant formulation may have an MPLA:phospholipid molar ratio of about 1:5.6 to about 1:880, or about 1:88 to about 1:220. In a preferred embodiment, the liposome composition of the adjuvant formulation comprises PC and PG, where PC is dimyristoyl phosphatidylcholine (DMPC) and PG is dimyristoyl phosphatidylglycerol (DMPG), and has an MPLA:phospholipid molar ratio of about 1:220, about 1:88 or about 1:5.6, preferably 1:88.

[0103] In a further embodiment, the adjuvant formulation may have a saponin content (total weight per ml of liposome suspension) of about 1 mg or less, about 0.9 mg or less, about 0.8 mg or less, about 0.7 mg or less, about 0.6 mg or less, about 0.5 mg or less, about 0.4 mg or less, about 0.3 mg or less, about 0.2 mg or less, about 0.1 mg or less, about 0.09 mg or less, about 0.08 mg or less, about 0.07 mg or less, about 0.06 mg or less, about 0.05 mg or less, about 0.04 mg or less, about 0.03 mg or less, about 0.02 mg or less, or about 0.01 mg or less. In a preferred embodiment, the adjuvant formulation comprises a saponin content of about 0.2-0.4 mg / ml.

[0104] Also provided is an immunogenic composition comprising an immunogen and an adjuvant formulation. The immunogenic composition may typically comprise a physiologically acceptable vehicle. The immunogen of the immunogenic composition may be selected from the group consisting of naturally occurring or artificially produced proteins, recombinant proteins, glycoproteins, peptides, carbohydrates, haptens, whole viruses, bacteria, protozoa, and virus-like particles. Also provided is a method of immunizing an animal, comprising administering the immunogenic composition.

[0105] Further provided is a method of reducing the toxicity of saponin as an adjuvant or preparing an adjuvant formulation, comprising adding a monophosphoryl lipid A (MPLA)-containing liposome composition to the saponin, the liposome composition comprising i) a lipid bilayer comprising a phospholipid whose hydrocarbon chain has a melting temperature in water of ≧23° C. and ii) cholesterol at a molar percent concentration greater than about 50% (mol / mol). The saponin may be selected from the group consisting of QS-7, QS-18, QS-21, and mixtures thereof. Preferably, the saponin is QS-21. The liposome composition may comprise cholesterol at a molar percent concentration of about 55% to about 71% (mol / mol), preferably about 55% (mol / mol).

[0106] 1.Definition An "immunogen" is an agent that can induce humoral and / or cell-mediated immune response. The immunogen as described herein can be an antigen, a hapten, or an inactivated pathogen. The immunogenic composition as described herein can be, for example, a vaccine formulation.

[0107] As used herein, the term "uniform" shall mean a final adjuvant formulation comprising liposomes having a size range of about 30 nm to 400 nm as determined by methods known in the art, including, but not limited to, dynamic light scattering (DLS), transmission electron cryomicroscopy (e.g., cryo-TEM or cryo-EM), nanoparticle tracking analysis (NTA, e.g., ViewSizer). A "uniform" adjuvant formulation may also mean a final adjuvant formulation comprising liposomes having a polydispersity index (PDI) of between about 0.05 to 0.5 or between about 0.05 to about 0.3, preferably about 0.3. Calculations used to determine size and PDI parameters can be found in ISO standard documents 13321:1996 E and ISO 22412:2008 (Worldwide MIDynamic Light Scattering, Common Terms Defined. Malvern Instruments Limited; Malvern, UK: 2011. Pages 1-6. Inform White Paper). As used herein, a "homogeneous" adjuvant formulation is also intended to mean a "monodisperse" adjuvant formulation.

[0108] As used herein, the term "heterogeneous" is intended to mean a final adjuvant formulation comprising liposomes having sizes that vary from about 30 nm to 10 micrometers or more, from about 30 nm to 4 micrometers, from about 30 nm to 1400 nm, preferably from about 30 nm to 1000 nm, as determined by methods known in the art, including but not limited to dynamic light scattering (DLS), transmission electron cryomicroscopy (e.g., cryo-TEM or cryo-EM), nanoparticle tracking analysis (NTA, e.g., ViewSizer). A "heterogeneous" adjuvant formulation may also refer to a final adjuvant formulation comprising liposomes having a polydispersity index (PDI) of >0.5. Calculations used to determine size and PDI parameters can be found in ISO standard documents 13321:1996 E and ISO 22412:2008 (Worldwide MIDynamic Light Scattering, Common Terms Defined. Malvern Instruments Limited; Malvern, UK: 2011. Pages 1-6. Inform White Paper). As used herein, a "heterogeneous" adjuvant formulation is also intended to mean a "polydisperse" adjuvant formulation.

[0109] "Liposome" as used herein refers to a closed bilayer membrane containing an enclosed aqueous volume. Liposomes may be unilamellar vesicles with a single membrane bilayer, or multilamellar vesicles containing multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is one in which the hydrophobic (non-polar) tails of the lipids are oriented toward the center of the bilayer and the hydrophilic (polar) heads are oriented toward the aqueous phase. Liposomes, as they are commonly used, consist of smectic mesophases, which may be of phospholipid or non-phospholipid smectic mesophases. Smectic mesophases are most accurately described by Small, HANDBOOK OF LIPID RESEARCH, Vol. 4, Plenum, NY, 1986, pp. 49-50. According to Small, "When a given molecule is heated, instead of immediately melting into an isotropic liquid, it may rather pass through an intermediate state called a mesophase or liquid crystal, characterized by residual order in some directions but a lack of order in others. In general, the molecules of liquid crystals are somewhat longer than they are wide and have polar or aromatic moieties at certain locations along the length of the molecule. Molecular shape and polar-polar, or aromatic, interactions allow the molecules to align in partially ordered arrays. These structures occur characteristically in molecules that have a polar group at one end. Liquid crystals with long-range order along the long molecular axis are called smectic, lamellar, or lamellar liquid crystals. In the smectic state, the molecules are arranged in monolayers or bilayers, perpendicular or tilted to the plane of the layers, and may have frozen or melted aliphatic chains."

[0110] Lipid A is a group of complex, heavily acylated and amidated diglucosamine diphosphate molecules that is the lipid moiety common to all lipopolysaccharides (LPS; also known as endotoxins) derived from Gram-negative bacteria. LPS covers virtually the entire outer surface of all Gram-negative bacteria, and lipid A tethers LPS to the outer lipid surface of the bacteria. The O-polysaccharide portion of LPS in wild-type smooth bacteria is linked to a relatively conserved core oligosaccharide expressed in rough mutants, which in turn is linked to lipid A via a highly conserved 2-keto-3-deoxyoctanoic acid sugar, a unique chemical structure sometimes required for bacterial survival and found only in LPS. See, e.g., Alving et al., 2012, Expert Rev. Vaccines 11:733-44. "Monophosphoryl lipid A" is a lipid A homologue in which the glucosamine-1-phosphate group on the polar head group has been removed. There are also many MPLA homologues.

[0111] The "mole percent concentration of cholesterol" of a liposomal composition as used herein refers to the ratio of cholesterol:total phospholipids (ie, phosphatidylcholine and phosphatidylglycerol) initially used in preparing the liposomal composition.

[0112] "Physiologically acceptable vehicle" as used herein refers to a vehicle suitable for in vivo administration (e.g., oral, transdermal or parenteral administration) or in vitro use, i.e., cell culture. An exemplary physiologically acceptable vehicle may be the physiologically acceptable component of liposomes as disclosed in U.S. Patent Nos. 4,186,183 and 4,302,459.

[0113] "Preferred" and "preferably" as used herein should be construed for European claim interpretation only. These terms should be excluded or deleted from the interpretation of sentences and paragraphs in which they appear for U.S. claim interpretation.

[0114] As used herein, "about" refers to ±5% of the stated value.

[0115] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. It should be noted that, as used herein, the singular forms "a", "and" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, and reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0116] 2. Saponin For this embodiment, the preferred saponin is Quil A, its derivatives, or any of its purified components (e.g., QS-7, QS-18, QS-21, or mixtures thereof). Quil A is a saponin preparation isolated from the South American tree Quillaja Saponaria Molina, which was first found to have adjuvant activity. Dalsgaard et al., 1974, Archiv. fur die gesanite Virusforschung, 44:243-254. Purified fragments of Quil A have been isolated by HPLC (EP 0 362 278) and include, for example, QS-7 and QS-21 (also known as QA7 and QA21, respectively). QS-21 is the 21st fraction purified from the sap of the Quillaja Saponaria tree. QS-21 has been shown to induce CD8+ cytotoxic T cell (CTL), Th1 cell and potent IgG2a antibody responses.

[0117] 3. Monophosphoryl lipid A (MPLA)-containing liposomes (L(MPLA)) Liposomes are closed bilayer membranes that contain an enclosed aqueous volume. Liposomes may be unilamellar vesicles with a single membrane bilayer, or multilamellar vesicles that contain multiple membrane bilayers, each separated from the next by an aqueous layer. The resulting membrane bilayer structure is such that the hydrophobic (non-polar) tails of the lipids are oriented toward the center of the bilayer and the hydrophilic (polar) heads are oriented toward the aqueous phase. Suitable hydrophilic polymers for surrounding liposomes include, but are not limited to, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide and hydrophilic peptide sequences as described in U.S. Patent No. 6,316,024, U.S. Patent No. 6,126,966, U.S. Patent No. 6,056,973 and U.S. Patent No. 6,043,094.Liposomes can be made without hydrophilic polymers.Therefore, liposome preparations can contain or not contain hydrophilic polymers.

[0118] Liposomes can be composed of any lipid or lipid combination known in the art.For example, the lipids that form the vesicles can be naturally occurring or synthetic lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, as disclosed in U.S. Patent Nos. 6,056,973 and 5,874,104.

[0119] The vesicle-forming lipid may be a glycolipid, a cerebroside or a cationic lipid such as 1,2-dioleyloxy-3-(trimethylamino)propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1[(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 3[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DCChol); or dimethyldioctadecylammonium (DDAB), as also disclosed in U.S. Pat. No. 6,056,973. Cholesterol can also be present in a suitable range to provide stability to liposome vesicles, as disclosed in U.S. Patent Nos. 5,916,588 and 5,874,104.Additional liposome technologies are described in U.S. Patent Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 4,684,479. These described liposomes and lipid-coated microbubbles and methods for producing them. Thus, one of skill in the art could generate liposomes for the purposes of this embodiment, taking into account both this disclosure and the disclosures of these other patents. For this embodiment, the liposome composition typically contains about 1 mM to about 150 mM phospholipid.

[0120] Any of the above exemplary liposomes may contain monophosphoryl lipid A (MPLA) or may be combined with other liposomes and lipid A (MPLA). MPLA alone may be toxic to humans and animals. However, when present in liposomes, no toxicity is detected. See, for example, Alving et al., 2012. MPLA serves as a powerful adjuvant and is useful for enhancing the immunogenicity of liposomes and peptides, proteins or haptens associated with liposomes.

[0121] In the present embodiment, monophosphoryl lipid A (MPLA)-containing liposomes (L(MPLA)) are originally called Walter Reed liposomes, but are now known as Army Liposome Formulation (ALF) as a vaccine adjuvant. See, for example, Alving et al., 2012. ALF adjuvant liposomes contain (1) a lipid bilayer containing phospholipids whose hydrocarbon chains have a melting temperature in water of ≧23° C., usually dimyristoyl phosphatidylcholine (DMPC, e.g., 1,2-dimyristoyl-sn-glycero-3-phosphocholine) and dimyristoyl phosphatidylglycerol (DMPG, e.g., 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)); (2) cholesterol (Chol) as a stabilizer; and (3) monophosphoryl lipid A (MPLA) as an immunostimulant. In human trials, ALF-type liposomal adjuvants have been found to be safe and potent in vaccine candidates against malaria, HIV-1, and cancer. See, for example, Fries et al., 1992, Proc. Natl. Acad. Sci. USA, 89:358-62; Alving, 2002, Vaccine, 20:S56-64. Certain compositions of ALF, in which QS-21 is added to ALF to form ALFQ, contain cholesterol at a molar percent concentration greater than about 50% (mol / mol), preferably about 55% to about 71% (mol / mol), or more preferably about 55% (mol / mol). Additionally, in this embodiment, the monophosphoryl lipid A (MPLA)-containing liposome (L(MPLA)) containing saponin (e.g., QS-21) may be a homogenous LiNA-2 adjuvant formulation or a heterogenous LiNA-2 adjuvant formulation as described herein.

[0122] For this embodiment, L(MPLA) may comprise a phosphatidylcholine (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearyl phosphatidylcholine (DSPC). L(MPLA) may comprise a phosphatidylglycerol (PG) selected from dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). The PC to PG ratio (mol / mol) of the liposome may be about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. The liposomes may have a MPLA content (total weight per ml of liposome suspension) of about 5 mg or less, about 4 mg or less, about 3 mg or less, about 2 mg or less, about 1 mg or less, about 0.9 mg or less, about 0.8 mg or less, about 0.7 mg or less, about 0.6 mg or less, about 0.5 mg or less, about 0.4 mg or less, about 0.3 mg or less, about 0.2 mg or less, about 0.1 mg or less, about 0.09 mg or less, about 0.08 mg or less, about 0.07 mg or less, about 0.06 mg or less, about 0.05 mg or less, about 0.04 mg or less, about 0.03 mg or less, about 0.02 mg or less, or about 0.01 mg or less. Alternatively, the liposomes may have an MPLA:phospholipid molar ratio of about 1:5.6 to about 1:880, preferably about 1:88 to about 1:220. Prior to addition of saponin, the liposomes may comprise multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). Small unilamellar vesicles may be about 50 to about 100 nm in diameter and multilamellar vesicles may be about 1 to about 4 μm in diameter.

[0123] 4. Adjuvant formulation containing MPLA-containing liposomes (L(MPLA) and saponin An adjuvant formulation known as AS01 (also known as AS01B or AS01E) was previously marketed by GlaxoSmithKline. In AS01, the lipid bilayer was composed of dioleoylphosphatidylcholine, cholesterol, MPLA, and neutral lipids that are "non-crystalline" at room temperature, such as QS-21. See U.S. Pat. No. 10,039,823. During the manufacture of AS01, small unilamellar liposomal vesicles (SUVs) are first created, and then purified QS-21 is added to the SUVs. QS-21 confers unique properties in that it binds to cholesterol in liposomes, where it creates holes or other permanent structural changes in the liposome. See, e.g., Paepenmuller et al., 2014, Int. J. Pharm., 475:138-46. A reduction in the amount of free QS-21 would likely reduce the local injection pain often caused by free QS-21. See, e.g., Waite et al., 2001, Vaccine, 19:3957-67; Mbawuike et al., 2007, Vaccine, 25:3263-69. The AS01 formulation was created for vaccines where the induction of a stronger T cell-mediated immune response is required. See Garcon et al., 2007, Expert. Rev. Vaccines, 6:723-39. The AS01 formulation is being developed as an adjuvant for various vaccines. See Garcon & Mechelen, 2011, Expert. Rev. Vaccines, 10:471-86. AS01 formulations may contain cholesterol (sterol) at a molar percentage concentration of 1-50% (mol / mol), preferably 20-25% (mol / mol), as described in US Pat. No. 10,039,823.

[0124] The present invention provides an adjuvant formulation produced by the method described herein, comprising a monophosphoryl lipid A (MPLA)-containing liposomal composition and at least one saponin, wherein the liposomal composition comprises i) a lipid bilayer comprising a phospholipid whose hydrocarbon chain has a melting temperature in water of ≧23° C. (e.g., dimyristoyl phosphatidylcholine (DMPC) and / or dimyristoyl phosphatidylglycerol (DMPG)) and ii) cholesterol at a molar percent concentration of greater than about 50% (mol / mol), or preferably about 55% to about 71% (mol / mol), or more preferably about 55% (mol / mol). At least these two characteristics are distinct from those of AS01 as discussed above. The saponin may be selected from QS-7, QS-18, QS-21, or a mixture thereof, or the saponin may preferably be QS-21. The adjuvant formulation may contain about 1 mg or less, about 0.9 mg or less, about 0.8 mg or less, about 0.7 mg or less, about 0.6 mg or less, about 0.5 mg or less, about 0.4 mg or less, about 0.3 mg or less, about 0.2 mg or less, about 0.1 mg or less, about 0.09 mg or less, about 0.08 mg or less, about 0.07 mg or less, about 0.06 mg or less, about 0.05 mg or less, about 0.04 mg or less, about 0.03 mg or less, about 0.02 mg or less, or about 0.01 mg or less of saponin per ml of liposome suspension. In a preferred embodiment, the adjuvant formulation contains a saponin content of about 0.2 to 0.4 mg / ml.

[0125] In one embodiment, the adjuvant formulation comprises a liposome composition comprising i) a lipid bilayer comprising a phospholipid and ii) cholesterol, wherein the molar ratio of cholesterol to phospholipid is greater than about 1.

[0126] In a preferred embodiment, the liposome composition of the adjuvant formulation comprises PC and PG, where PC is dimyristoyl phosphatidylcholine (DMPC) and PG is dimyristoyl phosphatidylglycerol (DMPG), with a molar ratio of PC to PG (mol / mol) of about 9:1.

[0127] In a further aspect, the liposome composition of the adjuvant formulation may have an MPLA:phospholipid molar ratio of about 1:5.6 to about 1:880, or about 1:88 to about 1:220. In a preferred embodiment, the liposome composition of the adjuvant formulation comprises PC and PG, where PC is dimyristoyl phosphatidylcholine (DMPC) and PG is dimyristoyl phosphatidylglycerol (DMPG), and has an MPLA:phospholipid molar ratio of about 1:220, about 1:88 or about 1:5.6, preferably 1:88.

[0128] In another embodiment, the invention provides an adjuvant formulation produced by the methods described herein, comprising: (a) as a phospholipid, at least one phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), and distearylphosphatidylglycerol (DSPG); (b) cholesterol; (c) monophosphoryl lipid A (MPLA); and (d) saponin; comprising unilamellar liposomes having a liposomal bilayer with a molar ratio of cholesterol (b) to phospholipid (a) greater than about 50:50; and the unilamellar liposomes have a median diameter size in the micrometer range as detected by light scattering analysis. In one embodiment, the saponin is QS-7, QS-18, QS-21, or a mixture thereof, preferably QS-21. In another embodiment, the ratio of cholesterol (b) to phospholipid (a) is about 55:45 to about 71:29. In another embodiment, the ratio of cholesterol (b) to phospholipid (a) is about 55:45. In another embodiment, dimyristoyl phosphatidylcholine (DMPC) is selected as the phospholipid, and in addition, dimyristoyl phosphatidylglycerol (DMPG) is selected as the phospholipid. In another embodiment, both PC and PG are selected as phospholipids, and the ratio of PC to PG (mol / mol) is about 0.5:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, or about 15:1. In another embodiment, the present invention provides a liposomal suspension comprising an adjuvant formulation as described herein and phosphate buffer solution (PBS), pH 7.4, comprising (i) 1.272 mM to 50 mM phospholipid (a), and (ii) about 5 mg / ml or less of MPLA (c). In another embodiment, the molar ratio of MPLA (c) to phospholipid (a) is about 1:5.6 to about 1:880.In a further aspect, the invention provides a liposomal suspension comprising an adjuvant formulation as described herein and phosphate buffer solution (PBS), pH 7.4, comprising (i) 1.272 mM to 50 mM phospholipid (a), and (ii) about 1 mg / ml or less of saponin (d). In another aspect, the molar ratio of MPLA (c) to phospholipid (a) is about 1:88 to about 1:220.

[0129] Examples of adjuvant formulations comprising an MPLA-containing liposome composition made by the methods described herein and at least one saponin (e.g., QS-21) include, but are not limited to, the adjuvant formulations described in U.S. Pat. No. 10,434,167 (e.g., ALFQ) and the two Liposomal Novel Adjuvant-2 (LiNA-2) adjuvant formulations described herein: a LiNA-2 homogeneous adjuvant formulation and a LiNA-2 heterogeneous adjuvant formulation. In one embodiment, LiNA-2 adjuvant comprises synthetic TLR4 agonist, monophosphoryl lipid A (MPLA), triterpenoid glycoside saponin (QS-21), 1,2-dimyristoyl-sn-glycero-3-phosphocholine also known as dimyristoyl phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) also known as dimyristoyl phosphatidylglycerol (DMPG), and cholesterol. In a preferred embodiment, LiNA-2 comprises MPLA (e.g., 3D-PHAD®), QS-21, DMPC, DMPG, and cholesterol. In another preferred embodiment, LiNA-2 comprises MPLA (e.g., 3D-PHAD®), QS-21, DMPC, DMPG, and cholesterol in a phosphate buffer containing sodium chloride (NaCl). In one embodiment, the LiNA-2 adjuvant is designed to be reconstituted into a lyophilized powder formulation for administration, hi another embodiment, the LiNA-2 is designed to be mixed with a liquid formulation for administration.

[0130] In a further aspect, the LiNA-2 homogeneous or LiNA-2 heterogeneous adjuvant formulation may be LiNA-2 at 1X concentration (1XLiNA-2) or LiNA-2 at 2X concentration (2XLiNA-2) when compared to the ALFQ concentration as described herein. In one embodiment, the adjuvant formulation is ALFQ comprising: (i) 7.0 mg / mL DMPC, (ii) 0.78 mg / ml DMPG, (iii) 5.4 mg / ml cholesterol, (iv) 0.2 mg / mL MPLA (3D-PHAD), and (v) 0.1 mg / ml QS-21. In another embodiment, the adjuvant formulation is 1XLiNA-2, which may be homogeneous or heterogeneous and comprises (i) 14±7 mg / mL DMPC, (ii) 1.6±0.8 mg / ml DMPG, (iii) 11±6 mg / ml cholesterol, (iv) 0.40±0.20 mg / mL MPLA (3D-PHAD), and (v) 0.20±0.10 mg / ml QS-21. In a further embodiment, the adjuvant formulation is 2XLiNA-2, which may be homogeneous or heterogeneous, and comprises (i) 28±14 mg / mL DMPC, (ii) 3.2±1.6 mg / ml DMPG, (iii) 22±11 mg / ml cholesterol, (iv) 0.80±0.40 mg / mL MPLA (3D-PHAD), and (v) 0.40±0.20 mg / ml QS-21.

[0131] 5. Use of an adjuvant formulation containing MPLA-containing liposomes (L(MPLA) and saponin The adjuvant formulation of this embodiment can be mixed with an immunogen to obtain an immunogenic composition, such as a vaccine. The immunogenic composition can include a physiologically acceptable vehicle, such as any one of those described in U.S. Pat. No. 5,888,519. The immunogenic composition can include naturally occurring or artificially created proteins, recombinant proteins, glycoproteins, peptides, carbohydrates, nucleic acids, haptens, whole viruses, bacteria, protozoa, or virus-like particles, or conjugates thereof, as immunogens. The immunogenic composition may suitably be used as a vaccine for chickenpox or shingles, human respiratory syncytial virus infection (RSV), cytomegalovirus infection (CMV), human metapneumovirus, human parainfluenza virus type 1 or 3, Lyme disease, Streptococcus pneumonia, Clostridioides difficile, Escherichia coli or Klebsiella pneumonia, influenza, HIV-1, hepatitis A, hepatitis B, human papillomavirus, meningococcal meningitis type A, meningococcal meningitis type B, meningococcal meningitis type C, tetanus, diphtheria, whooping cough, polio, Haemophilus influenzae type B, dengue, hand, foot and mouth disease, typhoid, Pneumococcus, Japanese encephalitis virus, anthrax, shingles, malaria, norovirus, or cancer. The immunogenic composition may be suitably used in a method for treating or preventing a disease or infection in a subject, preferably a human, caused by a pathogen associated with the infection, the pathogen being selected from the group consisting of Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus spp.genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae, Burkholderia cepacia cepacia and other Burkholderia spp., Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae, Campylobacter genus, Candida albicans, Candida spp., Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetiiburnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebola virus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr virus (EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Escherichia coli fimbrial antigen H, Fasciola hepatica and Fasciola gigantica, FFI prions, Filaria superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp. spp), GSS prion, Gannaritovirus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Hendra virusNipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus), Hortaea werneckii, human bocavirus (HBoV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 7 (HHV-7), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, granulomatis, Klebsiella pneumoniae, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowlerifowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae (influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii jirovecii, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rhinovirus, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, rotavirus, rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenespyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton species spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella-Zoster Virus (VZV), Variola major or minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0132] The present invention provides an immunogenic composition comprising an immunogen and an adjuvant formulation as described herein. EXAMPLES

[0133] In order that the present invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any manner. The following examples illustrate some embodiments of the present invention.

[0134] In the following examples, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), and synthetic monophosphoryl lipid A (MPLA) (3D-PHAD™) can be purchased from Avanti Polar Lipids (Alabaster, Ala., USA). Purified QS-21 can be purchased from Desert King International (San Diego, Calif., USA).

[0135] Also, in the following examples, the cholesterol content of the liposomal composition can be analyzed by established methods to ascertain the cholesterol, and indirectly, the phospholipid concentration. See, e.g., Zlatkis et al., 1953, J. Lab. Clin. Med., 41:486-492. The cholesterol concentration of the liposomal composition can be determined from a cholesterol standard curve.

[0136] Example 1 Preparation of homogeneous adjuvant formulations A solvent injection process was used to generate liposomes of various sizes. Liposomes were formed by dissolving lipids (DMPG, DMPC, cholesterol, and MPLA) in organic solvent (ethanol or isopropyl alcohol) by sonication, heat, or a combination of both (organic phase). The lipids in organic solvent (organic phase) were heated to 65°C. The organic phase was pumped into a buffer (aqueous phase) that was maintained at room temperature (22°C to 25°C) or heated (up to 60°C). The buffer (aqueous phase) was 10 mM phosphate at pH 6.2 containing 150 mM NaCl. The introduction of lipids (organic phase) could be slow, from 0.5 mL / min to 400 mL / min, or it could be a rapid addition of lipids (organic phase) to the buffer (aqueous phase).

[0137] By controlling the rate of lipid addition to the buffer along with changing the ratio of organic solvent to the aqueous phase (buffer or water), the size of the liposomes formed had a size range of 40 nm to 400 nm. The ratios tested ranged from 1:4 to 1:16. Liposome formation was controlled by stirring the buffer at 700 rpm along with controlling the temperature of the buffer at room temperature or in a heated state. The size of the liposomes formed during this process was reduced using a high-pressure extruder or a microfluidic homogenizer. The size was controlled by the selection of mesh sizes ranging from 50 nm to 120 nm or by using homogenization pressures between 17000 and 24000 PSI. After this downsizing process, the intermediate liposomes formed were in the range of 40 to 100 nm allowing for sterile filtration.

[0138] Table 1 below presents data from multiple batches of intermediate liposomes formed.

[0139] [Table 1]

[0140] The organic solvent in the liposomes can be removed before or after downsizing. Tangential flow filtration using hollow fiber or cassette membranes is used to remove ethanol. The concentration of the final product can be adjusted by controlling the ultrafiltration and diafiltration steps. The molecular weight cut-off (MWCO) of the membranes used in TFF ranges from 100 to 500 kDa. After TFF, the liposomes are resuspended in buffer and passed through a bioburden reduction filter (0.45 micron) and a sterile filter (0.22 micron).

[0141] The intermediate liposomes containing MPLA are compounded (combined, mixed, or denatured process) with a saponin (e.g., QS-21) in an aseptic (sterile environment) manner to produce the final adjuvant formulation. The size of the final adjuvant ranges from 50-200 nm with a polydispersity of 0.05-0.3. During compounding, the mixing speed was set at 300 rpm. The mixing process ranged from 1 hour to 24 hours, and mixing could be performed at room temperature or at 2-8°C.

[0142] Example preparation of organic phase: Twice the final concentration (1X) amount of lipids (DMPC, DMPG and cholesterol) were weighed and added to glass vials containing the required volume of ethanol in appropriate quantities (qs). 45 ml of 2X lipid solution was added to 360 ml of 10 mM phosphate buffer containing 150 mM NaCl at pH 6.2 in a 1:8 ratio. 50 ml glass syringes were heated at 50°C. The lipid masses were selected according to Table 2 to prepare 50 ml of lipid stock solutions: 40 mg of MPLA (3D-PHAD) was added to 2 ml of ethanol in each vial and sonicated in a bath sonicator for 30 minutes with heating at 48°C. These MPLA (3D-PHAD) solutions were then added to the beakers of the other weighed lipids and the total volume of lipid stock solutions was adjusted to 50 ml.

[0143] [Table 2]

[0144] Steps for preparing a batch 1. Pour 360ml of PB into a 500ml Pyrex media storage bottle containing a white magnet. 2. Set the syringe pump (Speed ​​= 900 μl / min, Syringe diameter = X mm (choose the appropriate diameter of the syringe based on the pump manual, Volume = 45.00 ml). 3. Wrap the syringe with the flat surface of a spiral heating pad and connect the pad to a temperature controller / heater. Set the heater temperature to 50°C. 4. Remove 50 mL of the ethanol lipid solution into a 50 mL wrapped glass syringe using a needle to remove any bubbles or gas inside. Remove the needle and immediately cap the syringe. Cut 25 cm of tubing size 16 and cap one end of it. 5. Remove the cap from the syringe and immediately connect the open end of the tube tip to the syringe. 6. Place the PB-containing medium storage bottle on a hot plate while stirring the contents with a magnet at approximately 900 rpm at room temperature (22°C to 25°C). 7. Open the other end of the tubing and place it into the medium storage bottle. 8. Place the tube in the lipid solution, being careful to avoid any air bubbles forming inside, and recap the end of the tube to ensure the lipid is ready to be added to the PB buffer. 9. Open the end of the tubing and place into the medium storage bottle. Begin infusion at 900 ul / min until 45.00 ml of lipid solution has been added to the reaction bottle. 10. Measure size by DLS. 950 μl Milli-Q water and 50 μl sample (DF=20).

[0145] The resulting samples were extruded using a 100 ml extruder. The first cycle was performed using one 100 nm membrane, the second and third cycles using 50 nm membranes in each cycle.

[0146] The pressures used for extrusion were between 400 and 500 psi for the second and third cycles and between 600 and 700 psi for the first cycle of extrusion.

[0147] TFF was performed on the extruded samples using 100, 300 or 500 kDa MWCO mPES hollow fiber membranes. 275 ml of sample was concentrated to 50 ml and 10 DV of washes were performed with it (using 500 ml of buffer) after which the sample was concentrated to 35 mL.

[0148] The extruded liposomes were loaded into a hollow fiber tangential flow system (500 kDa mPES membrane). The total loaded volume was concentrated to the final volume and combined with that of the lipid solution. Dia-volume washes (10-12) were performed to remove all ethanol. Final size and concentration measurements were made and the intermediate liposomes were then sterile filtered. The intermediate liposomes should be twice as concentrated as the final product. The intermediate liposomes were stored at 2-8°C and then sterile filtered.

[0149] An appropriate amount of QS-21 (0.5 mg / mL to 2 mg / mL) was weighed out and reconstituted using 10 mM phosphate buffer containing 150 mM sodium chloride at pH 6.2 and mixed with the formed liposome intermediate to achieve a final target concentration of either 0.2 mg / mL or 0.4 mg / mL of QS-21 in the adjuvant formulation. The solution of QS-21 was sterile filtered and combined with the intermediate liposomes to produce the final adjuvant formulation.

[0150] Table 3 summarizes the size and PDI of multiple batches of the final adjuvant formulation produced by the process described in this Example.

[0151] [Table 3]

[0152] Example 2 Preparation of homogeneous adjuvant formulations: A process using solvent injection to prepare 40-150 nm intermediate liposomes without downsizing A. Preparation of intermediate liposomes 40-150 nm Lipids (DMPG, DMPC, cholesterol and MPLA) were weighed into a new glass bottle and 100 ml of ethanol was added to make lipid stocks (organic phase) as described in Table 4. The lipid stocks were stirred and the glass bottles were heated in a water bath and in a beaker at 45° C. After the lipids were dissolved, the lipid stocks were diluted 2.5 times using ethanol.

[0153] [Table 4]

[0154] The resulting liposomes were size screened using a NanoAssemblr with the conditions listed in Table 5. A flow ratio of 4:7 was selected to further develop the syringe injection process described in Section B below.

[0155] [Table 5]

[0156] B. Syringe injection [Using intermediate liposomes with a diameter of 40-150 nm] The lipid solution in ethanol (organic phase) described in section A above was heated to 40-65 °C in a beaker placed on a hot plate and loaded into a 60 mL plastic / glass syringe (with 10 mL margin). The syringe was wrapped in a heating pad and the temperature was maintained between 45-55 °C. All 60 mL of lipid solution was loaded into the organic syringe. Water was heated to 45 °C in a beaker placed on a hot plate and loaded into a 120 mL plastic syringe. The syringe was maintained at 45 °C using a heating pad. The aqueous and organic phases were connected by an Upchurch Stainless Steel Tee assembly (Idex). Both syringes were attached to a pump and they were run together using the software generating 20 ml of liposomes according to the conditions in Table 6. The resulting liposomes were 40-140 nm with a PDI of 0.04-0.2.

[0157] The resulting liposomes were purified and concentrated by TFF to prepare liposome intermediates with sizes of 40-91 nm and PDIs of 0.05-0.1.

[0158] [Table 6]

[0159] C. Preparation of QS-21 11 mg of QS-21 was weighed into a 50 mL conical tube. 6.629 mL of PBS buffer (10 mM phosphate, 150 mM NaCl, pH 6.2) was added to make a 1.5 mg / mL QS-21 solution. The QS-21 solution was shaken until the QS-21 was completely dissolved. The QS-21 stock solution was filtered using a 0.22 uM PES steri-flip filter. The stock solution was collected in a 50 mL conical tube, covered in aluminum foil and stored at 5°C.

[0160] D. Preparation of Adjuvant Products 1 mL of liposome intermediate from process B was mixed with 0.219 mL of QS-21 solution and 0.421 ml of PBS buffer. The combined solution was immediately covered with aluminum foil to avoid light exposure. It was then shaken at 350 rpm at room temperature for 1 hour and then stored without stirring at room temperature for 23 hours. The final formulation with a size of 102 nm and a PDI of 0.18 was set aside as the final formulation at 2-8°C. The concentration of the final adjuvant formulation contained the desired range of QS-21 of 0.2-0.4 mg / mL.

[0161] The given process can be scaled up using a pump based setup where the flow rates and mixing parameters are controlled as was done in the syringe setup described in section B of this example.

[0162] Example 3 Methods for preparing heterogeneous adjuvant formulations A. Method I: Process using Solvent Injection and Microfluidizer 1) Preparation of intermediates Lipids were weighed in a new glass bottle and 100ml of lipid stock in ethanol was added. The amount of lipid is as per the table below. A magnetic stir bar was placed in the bottle. The glass bottle was heated in a water bath and in a beaker at 65°C. After the lipids were dissolved, the sample was diluted 2.5 times using ethanol.

[0163] [Table 7]

[0164] The lipid solution was heated to 65°C in a beaker placed on a hot plate and loaded into a 60 mL plastic syringe (with 10 mL margin). The syringe was maintained at 65°C with a heating pad wrapped around it and secured by a cable tie. 60 mL of lipid solution was loaded into an organic syringe. WFI was heated to 60°C in a beaker placed on a hot plate and loaded into two 100 mL plastic syringes (with 10 mL margin). The syringe was maintained at 60°C with a heating pad wrapped around it and secured by a cable tie.

[0165] The settings used in the Adagio software / system for the organic phase are described in Table 8.

[0166] [Table 8]

[0167] The lipid solution in ethanol (organic phase) described in section A above was heated to 65°C in a beaker placed on a hot plate and loaded into a 65mL plastic / glass syringe. The syringe was wrapped in a heating pad to maintain the temperature at approximately 65°C. All 60mL of lipid solution was loaded into the organic syringe. Water was heated to 60°C in a beaker placed on a hot plate and loaded into a 120mL plastic syringe. The syringe was maintained at 60°C using a heating pad. The aqueous and organic phases were connected by a Steel Upchurch Tee assembly. Both syringes were attached to a pump and they were run together using the software which produced 20mL of liposomes according to the conditions in Table 8. The initial 5% of the sample run is discarded. Ensure the collected sample is not turbid and collect in a glass container. The resulting liposomes were 40-70nm with a PDI of 0.04-0.2.

[0168] 1200 ml of sample with size about 45 nm and PDI about 0.04-0.1 combined from several different batches was gradually loaded into the TFF system in the concentration step to obtain 60 ml of liposomes. During the diafiltration step, the flow rate was reduced from 500 ml / min to 400, 300, 200 and 100 at 10X points, then kept stable at 10x to 20x at 100 ml / min, feed 14 psi, TMP 7.5 psi. Approximately 10 times, a dilution volume of PBS buffer, pH 6.2, was used for buffer exchange starting at 100 ml / min, and the flow rate was gradually increased to 200, 300, 400, 500 ml / min, then kept at 500 ml / min, feed 12 psi, TMP 8 psi. A flow rate of about 200 ml / min was collected for the retentate, but only 60-64 ml was obtained. The samples collected after TFF were filtered using a steriflip 50 mL filtration system with a 220 nm membrane to prepare liposomes with a size of approximately 55 nm and a PDI of approximately 0.13.

[0169] The liposome solution was further passed through the Y-shaped chamber of a microfluidizer for about 10 passes, followed by filtration to prepare an intermediate with a size of 58 nm and a PDI of 0.12.

[0170] 2) Preparation of QS-21 11 mg of QS-21 was weighed into a 50 mL conical tube. 6.629 mL of PBS buffer (10 mM phosphate, 150 mM NaCl, pH 6.2) was added to make a 1.5 mg / mL QS-21 solution. The QS-21 solution was shaken until the QS-21 was completely dissolved. The QS-21 stock solution was filtered using a 0.22 uM PES steri-flip filter. The stock solution was collected in a 50 mL conical tube, covered with aluminum foil and stored at 5°C.

[0171] 3) Preparation of adjuvant products 1 mL of intermediate and 0.219 mL of QS-21 solution were mixed with 0.421 ml of PBS buffer. The combined solution was immediately covered with aluminum foil to avoid light exposure. It was then shaken at 350 rpm at room temperature for 1 hour, and then stored without stirring at room temperature for 48 hours. The final formulation with a size of 645 nm and a PDI of 0.62 was set aside as the final formulation at 2-8°C. The prescribed process was scaled up using a pump based setup where the flow rate and mixing parameters were controlled as done in the syringe setup.

[0172] B. Method II: Ethyl acetate injection process 1) Preparation of intermediates The lipids were weighed and added to a glass vial containing the required volume of 155 mL of ethyl acetate and 5 mL of isopropanol (IPA). The lipids are incubated in a water bath at 55 °C for 30 min to create a clear solution. The clear lipid solution is loaded into a 50 mL glass syringe wrapped with a heating pad to maintain the temperature at 55 °C controlled by a temperature sensor. Using a syringe pump at a flow rate of 20 mL / min, 4.5 mL of lipid solution was added to 36 mL of PBS pH 6.2 buffer. The formulation was purified and concentrated to 5-6 mL by TFF followed by 10 DV of PBS pH 6.2. This was further concentrated to 1.7 mL by centrifugation to prepare the liposome intermediate.

[0173] [Table 9]

[0174] [Table 10]

[0175] 2) Preparation of adjuvant products 0.2 mL of 1.2 mg / mL QS-21 solution was added to 0.4 mL of intermediate, followed by 2.25 mL of PBS buffer pH 6.2. The bottle was shaken on a shaking bed at 300 rpm for 1 hour at room temperature. The solution was incubated at room temperature for an additional 24 hours. The final adjuvant formulation was set aside at 5° C.

[0176] [Table 11]

[0177] C. Method III: Rehydration of Lyophilized Lipids and Processing Using a Microfluidizer 1) Preparation of intermediates Preparation of the formulation involves weighing the lipids MPLA (3D-PHAD), DMPC, DMPG, and cholesterol, dissolving them in pre-heated (55-65°C) solvent-free tert-butyl alcohol (TBA), and stirring at 300 rpm for >30 min to completely dissolve. The final solution is loaded into the lyophilizer for the freeze / dry process. After completion of the freezing step, the lyophilized lipids are then resuspended in 1 / 3 volume of 55-60°C pre-heated buffer (PBS, pH 6.2) for hydration. The concentration can be controlled by varying the volume of the buffer. Rehydration is continued at 55-60°C with stirring at 300 rpm for at least 1 h to form large liposomes ranging from nm to 10 microns. Downsizing of the liposomes is performed using an LM10 microfluidizer, applying approximately 18,640 psi pressure (setting = 17,500 psi) under a 35-45 °C water bath, passing the entire volume of the liposome solution over approximately 5-12 passes to generate small liposomes (50-100 nm). Filtration of the final liposome intermediate using a 0.22 um PES sterile filter is used to generate a homogenous liposome population with particle size approximately 40-70 nm with low polydispersity (PDI approximately 0.2). The final filtered liposomes as intermediates can be stored at 5 °C or used further to generate the final adjuvant product.

[0178] 2) Preparation of adjuvant products To further manufacture the final formulation, the intermediate is equilibrated at room temperature and compounded with the addition of PBS buffer and QS-21 solution in PBS buffer (i.e., 1.5 mg / mL QS-21) to make the final adjuvant formulation. Shaking at 300 rpm for 1 hour at room temperature, followed by storage at room temperature for an additional 24 hours (protected from light), produces a final heterogeneous adjuvant product with a particle size >300 nm and PDI >0.5. The final product is stored at 5° C. and protected from light. Size and PDI are reproducible in three replicate preparations, shown in Table 12.

[0179] [Table 12]

[0180] Example 4 Adjuvant formulations The present invention provides a method for producing scalable quantities of MPLA-liposome adjuvant formulations containing saponins (e.g., QS-21). Exemplary MPLA-liposome adjuvant formulations containing saponins (e.g., QS-21) include, but are not limited to, the MPLA-liposome formulations (e.g., ALFQ) described in U.S. Pat. No. 10,434,167, as well as the two LiNA-2 (Liposomal Novel Adjuvant-2) adjuvant formulations described herein, namely, a LiNA-2 homogeneous adjuvant formulation and a LiNA-2 heterogeneous adjuvant formulation, which may be 1XLiNA-2 concentration or 2XLiNA-2 concentration. The compositions of these adjuvant formulations are described in Table 13 below:

[0181] [Table 13]

[0182] Example 5 Immunogenicity of C. difficile vaccine antigens formulated with different LiNA-2 adjuvants The relative immunogenicity of C. difficile toxoid antigen formulated with aluminum hydroxide (Al(OH)3) and various LiNA-2 adjuvant formulations (homogeneous and heterogeneous) was compared in rats. The homogeneous and heterogeneous LiNA-2 adjuvants are described herein and in Table 14. The final rat LiNA-2 adjuvant dose was prepared by diluting a 1X concentration of LiNA-2 in a 1:5 dilution using PBS buffer, pH 6.2.

[0183] [Table 14]

[0184] Functional cytotoxic activity of sera was measured at multiple time points post-immunization using the Toxin Neutralization Assay (TNA) described below.

[0185] Wistar Han rats (10 per group, 8-10 weeks old, Charles River Laboratories) were immunized intramuscularly (IM) according to the study design in Table 15. Group 1 received C. difficile vaccine antigen formulated with Al(OH)3. Groups 2 and 3 received C. difficile vaccine antigen formulated with homogeneous and heterogeneous LiNA-2 adjuvants, respectively.

[0186] Serum was collected at multiple time points and the ability to neutralize toxin cytotoxic activity was measured in TNA. Neutralizing titers in sera from individual animals for toxin B are shown in Figure 1 and demonstrate that toxoids formulated with homogeneous and heterogeneous LiNA-2 elicited similar immune responses capable of neutralizing toxin B cytotoxicity.

[0187] [Table 15]

[0188] Toxin Neutralization Assay (TNA) The immune response induced by administering the compositions of the invention can be determined using a toxin neutralization assay (TNA), an ELISA, or more preferably, a cytotoxicity assay, such as those described in WIPO Patent Application WO / 2012 / 143902, U.S. Pat. No. 9,187,536, and WIPO Patent Application WO / 2014 / 060898, each of which are incorporated herein by reference in their entirety.

[0189] Toxin neutralization assays (TNA) can be used to quantify neutralizing antibodies against C. difficile toxins. In this assay, serially diluted serum can be incubated with a fixed amount of C. difficile toxin A or B. Test cells (e.g., Vero cells) can then be added and the serum-toxin-cell mixture can be incubated under appropriate conditions (e.g., at 37°C for 6 days). The ability of serum to neutralize the cytotoxic effects of C. difficile toxins can be determined by and correlated with cell viability. The assay utilizes the accumulation of acid metabolites in closed culture wells as an indicator of normal cellular respiration. Cells exposed to toxins experience a decrease in metabolism and CO2 production; as a result, the pH increases (e.g., to 7.4 or higher) as indicated by phenol red pH indicator in the cell culture medium. At this pH, the medium appears red. However, cells exposed to control or toxins neutralized by antibodies metabolize and produce normal amounts of CO2; as a result, the pH is maintained (e.g., at or below 7.0), at which the medium appears yellow. Thus, C. difficile toxin neutralizing antibodies correlate with the ability of the sera to neutralize the metabolic effects of C. difficile toxins on cells, as evidenced by their ability to maintain a certain pH (e.g., at or below 7.0). The color change in the medium can be measured using a plate reader (e.g., at 562 nm to 630 nm) to further calculate the antitoxin neutralizing antibody titer for 50% inhibition of C. difficile toxin-mediated cytotoxicity. In one embodiment, the composition induces a toxin-neutralizing antibody titer in a subject after receiving a dose of the composition that is at least 1-fold higher, e.g., at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 32-fold, etc., or more, than the toxin-neutralizing antibody titer in the subject before receiving said dose, when measured under identical conditions in a toxin neutralization assay.

[0190] Briefly, in this example, 384-well microtiter plates were seeded with IMR-90 cells, which served as targets for toxin-mediated cytotoxicity. Each test serum sample was analyzed separately for its ability to neutralize toxin A or toxin B. Four serial dilutions of test serum were mixed with a fixed concentration of toxin A (TcdA) or toxin B (TcdB) for 60 minutes in a humidified incubator (37°C / 5% CO2) to allow neutralization of the toxin to occur. All plates included reference standards and quality controls, which consisted of antitoxin antibodies of known titers to monitor assay performance. After 60 minutes of incubation, the toxin-antiserum mixture was applied to the IMR-90 cell monolayer, and the plate was further incubated for 72 hours. The viability of the IMR-90 cell monolayer was then tested using the luciferase-based CellTiter-Glo® reagent, which provides a measurement of ATP levels in metabolically active cells, and reported as relative luminescence units (RLU). High ATP levels indicate high cell viability and antibody-mediated neutralization of TcdA or TcdB. Neutralizing antibody concentrations were determined by comparing the RLU values ​​of test samples to calibration curves from antitoxin A or B reference standards using a custom-written Statistical Analysis System (SAS®) program. Functional antibody concentrations were expressed as arbitrary units per mL of serum (or neutralization units / mL). The lower limit of quantification (LLOQ) for the TcdA and TcdB TNA assays is 75.9 and 249.7 neutralization units / mL of serum, respectively.

Claims

1. 1. A method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition, the liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the at least one phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) concentrating the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5; thereby producing a homogenous adjuvant formulation.

2. 2. The method of claim 1, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof.

3. The method of claim 2, wherein the saponin is QS-21.

4. 2. The method of claim 1, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat or a combination thereof.

5. 5. The method of claim 4, wherein the organic solvent is ethanol or isopropyl alcohol.

6. The process of claim 4, wherein the organic phase is heated to a temperature between 45°C and 65°C.

7. The method of claim 1 , wherein the aqueous phase comprises water or a buffer.

8. 8. The method of claim 7, wherein the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl.

9. 8. The process of claim 7, wherein the aqueous phase is at a temperature between 20°C and 60°C.

10. 2. The method of claim 1, wherein the flow rate in step (ii) is from 0.5 mL / min to 400 mL / min or is rapid addition.

11. 2. The method of claim 1, wherein the intermediate liposomes of step (iii) are stirred at a speed of 700 rpm to 900 rpm.

12. 2. The process of claim 1, wherein the ratio of organic phase to aqueous phase in step (ii) is in the range of 1:4 to 1:

16.

13. The method of claim 1, wherein the size of the intermediate liposomes in step (iv) is downsized by using a high-pressure extruder or a microfluidic homogenizer.

14. 14. The method of claim 13, wherein the size of the liposomes is downsized in step (iv) by using a membrane size ranging from 50 nm to 120 nm or a homogenization pressure between 17000 PSI to 24000 PSI, or a combination thereof.

15. 2. The method of claim 1, wherein the organic solvent is removed before the downsizing in step (iv) or after the downsizing in step (iv).

16. 2. The method of claim 1, wherein the removal of the organic phase of the intermediate liposomes in step (iv) is by tangential flow filtration (TFF).

17. 17. The method of claim 16, wherein the TFF is TFF diafiltration.

18. 17. The method of claim 16, wherein the TFF comprises a membrane with a molecular weight cut off (MWCO) in the range of 100-500 kDa.

19. 10. The method of claim 1, wherein the concentrating of step (v) is by ultrafiltration.

20. 20. The method of claim 19, wherein the ultrafiltration comprises a bioburden reduction filter and a sterilizing filter.

21. 10. The method of claim 1, wherein the blending in step (vi) is at a mixing speed of 300 rpm.

22. 10. The method of claim 1, wherein the compounding in step (vi) is in the range of 1 hour to 24 hours.

23. 2. The method of claim 1, wherein the combining in step (vi) is carried out at room temperature or at 2-8°C.

24. 2. The method of claim 1, wherein the final adjuvant formulation has a size range of about 30 nm to 400 nm.

25. The method of claim 1, wherein the final adjuvant formulation has a polydispersity of 0.05 to 0.

5.

26. 2. The method of claim 1, wherein the injecting in step (ii) is by pump or syringe injection.

27. 1. A method for producing a homogenous adjuvant formulation comprising a liposomal bilayer comprising: (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising: (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the at least one phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent or mixture of organic solvents to form an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) filtering the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (v) with saponin to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity of 0.05-0.5; thereby producing a homogenous adjuvant formulation.

28. 28. The method of claim 27, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof.

29. 29. The method of claim 28, wherein the saponin is QS-21.

30. 28. The method of claim 27, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, stirring or a combination thereof.

31. 31. The method of claim 30, wherein the organic solvent is ethanol or isopropyl alcohol or other organic solvent.

32. The process according to claim 30, wherein the organic phase is heated to a temperature between 45°C and 65°C, preferably between 50°C and 65°C or between 45°C and 55°C.

33. 28. The method of claim 27, wherein the aqueous phase comprises water or a buffer.

34. 34. The method of claim 33, wherein the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl.

35. 34. The method of claim 33, wherein the aqueous phase is at a temperature between 20°C and 65°C.

36. 28. The method of claim 27, wherein the flow rate in step (ii) is 12 mL / min.

37. 28. The method of claim 27, wherein the intermediate liposomes of step (vi) are stirred at a speed of 100 rpm to 1000 rpm.

38. 28. The process of claim 27, wherein the ratio of organic phase to aqueous phase in step (ii) is in the range of 1:2 to 1:

16.

39. 28. The method of claim 27, wherein the removal of the organic phase of the intermediate liposomes in step (iv) is by tangential flow filtration (TFF).

40. 40. The method of claim 39, wherein the TFF is TFF diafiltration.

41. 41. The method of claim 40, wherein the TFF comprises a membrane with a molecular weight cut off (MWCO) in the range of 100-500 kDa.

42. 28. The method of claim 27, wherein the concentrating in step (iii) is by ultrafiltration.

43. 28. The method of claim 27, wherein the filtration of step (v) comprises a bioburden reduction filter and a sterilizing filter.

44. 28. The method of claim 27, wherein the blending in step (vi) is at room temperature for 1 hour with a mixing speed of 350 rpm.

45. 28. The method of claim 27, wherein the final adjuvant formulation has a size range of about 50 nm to 200 nm.

46. 28. The method of claim 27, wherein the final adjuvant formulation has a polydispersity of 0.05 to 0.

5.

47. 28. The method of claim 27, wherein the injecting in step (ii) is by pump or syringe injection.

48. 1. A method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising: (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising: (i) at least one phospholipid selected from at least one phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the at least one phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid solution as an organic phase; (ii) injecting the organic and aqueous phases of step (i) together at a specific flow rate and in a specific ratio of organic solvent phase to aqueous phase to form liposomes; (iii) concentrating the intermediate liposomes of step (ii); (iv) removing the organic phase of the intermediate liposomes of step (iii); (v) treating the intermediate liposomes of step (iv) with a microfluidizer at about 17,500 psi for 10 passes; (vi) filtering the intermediate liposomes of step (v) using a 0.22 um membrane; (vii) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

49. 49. The method of claim 48, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof.

50. 50. The method of claim 49, wherein the saponin is QS-21.

51. 49. The method of claim 48, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, stirring or a combination thereof.

52. 52. The method of claim 51 , wherein the organic solvent is ethanol or isopropyl alcohol or a mixture thereof.

53. 52. The method of claim 51 , wherein the lipid formulation organic phase is heated to a temperature of from 45° C. to 65° C.

54. 49. The method of claim 48, wherein the aqueous phase comprises water or a buffer.

55. 55. The method of claim 54, wherein the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl.

56. 55. The method of claim 54, wherein the aqueous phase is at a temperature between 45°C and 60°C.

57. 49. The method of claim 48, wherein in step (ii), the organic phase has a flow rate of 1.333 ml / min and the aqueous phase has a flow rate of 10.667 ml / min (organic:aqueous = 1:8, using two syringes).

58. 49. The method of claim 48, wherein the intermediate liposomes of step (iii) are stirred at a speed of 100 rpm to 900 rpm.

59. 49. The process of claim 48, wherein the ratio of organic phase to aqueous phase in step (ii) is in the range of 1:4 to 1:

8.

60. 49. The method of claim 48, wherein the removal of the organic phase of the intermediate liposomes in steps (iii) and (iv) is by tangential flow filtration (TFF).

61. 61. The method of claim 60, wherein the size of the intermediate liposomes in step (iv) is manipulated by using a microfluidizer.

62. 49. The method of claim 48, wherein the organic solvent is removed prior to the microfluidizing of step (v).

63. 61. The method of claim 60, wherein the TFF is TFF ultrafiltration and diafiltration.

64. 61. The method of claim 60, wherein the TFF comprises a membrane with a molecular weight cut off (MWCO) in the range of 100-500 kDa.

65. 49. The method of claim 48, wherein the concentrating in step (iii) is by ultrafiltration.

66. 49. The method of claim 48, wherein a buffer is added to the formulation of step (vi).

67. 49. The method of claim 48, wherein the combining in step (vii) is at room temperature for 1 hour to 48 hours with a mixing speed of 350 rpm or is stirred for 1 hour.

68. 68. The method of claim 67, wherein after the compounding of step (vii), the intermediate liposomes are stored at room temperature without stirring for up to 48 hours.

69. 49. The method of claim 48, wherein the final adjuvant formulation has a size of about 300-1000 nm.

70. 49. The method of claim 48, wherein the final adjuvant formulation has a polydispersity of 0.4 to 1.

71. 49. The method of claim 48, wherein the injecting in step (ii) is by Nanoassemblr or pump or syringe injecting.

72. 1. A method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising: (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising: (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the at least one phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) dissolving phospholipids, cholesterol and MPLA in an organic solvent to form a lipid formulation as an organic phase; (ii) injecting the organic phase of step (i) into an aqueous phase at a specific flow rate and a specific ratio of organic phase to aqueous phase to form liposomes; (iii) agitating the liposomes of step (ii) to form intermediate liposomes; (iv) concentrating the intermediate liposomes of step (iii); (v) removing the organic phase of the intermediate liposomes of step (iv); (vi) combining the intermediate liposomes of step (vi) with saponin; thereby producing a heterogeneous adjuvant formulation.

73. 73. The method of claim 72, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof.

74. 74. The method of claim 73, wherein the saponin is QS-21.

75. 73. The method of claim 72, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, stirring or a combination thereof.

76. 76. The method of claim 75, wherein the organic solvent comprises ethyl acetate and isopropyl alcohol.

77. 76. The method of claim 75, wherein the lipid formulation organic phase is heated to a temperature of from 50°C to 65°C.

78. 73. The method of claim 72, wherein the aqueous phase comprises water or a buffer.

79. 79. The method of claim 78, wherein the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl.

80. 79. The method of claim 78, wherein the aqueous phase is at a temperature of from 20°C to 25°C.

81. 73. The method of claim 72, wherein the flow rate in step (ii) is 20 mL / min.

82. 73. The method of claim 72, wherein the intermediate liposomes of step (iii) are stirred at a speed of 100 rpm to 900 rpm.

83. 73. The process of claim 72, wherein the ratio of organic phase to aqueous phase in step (ii) is in the range of 1:4 to 1:

8.

84. 73. The method of claim 72, wherein the organic solvent is removed prior to combining in step (vi).

85. 73. The method of claim 72, wherein the removal of the organic phase of the intermediate liposomes in steps (iv) and (v) is by tangential flow filtration (TFF).

86. 86. The method of claim 85, wherein the TFF is TFF ultrafiltration and diafiltration.

87. 86. The method of claim 85, wherein the TFF comprises a membrane with a molecular weight cut off (MWCO) in the range of 100-500 kDa.

88. 73. The method of claim 72, wherein the concentrating in step (vi) is by ultrafiltration.

89. 73. The method of claim 72, wherein a buffer is added to the formulation of step (vi).

90. 73. The method of claim 72, wherein the blending in step (vi) is at room temperature for 1 hour with a mixing speed of 300 rpm.

91. 91. The method of claim 90, wherein after the compounding in step (vii), the intermediate liposomes are stored at room temperature without stirring for 24 hours.

92. 73. The method of claim 72, wherein the final adjuvant formulation has a size range of 300 nm to 1000 nm.

93. 73. The method of claim 72, wherein the final adjuvant formulation has a polydispersity of 0.4 to 1.

0.

94. 73. The method of claim 72, wherein the injecting in step (ii) is by pipette, pump or syringe injection.

95. 1. A method for producing a heterogeneous adjuvant formulation comprising a liposomal bilayer comprising: (a) monophosphoryl lipid A (MPLA); (b) saponin; and (c) a liposomal composition comprising: (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the at least one phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and combinations thereof; and (ii) cholesterol, wherein the molar percent concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol); The method further comprising: (i) preparing a lyophilized organic phase comprising phospholipids, cholesterol and MPLA; (ii) rehydrating the lyophilized organic phase of step (i) with an aqueous phase to form intermediate liposomes; (iii) downsizing the intermediate liposomes of step (ii) using a microfluidizer; (iv) combining the intermediate liposomes of step (iii) with saponin; thereby producing a heterogeneous adjuvant formulation.

96. 96. The method of claim 95, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21, or a mixture thereof.

97. 97. The method of claim 96, wherein the saponin is QS-21.

98. 96. The method of claim 95, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, stirring or a combination thereof.

99. 99. The method of claim 98, wherein the organic solvent comprises tert-butyl alcohol (TBA) or a mixture thereof.

100. 100. The method of claim 99, wherein the lipid organic phase is heated to a temperature between 25°C and 65°C.

101. 101. The method of claim 100, wherein the organic phase solution is freeze-dried.

102. 96. The method of claim 95, wherein the aqueous phase comprises water or a buffer.

103. 103. The method of claim 102, wherein the buffer comprises 10 mM phosphate at pH 6.2 containing 150 mM NaCl.

104. 103. The method of claim 102, wherein the aqueous phase is at a temperature of from 20°C to 70°C.

105. 96. The method of claim 95, wherein the intermediate liposomes of step (ii) are stirred at a speed of 100 rpm to 1000 rpm.

106. 96. The method of claim 95, wherein the size of the intermediate liposomes in step (iii) is downsized with a microfluidizer and a pressure of 18,640 PSI.

107. 96. The method of claim 95, wherein the organic solvent is removed prior to the rehydration of step (ii).

108. 96. The method of claim 95, wherein the organic solvent is removed by lyophilization.

109. 96. The method of claim 95, wherein a buffer is added to the formulation of step (iv).

110. 96. The method of claim 95, wherein the blending in step (iv) is at room temperature for 1 hour at a mixing speed of 300 rpm or with stirring.

111. 111. The method of claim 110, wherein after the compounding in step (iv), the intermediate liposomes are stored at room temperature without stirring for 24 hours.

112. 96. The method of claim 95, wherein the final adjuvant formulation has a size >300 nm.

113. 96. The method of claim 95, wherein the final adjuvant formulation has a polydispersity > 0.

4.

114. 114. An adjuvant formulation produced by any one of the methods of claims 1 to 113.

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