Method for preparing adjuvant

Abstract

The present invention provides a method of preparing a homogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) a monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), and (ii) cholesterol, the liposomal bilayer comprising (a) a monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposomal composition comprising (i) at least one phospholipid selected from the group consisting of phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), and combinations thereof, and wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) wherein the cholesterol has a molar percentage concentration of greater than 50% (mol / mol) in the liposome composition, and a homogeneous adjuvant formulation prepared from the method.

Description

Technical Field

[0001] The present invention provides a method for preparing a homogeneous adjuvant formulation comprising a liposomal bilayer comprising (a) a monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG), wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, and (ii) cholesterol, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol), and the present invention further provides a homogeneous adjuvant formulation prepared by the method. Background Technology

[0002] A commercially available liposomal adjuvant formulation (designated AS01) containing both monophosphoryl lipid A (MPLA) and QS-21 saponin was used in the United States in 2017 as an adjuvant for the shingles vaccine in adults over 50 years of age. The U.S. Army has developed another liposomal formulation containing both MPLA and QS-21 saponin, designated ALFQ (Army Liposome Formulation Q). ALFQ has demonstrated efficacy as a liposomal vaccine adjuvant in rodent and non-human primate studies, and has also shown to be pyrogen-free and non-toxic in preclinical studies. The improved safety profile of ALFQ is attributed to the irreversible binding of liposomal cholesterol to free QS-21 to form a complex that prevents hemolysis caused by QS-21 binding to erythrocytes. When soluble QS-21 is added to a suspension of liposome intermediates, the ALFQ size increases from 50 to 100 nm to approximately 30,000 nm during ALFQ fabrication. As described in the literature, ALFQ liposome intermediates are prepared via a rehydration method (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 this method, lipids are mixed and dissolved in an organic solvent, dried under vacuum, and then liposomes are formed in PBS and their size is reduced to 30 to 100 nm using a microfluidizer. In the most widely used methods, a lipid film (from an organic solvent) is deposited on the container wall, an aqueous solution of the material to be encapsulated is added, and the container is stirred (Bangham et al., J. Mol. Biol. 13 (1965) 238-252). Under suitable conditions, this method results in the formation of multilayered microliposomes of liposomes. However, due to the size limitations of the equipment used (i.e., rotary evaporators, such as Rotavap), this method is generally not scalable for manufacturing.

[0003] In another method, an organic solution of lipids is freeze-dried to obtain a freeze-dried product with the physical property of readily hydrating with an aqueous solution of the material to be encapsulated. This manufacturing method is limited by the batch-to-batch variability in drug encapsulation in liposomes. For example, Conrad et al. (Biochim. Biophys. Acta 332 (1974) 36-46) demonstrated a standard deviation of 12% to 13% in encapsulation efficiency between liposome formulations prepared independently using this method.

[0004] Therefore, there is a need for reproducible methods that can control the size and polydispersity of liposome adjuvant formulations containing MPLA and saponins (including but not limited to QS-21). Additionally, there is a need for methods that are robust enough for large-scale use (i.e., scalable for clinical and commercial manufacturing). Summary of the Invention

[0005] This invention provides a method for preparing homogeneous adjuvant formulations as described herein, which is 1) more reproducible and fully controlled, enabling the production of homogeneous liposome adjuvant formulations with defined size and polydispersity, and 2) more scalable than conventional batch methods (such as those described above, e.g., rehydration) or batch mixing. This invention leverages the advantages of semi-continuous flow chemistry, namely, by forming liposomes in a microfluidic mixer, where process parameters such as chemical composition, flow state, flow rate, and temperature can be precisely controlled to obtain reproducible products (i.e., homogeneous). The semi-continuous flow method is more scalable because it is not limited by container size (e.g., as in the example of a rotary evaporator), but rather allows for the addition of more microfluidic mixers in parallel, and the volumetric flow rate to be increased as needed to achieve desired clinical or commercial-scale yields. These advantages enable the establishment of process control strategies that comply with FDA guidelines for Good Manufacturing Practices (CGMP), including both material quality inspection and equipment monitoring, to ensure that materials and finished products meet predetermined quality requirements and are thus consistently and reliably achieved during the manufacturing process (see https: / / www.fda.gov / files / drugs / published / Process-Validation--General-Principles-and-Practices.pdf, pages 5 and 9). Another advantage of this invention is that the liposome adjuvant formulations formed according to the methods described herein do not require additional size reduction steps, unlike those described in PCT International Application No. PCT / IB2023 / 052255. Accordingly, this invention provides a reproducible method for large-scale (i.e., scalable) manufacturing of quantities sufficient for clinical and commercial use, which controls the size and polydispersity of homogeneous liposome adjuvant formulations containing MPLA and saponins.

[0006] The present invention provides a first method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, the liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase into the aqueous phase from step (ii) to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0007] In one embodiment, the first method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase into the aqueous phase from step (ii) to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0008] The present invention also provides a second method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) The intermediate liposomes from step (ii) are mixed with saponin, wherein the saponin is first dissolved in a buffer solution or water to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0009] In one embodiment, the second method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) The intermediate liposomes from step (ii) are mixed with saponin, wherein the saponin is first dissolved in a buffer solution or water to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0010] The present invention further provides a third method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, the liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the 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, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) Remove the organic phase of the intermediate liposome from step (ii); (iv) Concentrate the intermediate liposome from step (iii); (v) The intermediate liposome from step (iv) is sterile filtered; (vi) Sterilely filter the saponin; and (vii) The intermediate liposomes from step (v) are sterilized and mixed with the saponin from step (vi) to form a final adjuvant formulation having a size range of about 30 to 400 nm and a polydispersity index of 0.05 to 0.50. Homogeneous adjuvant formulations were thus prepared.

[0011] In one embodiment, the third method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) Remove the organic phase of the intermediate liposome from step (ii); (iv) Concentrate the intermediate liposome from step (iii); (v) The intermediate liposome from step (iv) is sterile filtered; (vi) Sterilely filter the saponin; and (vii) The intermediate liposomes from step (v) are sterilized and mixed with the saponin from step (vi) to form a final adjuvant formulation having a size range of about 30 to 400 nm and a polydispersity index of 0.05 to 0.50. Homogeneous adjuvant formulations were thus prepared.

[0012] The present invention also provides adjuvant formulations prepared by any of the methods described herein. Attached Figure Description

[0013] Figure 1 A schematic diagram showing a method for forming homogeneous liposome adjuvants, wherein saponins are added to the aqueous phase.

[0014] Figure 2 A schematic diagram showing a method for forming homogeneous liposome adjuvants, wherein saponins are added online to intermediate liposomes.

[0015] Figure 3 This provides an overview of the method for manufacturing homogeneous liposome adjuvants as described in Example 3.

[0016] Figure 4 Depicting Clostridium difficile formulated with different LiNA-2 adjuvants (homogeneous and heterogeneous) C. difficile The neutralizing titer in individual animals (rats) immunized with toxoid antigens was as described in Example 5.

[0017] Figure 5 A schematic cross-sectional view of the coaxial mixer in the axial plane.

[0018] Figure 6 Dynamic light scattering (DLS) response surface plots are depicted using data obtained from intermediate liposomes prepared via a coaxial mixer, as described in Example 6. In this figure, the z-mean particle size is depicted as a function of the total flow rate relative to the aqueous phase:organic phase ratio (v / v). Detailed Implementation

[0019] This invention relates to a method for preparing a homogeneous liposome adjuvant formulation, the adjuvant formulation comprising a liposome composition containing monophosphoryl lipid A (MPLA) and at least one saponin (e.g., QS-21). This adjuvant formulation comprises a liposome composition containing monophosphoryl lipid A (MPLA) and at least one saponin, wherein the liposome composition comprises i) a lipid bilayer containing phospholipids and ii) cholesterol, wherein the molar percentage concentration of the liposome composition is greater than about 50% (mol / mol), and wherein the homogeneous liposome adjuvant formulation has a size range of about 30 to 400 nm and a polydispersity index of 0.5 to 5.0. The saponin may be selected from QS-7, QS-18, QS-21, or mixtures thereof. The saponin is preferably QS-21. Therefore, the homogeneous adjuvant formulation comprises a liposome composition containing monophospholipid A (MPLA) and at least one saponin, wherein the liposome composition comprises i) a lipid bilayer containing phospholipids and ii) cholesterol, wherein the molar ratio of cholesterol to phospholipids is greater than about 1, and further wherein the homogeneous liposome adjuvant formulation has a size range of about 30 to 400 nm and a polydispersity index of 0.5 to 5.0. The saponin may be selected from QS-7, QS-18, QS-21, or mixtures thereof. The saponin is preferably QS-21.

[0020] This article describes several methods for preparing homogeneous liposome adjuvant formulations, which are scalable for manufacturing. The homogeneous method is a controlled and robust approach for producing size-controlled liposomes.

[0021] Examples 1 through 3 describe scalable methods for manufacturing homogeneous liposome adjuvant formulations. The methods described herein produce homogeneous formulations of liposomes having a size range of less than 1 micrometer and a controlled polydispersity index (PDI) range of 0.05 to 0.5. The methods described herein are reproducible and fully controlled, enabling the manufacture of homogeneous liposome adjuvant formulations with defined sizes.

[0022] This document also describes a multi-solvent injection method via microfluidics. As used herein, “microfluidic mixing” refers to the thorough and rapid mixing of multiple streams in a microscale device (such as microchannels within a microfluidic mixer). In such devices, sample mixing is achieved primarily by enhancing the diffusion effect between different types of streams (e.g., organic and aqueous phases) to prepare intermediate liposomes or final liposome adjuvant drug products. This method involves mixing a lipid solution in ethanol or other organic solvents (e.g., isopropanol) with an aqueous buffer. In Example 1, saponin is dissolved in an aqueous phase to form the final liposome adjuvant, as a continuous process. In Example 2, saponin in a buffer solution is added online with intermediate liposomes formed from mixing the organic and aqueous phases to form the final liposome adjuvant, as a continuous process. In Example 3, intermediate liposomes are aseptically combined (i.e., mixed) with saponin QS-21 in a buffer solution to form the final adjuvant drug product. These processes are performed at room temperature. The resulting intermediate liposomes have a size of 30 nm to 400 nm and a PDI of <0.5. The size and PDI of the intermediate and final adjuvant drug products can be controlled by adjusting parameters of the solvent injection, such as temperature, flow rate or the ratio of organic to aqueous phase flow rate and mixer type.

[0023] Overall, this article provides a novel method for preparing homogeneous adjuvant formulations comprising an MPLA-containing liposomal composition containing a saponin (e.g., QS-21). Compared to conventional methods, these methods offer control over the size and polydispersity of the liposomal adjuvant formulations, are readily scalable for clinical and commercial manufacturing, and are reproducible. Furthermore, this article provides novel MPLA-containing homogeneous liposomal compositions containing a saponin (e.g., QS-21).

[0024] Accordingly, the present invention provides a first method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, the liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase into the aqueous phase from step (ii) to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0025] In one embodiment of the first method for preparing 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.

[0026] In another embodiment of the first method for preparing the homogeneous adjuvant composition, the amount of saponin is from about 0.07 mg / ml to about 0.35 mg / ml. In one aspect, the amount of saponin is about 0.07 mg / ml. In a preferred aspect, the amount of saponin is 0.07 mg / ml.

[0027] In another embodiment of the first method for preparing a homogeneous adjuvant composition, the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0028] In another embodiment of the first method for preparing a homogeneous adjuvant composition, in step (i), phospholipids, cholesterol, and MPLA are dissolved in an organic solvent by ultrasonication, heating, or a combination thereof, preferably by heating. In one aspect, the organic solvent is ethanol or isopropanol. In another aspect, the organic phase is heated to a temperature between 45°C and 65°C. In one aspect, the organic phase is heated at 65°C.

[0029] In one embodiment of the first method for preparing a homogeneous adjuvant composition, the buffer solution in step (ii) comprises a chelating agent, such as EDTA. In one embodiment of the first method for preparing a homogeneous adjuvant composition, the buffer solution in step (ii) comprises phosphate-buffered saline (PBS). In one embodiment of the first method for preparing a homogeneous adjuvant composition, the buffer solution in step (ii) comprises phosphate-buffered saline (PBS) and EDTA. In another embodiment of the first method for preparing a homogeneous adjuvant composition, the buffer solution in step (ii) comprises 10 mM phosphate and 150 mM NaCl at pH 6.2. In another embodiment of the first method for preparing a homogeneous adjuvant composition, the buffer solution in step (ii) has a pH of 6.2 and comprises 10 mM phosphate, 150 mM NaCl, and EDTA.

[0030] In another aspect, the aqueous phase is at a temperature between 20°C and 25°C. In another aspect, the flow rate of step (ii) is between 12 mL / min and 240 mL / min or up to 3 L / min.

[0031] In another embodiment of the first method for preparing the homogeneous adjuvant composition, the mass ratio of water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:1. In a preferred embodiment, the mass ratio is 3:1.

[0032] In another embodiment of the first method for preparing the homogeneous adjuvant composition, the microfluidic mixer in step (iii) is injected using a pump or syringe. In one aspect, the microfluidic mixer in step (iii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer. In another aspect, the microfluidic mixer in step (iii) has an inner diameter ranging from 300 µm to 1,000 µm.

[0033] In another embodiment of the first method for preparing a homogeneous adjuvant composition, the removal of the organic phase containing saponin liposomes in step (iv) is performed by tangential flow filtration (TFF). In another aspect, TFF is TFF percolation. In another embodiment of the first method for preparing a homogeneous adjuvant composition, the concentration in step (v) is performed by TFF, wherein the TFF comprises percolation, ultrafiltration, or both.

[0034] In another aspect, TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa. In one aspect, the membrane is a hollow fiber membrane, a cassette membrane, or a rotary centrifuge membrane.

[0035] In another embodiment of the first method for preparing the homogeneous adjuvant composition, the filtration in step (vi) includes a bioload-reducing filter and a sterile filter. In one aspect, the bioload-reducing filter has a diameter of 0.45 micrometers. In another aspect, the sterile filter has a diameter of 0.22 micrometers.

[0036] In another embodiment of the first method for preparing a homogeneous adjuvant composition, the concentration in step (v) and the filtration in step (vi) occur at room temperature.

[0037] In one embodiment, the first method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase into the aqueous phase from step (ii) to form saponin-containing liposomes. (iv) Remove the organic phase containing the saponin liposomes from step (iii); and (v) The saponin-containing liposomes from step (iv) are concentrated to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0038] In one embodiment, the first method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase into the aqueous phase from step (ii) to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0039] The present invention also provides a second method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) The intermediate liposomes from step (ii) are mixed with saponin, wherein the saponin is first dissolved in a buffer solution or water to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0040] In one embodiment of the second method for preparing the 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.

[0041] In another embodiment of the second method for preparing a homogeneous adjuvant composition, the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0042] In another embodiment of the second method for preparing a homogeneous adjuvant composition, in step (i), phospholipids, cholesterol, and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating, or a combination thereof, preferably by heating. In one aspect, the organic solvent is ethanol or isopropanol. In another aspect, the organic phase is heated to a temperature between 45°C and 65°C. In one aspect, the organic phase is heated at 65°C.

[0043] In one embodiment of the second method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains a chelating agent, such as EDTA. In one embodiment of the second method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains phosphate-buffered saline (PBS). In one embodiment of the second method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains phosphate-buffered saline (PBS) and EDTA. In another embodiment of the second method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains 10 mM phosphate and 150 mM NaCl at pH 6.2. In another embodiment of the second method for preparing a homogeneous adjuvant composition, the buffer in step (ii) has a pH of 6.2 and contains 10 mM phosphate, 150 mM NaCl, and EDTA.

[0044] In another aspect, the aqueous phase in step (ii) is at a temperature between 20°C and 25°C. In another aspect, the flow rate in step (ii) is between 12 mL / min and 240 mL / min or up to 3 L / min.

[0045] In another embodiment of the second method for preparing the homogeneous adjuvant composition, the mass ratio of water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:1. In a preferred embodiment, the mass ratio is 3:1.

[0046] In another embodiment of the second method for preparing the homogeneous adjuvant composition, the microfluidic mixer in step (ii) is injected using a pump or syringe. In one aspect, the microfluidic mixer in step (ii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer. In another aspect, the microfluidic mixer in step (ii) has an inner diameter ranging from 300 µm to 1,000 µm.

[0047] In another embodiment of the second method for preparing a homogeneous adjuvant composition, the removal of the organic phase containing saponin liposomes in step (iv) is performed by tangential flow filtration (TFF). In another aspect, the TFF is TFF percolation. In another embodiment of the second method for preparing a homogeneous adjuvant composition, the concentration in step (v) is performed by TFF, wherein the TFF comprises percolation, ultrafiltration, or both.

[0048] In another aspect, TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa. In one aspect, the membrane is a hollow fiber membrane, a boxed membrane, or a rotary centrifuge membrane.

[0049] In another embodiment of the second method for preparing the homogeneous adjuvant composition, the filtration in step (vi) includes a bioload-reducing filter and a sterile filter. In one aspect, the bioload-reducing filter has a diameter of 0.45 micrometers. In another aspect, the sterile filter has a diameter of 0.22 micrometers.

[0050] In another embodiment of the second method for preparing a homogeneous adjuvant composition, the concentration in step (v) and the filtration in step (vi) occur at room temperature.

[0051] In one embodiment, the second method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) The intermediate liposomes from step (ii) are mixed with saponin, wherein the saponin is first dissolved in a buffer solution or water to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0052] The present invention further provides a third method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, the liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) Remove the organic phase of the intermediate liposome from step (ii); (iv) Concentrate the intermediate liposome from step (iii); (v) The intermediate liposome from step (iv) is sterile filtered; (vi) Sterilely filter the saponin; and (vii) The intermediate liposomes from step (v) are sterilized and mixed with the saponin from step (vi) to form a final adjuvant formulation having a size range of about 30 to 400 nm and a polydispersity index of 0.05 to 0.50. Homogeneous adjuvant formulations were thus prepared.

[0053] In one embodiment of the third method for preparing the 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.

[0054] In another embodiment of the third method for preparing a homogeneous adjuvant composition, the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0055] In another embodiment of the third method for preparing a homogeneous adjuvant composition, in step (i), phospholipids, cholesterol, and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating, or a combination thereof, preferably by heating. In one aspect, the organic solvent is ethanol or isopropanol. In another aspect, the organic phase is heated to a temperature between 45°C and 65°C. In one aspect, the organic phase is heated at 65°C.

[0056] In one embodiment of the third method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains a chelating agent, such as EDTA. In one embodiment of the third method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains phosphate-buffered saline (PBS). In one embodiment of the third method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains phosphate-buffered saline (PBS) and EDTA. In another embodiment of the third method for preparing a homogeneous adjuvant composition, the buffer in step (ii) contains 10 mM phosphate and 150 mM NaCl at pH 6.2. In another embodiment of the third method for preparing a homogeneous adjuvant composition, the buffer in step (ii) has a pH of 6.2 and contains 10 mM phosphate, 150 mM NaCl, and EDTA.

[0057] In another aspect, the aqueous phase in step (ii) is at a temperature between 20°C and 25°C. In another aspect, the flow rate in step (ii) is between 12 mL / min and 240 mL / min or up to 3 L / min.

[0058] In another embodiment of the third method for preparing the homogeneous adjuvant composition, the mass ratio of water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:1. In a preferred embodiment, the mass ratio is 3:1.

[0059] In another embodiment of the third method for preparing the homogeneous adjuvant composition, the microfluidic mixer in step (ii) is injected using a pump or syringe. In one aspect, the microfluidic mixer in step (ii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer. In another aspect, the microfluidic mixer in step (ii) has an inner diameter ranging from 300 µm to 1,000 µm.

[0060] In another embodiment of the third method for preparing the homogeneous adjuvant composition, the organic phase of the removal of the intermediate liposomes in step (iii) is achieved by tangential flow filtration (TFF). In another aspect, the TFF is TFF percolation, ultrafiltration, or both. In yet another aspect, the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa. In one aspect, the membrane is a hollow fiber membrane, a boxed membrane, or a rotary centrifuge membrane.

[0061] In another embodiment of the third method for preparing the homogeneous adjuvant composition, the aseptic filtration in steps (v) and (vi) includes a bioload reduction filter and a sterile filter. In one aspect, the bioload reduction filter has a diameter of 0.45 micrometers. In another aspect, the sterile filter has a diameter of 0.22 micrometers.

[0062] In one embodiment, the third method prepares a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1. The method comprises the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) Remove the organic phase of the intermediate liposome from step (ii); (iv) Concentrate the intermediate liposome from step (iii); (v) The intermediate liposome from step (iv) is sterile filtered; (vi) Sterilely filter the saponin; and (vii) The intermediate liposomes from step (v) are sterilized and mixed with the saponin from step (vi) to form a final adjuvant formulation having a size range of about 30 to 400 nm and a polydispersity index of 0.05 to 0.50. Homogeneous adjuvant formulations were thus prepared.

[0063] The present invention also provides homogeneous adjuvant formulations prepared by any of the methods described herein.

[0064] In one aspect, the liposome composition of the adjuvant formulation may contain cholesterol at a molar percentage concentration of more than 50% (mol / mol), about 55% to about 71% (mol / mol), or preferably about 55% (mol / mol). In another aspect, the liposome composition of the adjuvant formulation may contain phosphatidylcholine phosphatidylcholine (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearatel phosphatidylcholine (DSPC). In another aspect, the liposome composition of the adjuvant formulation may contain phosphatidylglycerol phosphatidylcholine (PG) selected from the group consisting of dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearatel phosphatidylglycerol (DSPG). In a further aspect, the liposome composition of the adjuvant formulation may comprise a combination of: (i) phosphatidylcholine phosphatidylcholine (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC) and distearyl phosphatidylcholine (DSPC), and (ii) phosphatidylglycerol phosphatidylcholine (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 multilayered liposomes (MLVs) or small monolayered liposomes (SUVs), wherein the small monolayered liposomes have a diameter of about 50 to about 100 nm, and wherein the multilayered liposomes have a diameter of about 30 nm to about 400 nm. In a preferred embodiment, the liposome composition of the adjuvant formulation comprises PC and PG, wherein PC is dimyristoyl phosphatidylcholine (DMPC) and PG is dimyristoyl phosphatidylglycerol (DMPG), having a PC to PG molar ratio (mol / mol) of approximately 9:1.

[0065] In another aspect, the liposome composition of the adjuvant formulation may contain 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 MPLA (total weight per ml of liposome suspension). The liposome composition of the adjuvant formulation may have a molar ratio of MPLA to phospholipid 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, wherein PC is dimyristoylphosphatidylcholine (DMPC) and PG is dimyristoylphosphatidylglycerol (DMPG), having a molar ratio of MPLA to phospholipid of about 1:220, about 1:88, or about 1:5.6, preferably 1:88.

[0066] In a further aspect, 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 aspect, the adjuvant formulation contains a saponin content of about 0.15 to 0.4 mg / ml.

[0067] In another embodiment, the adjuvant formulation is a homogeneous adjuvant composition comprising liposomes having a size range between about 1 nm and about 500 nm. In some embodiments, the liposomes within the adjuvant formulation have a size range between about 10 nm, about 20 nm, about 30 nm, about 40 nm, or about 50 nm and about 400 nm. In other embodiments, the liposomes within the adjuvant formulation have a size range between about 10 nm, about 20 nm, about 30 nm, about 40 nm, or about 50 nm and about 300 nm. In other embodiments, the liposomes within the adjuvant formulation have a size range between about 10 nm, about 20 nm, about 30 nm, about 40 nm, or about 50 nm and about 200 nm. In some embodiments, the liposomes within the adjuvant formulation have a size less than about 300 nm, about 250 nm, about 200 nm, about 150 nm, or about 100 nm. In a particular embodiment, the liposomes within the adjuvant formulation have a size of less than about 200 nm. In another particular embodiment, the liposomes within the adjuvant formulation have a size between about 100 nm and about 150 nm.

[0068] In a further aspect, the adjuvant formulation is a homogeneous adjuvant composition comprising liposomes having a polydispersity index (PDI) between about 0.05, about 0.1, about 0.015, or about 0.2 and about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5. In some embodiments, the adjuvant formulation comprises liposomes having a PDI of less than about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5. In a particular embodiment, the liposomes within the adjuvant formulation have a PDI of less than about 0.3. In another particular embodiment, the liposomes within the adjuvant formulation have a PDI between about 0.05 and about 0.2.

[0069] In another aspect, the adjuvant formulation is a heterogeneous adjuvant composition comprising liposomes having a size range between about 1 nm and about 10 µM. In some embodiments, the liposomes within the adjuvant formulation have a size range between about 30 nm and about 4 µM. In other embodiments, the liposomes within the adjuvant formulation have a size range between about 30 nm and about 1400 nm. In still other embodiments, the liposomes within the adjuvant formulation have a size range between about 30 nm and about 1000 nm. In some embodiments, the liposomes within the adjuvant formulation have a size range between about 100 nm, about 200 nm, about 300 nm, about 400 nm, or about 500 nm and about 1000 nm. In a particular embodiment, the liposomes within the adjuvant formulation have a size range between about 300 nm and about 1000 nm. In other embodiments, the liposomes within the adjuvant formulation have a size greater than about 500 nm, about 400 nm, about 300 nm, about 200 nm, or about 100 nm. In a particular embodiment, the liposomes within the adjuvant formulation have a size greater than 300 nm.

[0070] In another embodiment, the adjuvant formulation is a heterogeneous adjuvant composition comprising liposomes having a polydispersity index (PDI) between about 0.4 and about 1. In some embodiments, the adjuvant formulation comprises liposomes having a PDI of about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, or greater. In a particular embodiment, the liposomes within the adjuvant formulation have a PDI greater than about 0.4. In another particular embodiment, the liposomes within the adjuvant formulation have a PDI greater than about 0.5.

[0071] The present invention also provides immunizing compositions comprising an immunogen and said adjuvant formulation. Immunizing compositions may generally contain a physiologically acceptable mediator. The immunogen of the immunizing composition may be selected from the group consisting of naturally occurring or artificially produced proteins, recombinant proteins, glycoproteins, peptides, carbohydrates, haptens, intact viruses, bacteria, protozoa, and virus-like particles. The present invention also provides a method of immunizing an animal, comprising administering an immunizing composition.

[0072] The present invention further provides a method for reducing the toxicity of saponins as adjuvants or for preparing adjuvant formulations, comprising adding a liposome composition containing monophosphoryl lipid A (MPLA) to saponins, wherein the liposome composition comprises i) a lipid bilayer containing phospholipids, wherein the hydrocarbon chain has a melting temperature of ≥23°C in water, and ii) cholesterol having a molar percentage concentration greater than about 50% (mol / mol). The saponins may be selected from the group consisting of QS-7, QS-18, QS-21, and mixtures thereof. QS-21 is preferred. The liposome composition may contain cholesterol having a molar percentage concentration of about 55% to about 71% (mol / mol), preferably about 55% (mol / mol).

[0073] 1. Definition An "immunogen" is an agent capable of inducing a humoral and / or cellular immune response. Immunogens as described herein can be antigens, haptens, or inactivated pathogens. Immunogenic compositions as described herein can be, for example, vaccine formulations.

[0074] As used herein, the term "homogeneous" should mean a final adjuvant formulation comprising liposomes having a size range of about 30 nm to 400 nm, determined by methods known in the art, including but not limited to dynamic light scattering (DLS), transmission electron microscopy or cryo-electron microscopy (e.g., cryo-TEM or cryo-EM), and nanoparticle tracking analysis (NTA, e.g., ViewSizer). "Homogeneous" adjuvant formulations can also mean a final adjuvant formulation comprising liposomes having a polydispersity index (PDI) between about 0.05 and 0.5, or between about 0.05 and about 0.3, preferably about 0.3. Polydispersity is measured by the polydispersity index (PDI). Calculations for determining size and PDI parameters can be found in ISO standard documents 13321:1996 E and ISO 22412:2008 (Worldwide MI). Dynamic Light Scattering, Common Terms Defined. Malvern Instruments Limited; Malvern, UK: 2011. Pages 1-6, Information White Paper. The term "homogeneous" adjuvant formulation as used herein should also mean "monodispersive" adjuvant formulation.

[0075] The following terms used herein have the same meaning and may be used interchangeably: "homogeneous adjuvant formulation", "homogeneous liposome adjuvant formulation", "homogeneous liposome adjuvant", "homogeneous LiNA-2", "liposome adjuvant drug product", "final adjuvant drug product", "final liposome adjuvant drug product", "final homogeneous adjuvant drug product", "final adjuvant formulation" and the like.

[0076] As used herein, the term "buffer solution" should be understood to mean any solution that resists changes in pH when an acid or base is added thereto. In some embodiments, the buffer solutions disclosed herein contain a chelating agent, such as EDTA.

[0077] As used herein, the term "size" refers to the diameter of a particle or swarm of particles. The diameter can be determined using various methods available in the art, such as dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA, e.g., ViewSizer). In some embodiments, the diameter is provided as the z-mean of the swarm. In some embodiments, the diameter is provided as the average value of the swarm. In some embodiments, the diameter is provided as D10, D50, D90, etc., of the swarm. For example, in some embodiments, the D90 value of the swarm is the value when 90% of the particles in the swarm have a smaller diameter.

[0078] As used herein, "liposome" refers to a closed bilayer membrane containing a volume of captured water. Liposomes can also be monolayered liposomes with a single membrane bilayer or multilayered liposomes with multiple membrane bilayers, each membrane bilayer separated from the next by an aqueous layer. The resulting membrane bilayer structure oriented the hydrophobic (nonpolar) tail of the lipid towards the center of the bilayer, while the hydrophilic (polar) head is oriented towards the aqueous phase. As is conventionally used, liposomes consist of a smectic mesophase, which can be composed of phospholipids or nonphospholipids. The smectic mesophase is most accurately described in Small's *HANDBOOKOF LIPID RESEARCH*, Vol. 4, Plenum, NY, 1986, pp. 49-50. According to Small, "When the given molecules are heated, they do not directly melt into an isotropic liquid, but rather through an intermediate state called an intermediate phase or liquid crystal, characterized by residual order in some directions but lack of order in others. Liquid crystal molecules are typically slightly longer than the width of the molecules and have polar or aromatic portions somewhere along the length of the molecules. The molecular shape and polar-polar or aromatic interactions allow the molecules to align in partially ordered arrays. These structures are characteristically found in molecules with polar groups at one end. Liquid crystals with long-range order along the long axis of the molecules are called smectic phases, layered or lamellar liquid crystals. In the smectic phase state, the molecules can be monolayered or bilayered, orthogonal or tilted to the layer plane, and have frozen or molten aliphatic chains." Lipid A is a complex group of numerous acylated and amidated diglucosamine diphosphate molecules and a lipid moiety common to all lipopolysaccharides (LPS; also known as endotoxins) from Gram-negative bacteria. LPS covers almost the entire outer surface of all Gram-negative bacteria, and lipid A anchors LPS to the bacterial outer lipid surface. In wild-type smooth bacteria, the O-polysaccharide moiety of LPS 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, sometimes a unique chemical structure required for bacterial survival and found only in LPS. See, for example, Alving et al., 2012, Expert Rev. Vaccines 11: 733-44. "Monophosphoryl lipid A" is an analogue of lipid A in which the glucosamine-1-phosphate group at the polar head end has been removed. Numerous analogues of MPLA also exist.

[0079] As used herein, a “microfluidic mixer” can be a Y-joint, T-joint, or coaxial microfluidic mixer. In one aspect, the microfluidic mixer in the step has an inner diameter ranging from 300 µm to 1,000 µm. The microfluidic mixer forms liposomes through a semi-continuous flow process. Examples of microfluidic mixers include, but are not limited to, Y-joint mixers from Precision Nanosystems, T-joint mixers from IDEX, or coaxial mixers, or any other configuration in which turbulence is generated. “Turbulence” should be understood as a flow in which fluids undergo irregular fluctuations or mixing, as opposed to laminar flow where fluids move along a smooth path or in a layer. In turbulence, the fluid velocity at a given point changes continuously in both magnitude and direction.

[0080] In one aspect, the microfluidic mixer used herein is a coaxial mixer as described in International Publication No. WO 2024 / 057209 (Darvari et al.). In one embodiment, the microfluidic mixer used herein is... Figure 5 The coaxial mixer depicted herein represents a coaxial flow device (1) extending along the main longitudinal axis X. Figure 5The coaxial mixer depicted includes a first (outer) tube (3) having an inlet (4) for controlled flow of an organic or aqueous phase and a second (inner) tube (5) having an inlet (6) for controlled flow of another phase, either organic or aqueous. The first tube (3) has a mixing section (7) for continuous mixing of the organic and aqueous phases and an outlet (9) for flow generated from the mixed solution containing the organic and aqueous phases. The second tube (5) is coaxially arranged within the first tube (3) along the longitudinal axis X and has an outlet (10) opening axially toward the mixing section (7) of the first tube (3). The mixing section (7) further includes a controlled micro-mixing environment (15) between the outlet (10) of the second tube (5) and the turbulent mixing section (11) for unobstructed merging flow. The disruptive physical component extends along the mixing section (7) for a certain length, in this case extending from the downstream end of the controlled micro-mixing environment section (15) in the turbulent mixing section (11) to the outlet (9) of the first pipe (3), and includes alternating spiral flow paths (21) arranged in the form of spiral grooves on the inner surface of the first pipe (3).

[0081] The “mole percentage concentration of cholesterol” used in this article refers to the ratio of cholesterol to total phospholipids (i.e., phosphatidylcholine and phosphatidylglycerol) originally used to prepare the liposome composition.

[0082] As used herein, “physiologically acceptable mediator” means a mediator suitable for in vivo administration (e.g., oral, transdermal, or parenteral administration) or in vitro administration (i.e., cell culture). Exemplary physiologically acceptable mediators may be those physiologically acceptable components of liposomes, as disclosed in U.S. Patent Nos. 4,186,183 and 4,302,459.

[0083] As used herein, “preferred” and “preferred” should be interpreted only for the purposes of the syntactic structure of the European patent application. These terms should be read from or omitted from the syntactic structure of sentences and paragraphs in which they appear for the purposes of the syntactic structure of the U.S. patent application.

[0084] As used in this article, “about” means ±5% of the reference value.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. It must be noted that the singular forms “a,” “an,” and “the” used herein include a plurality of indicators unless the context clearly specifies otherwise. Thus, for example, reference to “antibody” includes a plurality of such antibodies, and reference to “dosage” includes one or more dosages known to those skilled in the art and their equivalents, and so on.

[0086] 2. Saponins Regarding embodiments of the present invention, suitable saponins are Quil A, its derivatives, or any purified components thereof (e.g., QS-7, QS-18, QS-21, or mixtures thereof). Quil A is derived from the South American soapberry tree, Molina (Gnaphalium affine). Quillaja Saponaria Molina The saponin preparations isolated from the plant were first found to have adjuvant activity. (Dalsgaard et al., 1974, Archiv. für die gesanite Virusforschung, 44: 243-254). The purified fragments of Quil A have been separated by HPLC (EP 0 362 278), including, for example, QS-7 and QS-21 (also known as QA7 and QA21, respectively). QS-21 is derived from the soapberry tree (…). Quillaja Saponaria The 21st fraction of the purified sap of the tree. QS-21 has shown to induce responses in CD8+ cytotoxic T cells (CTL), Th1 cells, and major IgG2a antibodies.

[0087] 3. Liposomes containing monophosphoryl lipid A (MPLA) (L(MPLA)) Liposomes are closed bilayer membranes containing the volume of captured water. Liposomes can also be monolayered microparticles with a single membrane bilayer or multilayered microparticles with multiple membrane bilayers, each membrane bilayer being separated from the next membrane bilayer by an aqueous layer. The resulting membrane bilayer structure causes the hydrophobic (nonpolar) tail of the lipid to be oriented towards the center of the bilayer, while the hydrophilic (polar) head is oriented towards the aqueous phase. Suitable hydrophilic polymers for surrounding liposomes include, but are not limited to, PEG, polyvinylpyrrolidone, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol, polyaspartamide, and hydrophilic peptide sequences, as described in U.S. Patent Nos. 6,316,024, 6,126,966, 6,056,973, and 6,043,094. Liposomes may not be manufactured using hydrophilic polymers. Therefore, liposome formulations may or may not contain hydrophilic polymers.

[0088] Liposomes may be composed of any lipid or combination of lipids known in the art. For example, the lipids forming microliposomes may be naturally occurring or synthetic lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and myelin, as disclosed in U.S. Patent Nos. 6,056,973 and 5,874,104.

[0089] The lipids that form microlipids can also be glycolipids, cerebrosides, or cationic lipids, such as 1,2-dioleoyloxy-3-(trimethylamino)propane (DOTAP); N-[1-(2,3-di-tetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1(2,3-dioleoyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DCChol); or dimethyl dioctadecylammonium (DDAB), as disclosed in U.S. Patent No. 6,056,973. Cholesterol may also be present in appropriate ranges to impart stability to liposome microparticles, 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 patents describe liposomes and lipid-coated microvesicles, and methods of manufacturing the same. Therefore, in consideration of this disclosure and the disclosures of these other patents, those skilled in the art can prepare liposomes for the purposes of embodiments of the present invention. In embodiments of the present invention, the liposome composition typically contains about 1 mM to about 150 mM of phospholipids.

[0090] Any of the exemplary liposomes described above comprises monophosphoryl lipid A (MPLA) or may be combined with other liposomes and lipid A (MPLA). MPLA alone can be toxic to humans and animals. However, no toxicity has been detected when it is present in liposomes. See, for example, Alving et al., 2012. MPLA acts as a potent adjuvant and is suitable for enhancing the immunogenicity of liposomes and liposome-associated peptides, proteins, or haptens.

[0091] Regarding embodiments of the present invention, a liposome (L(MPLA)) containing monophospholipid A (MPLA) comprises (1) a lipid bilayer containing phospholipids, wherein the hydrocarbon chains have a melting temperature of ≥23°C in water, the phospholipids typically being dimyristoylphosphatidylcholine (DMPC, e.g., 1,2-dimyristoyl-sn-glycerol-3-phosphocholine) and dimyristoylphosphatidylglycerol (DMPG, e.g., 1,2-dimyristoyl-sn-glycerol-3-phospho-(1'-rac-glycerol)); (2) cholesterol as a stabilizer; and (3) monophospholipid A (MPLA) as an immunostimulant. Certain compositions contain a molar percentage concentration greater than about 50% (mol / mol), preferably about 55% to about 71% (mol / mol), or more preferably about 55% (mol / mol) of cholesterol. Additionally, in embodiments of the present invention, liposomes (L(MPLA)) containing monophosphoryl lipid A (MPLA) including saponins (e.g., QS-21) can be homogeneous LiNA-2 adjuvant formulations, as described herein.

[0092] In embodiments of the present invention, L(MPLA) may comprise phosphatidylcholine (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearyl phosphatidylcholine (DSPC). L(MPLA) may also comprise phosphatidylglycerol (PG) selected from the group consisting of dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). The PC to PG ratio (mol / mol) of the liposomes 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 an 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. Before the addition of saponin, liposomes may contain small monolayer liposomes (SUVs) or bilayer liposomes. The small monolayer or bilayer liposomes may have a diameter of about 50 to about 100 nm.

[0093] 4. Adjuvant formulations containing MPLA-containing liposomes (L(MPLA)) and saponins. An adjuvant formulation known as AS01 (also referred to as AS01B or AS01E) was previously introduced by GlaxoSmithKline. In AS01, the lipid bilayer consists of neutral lipids that are “non-crystalline” at room temperature, such as dioleoylphosphatidylcholine, cholesterol, MPLA, and QS-21. See U.S. Patent No. 10,039,823. During the manufacture of AS01, small monolayer liposome microparticles (SUVs) are first generated, and then purified QS-21 is added to the SUVs. QS-21 imparts unique properties because it binds to cholesterol in liposomes, where QS-21 causes perforation (pores) or other permanent structural changes in the liposomes. See, for example, Paepenmuller et al., 2014, Int. J. Pharm., 475: 138-46. Reducing the amount of free QS-21 may result in a reduction in local injection pain that is typically caused by free QS-21. See, for example, Waite et al., 2001, Vaccine, 19:3957-67; Mbawuike et al., 2007, Vaccine, 25:3263-69. AS01B formulations are being developed for use in vaccines where a stronger T-cell-mediated immune response needs to be induced. See Garçon et al., 2007, Expert. Rev. Vaccines, 6:723-39. AS01 formulations are being developed as adjuvants for various vaccines. See Garcon & Mechelen, 2011, Expert. Rev. Vaccines, 10:471-86. AS01 formulations may contain 1 to 50% (mol / mol), preferably 20 to 25% (mol / mol), of cholesterol (sterols), as described in U.S. Patent No. 10,039,823.

[0094] This invention provides adjuvant formulations prepared by the methods described herein, comprising a liposomal composition containing monophosphoryl lipid A (MPLA) and at least one saponin, wherein the liposomal composition comprises i) a lipid bilayer comprising phospholipids (e.g., dimyristoylphosphatidylcholine (DMPC) and / or dimyristoylphosphatidylglycerol (DMPG)), wherein the hydrocarbon chain has a melting temperature of ≥23°C in water, and ii) cholesterol having a molar percentage 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 two of these characteristics differ from those of AS01 discussed above. The saponin may be selected from QS-7, QS-18, QS-21 or mixtures thereof, or preferably QS-21. The adjuvant formulation may contain saponin in amounts 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 per ml of liposome suspension. In a preferred aspect, the adjuvant formulation contains a saponin content of about 0.2 to 0.4 mg / ml.

[0095] In one aspect, the adjuvant formulation comprises a liposome composition comprising i) a lipid bilayer containing phospholipids and ii) cholesterol, wherein the molar ratio of cholesterol to phospholipids is greater than about 1.

[0096] In a preferred aspect, the liposome composition of the adjuvant formulation comprises PC and PG, wherein PC is dimyristoyl phosphatidylcholine (DMPC) and PG is dimyristoyl phosphatidylglycerol (DMPG), having a molar ratio of PC to PG (mol / mol) of about 9:1.

[0097] In a further aspect, the liposome composition of the adjuvant formulation may have a molar ratio of MPLA to phospholipids 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, wherein PC is dimyristoylphosphatidylcholine (DMPC) and PG is dimyristoylphosphatidylglycerol (DMPG), having a molar ratio of MPLA to phospholipids of about 1:220, about 1:88, or about 1:5.6, preferably 1:88. In one embodiment, the liposome composition of the adjuvant formulation comprises DMPC, DMPG, and 3D-PHAD, and has a 3D-PHAD to phospholipid molar ratio between about 1:5 and about 1:6, for example, 1:5.6. In one embodiment, the liposome composition of the adjuvant formulation comprises DMPC, DMPG, and 3D-PHAD, and has a 3D-PHAD:phospholipid molar ratio between about 1:200 and about 1:240, for example, 1:220. In another embodiment, the liposome composition of the adjuvant formulation comprises DMPC, DMPG, and 3D-PHAD, and has a 3D-PHAD:phospholipid molar ratio between about 1:80 and about 1:95. In yet another particular embodiment, the liposome composition of the adjuvant formulation comprises DMPC, DMPG, and 3D-PHAD, and has a 3D-PHAD:phospholipid molar ratio of about 1:88.

[0098] In another embodiment, the present invention provides an adjuvant formulation prepared by the method described herein, wherein the adjuvant formulation comprises a monolayer liposome having a liposomal bilayer composed of: (a) at least one phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) as a phospholipid selected from the group consisting of: dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG) and distearate phosphatidylglycerol (DSPG); (b) cholesterol; (c) monophosphatidyllipid A (MPLA); and (d) saponin; wherein the molar ratio of cholesterol (b) to phospholipid (a) is greater than about 50:50; and wherein the monolayer liposome has a median diameter size in the micrometer range as detected by light scattering analysis. In one aspect, the saponin is QS-7, QS-18, QS-21, or a mixture thereof, preferably QS-21. In another aspect, the molar ratio of cholesterol (b) to phospholipid (a) is about 55:45 to about 71:29. In one aspect, the molar ratio of cholesterol (b) to phospholipid (a) is about 55:50, about 55:45, about 55:40, about 55:35, or about 55:30. In another aspect, the molar ratio of cholesterol (b) to phospholipid (a) is about 55:45. In another aspect, dimyristoylphosphatidylcholine (DMPC) is selected as the phospholipid, wherein dimyristoylphosphatidylglycerol (DMPG) is also selected as the phospholipid. In another aspect, both PC and PG are selected as phospholipids, wherein the PC to PG ratio (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 aspect, the present invention provides a liposome suspension comprising the adjuvant formulation described herein and phosphate-buffered saline (PBS), pH 7.4, wherein the liposome suspension comprises (i) 1.272 mM to 50 mM of phospholipid (a) and (ii) about 5 mg / ml or less of MPLA (c). In another aspect, the molar ratio of MPLA (c) to phospholipid (a) is about 1:5.6 to about 1:880. In a further aspect, the present invention provides a liposome suspension comprising the adjuvant formulation described herein and phosphate-buffered saline (PBS), pH 7.4, wherein the liposome suspension comprises (i) 1.272 mM to 50 mM of phospholipids (a) and (ii) about 1 mg / ml or less of saponins (d). In another aspect, the molar ratio of MPLA (c) to phospholipids (a) is about 1:88 to about 1:220.

[0099] Examples of adjuvant formulations comprising an MPLA-containing liposome composition and at least one saponin (e.g., QS-21) prepared by the methods described herein include the homogeneous adjuvant formulations described herein, namely the Liposomal Novel Adjuvant-2 (LiNA-2) homogeneous adjuvant formulation. In one aspect, the LiNA-2 homogeneous adjuvant comprises a synthetic TLR4 agonist, monophospholipid A (MPLA), a triterpenoid glycoside saponin (QS-21), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycerol-3-phospho-(1'-rac-glycerol) (DMPG), and cholesterol. In a preferred aspect, the LiNA-2 homogeneous adjuvant comprises MPLA (e.g., 3D-PHAD). ® The LiNA-2 adjuvant contains QS-21, DMPC, DMPG, and cholesterol. In some embodiments, the LiNA-2 adjuvant comprises a phosphate buffer. In some embodiments, the LiNA-2 adjuvant comprises a phosphate buffer at a concentration between about 1 mM and about 100 mM. In some embodiments, the LiNA-2 adjuvant comprises a phosphate buffer at a concentration between about 1 mM and 10 mM. In some embodiments, the LiNA-2 adjuvant comprises a phosphate buffer at concentrations of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In a particular embodiment, the LiNA-2 adjuvant comprises about 10 mM of phosphate buffer. In another preferred aspect, the LiNA-2 homogeneous adjuvant comprises MPLA (e.g., 3D-PHAD) in a phosphate buffer containing sodium chloride (NaCl). ® The LiNA-2 adjuvant comprises 3D-PHAD®, QS-21, DMPC, DMPG, and cholesterol. In a further specific embodiment, the LiNA-2 adjuvant comprises 3D-PHAD®, QS-21, DMPC, DMPG, cholesterol, and 10 mM phosphate buffer.

[0100] In some embodiments, the LiNA-2 adjuvant comprises sodium chloride. In some embodiments, the LiNA-2 adjuvant comprises sodium chloride between about 50 mM and about 500 mM. In other embodiments, the LiNA-2 adjuvant comprises about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, or about 250 mM sodium chloride. In a particular aspect, the LiNA-2 adjuvant comprises about 150 mM sodium chloride. In one embodiment, the LiNA-2 adjuvant comprises 3D-PHAD®, QS-21, DMPC, DMPG, cholesterol, sodium chloride, and phosphate buffer. In a further particular embodiment, the LiNA-2 adjuvant comprises 3D-PHAD®, QS-21, DMPC, DMPG, cholesterol, 150 mM sodium chloride, and 10 mM phosphate buffer.

[0101] In one aspect, the homogeneous LiNA-2 adjuvant is designed to reconstitute a freeze-dried powder formulation for application. In another aspect, the homogeneous LiNA-2 adjuvant is designed to be mixed with a liquid formulation for application.

[0102] In a further aspect, compared to ALFQ concentrations (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), the homogeneous adjuvant formulation of LiNA-2 may be a 1X concentration of LiNA-2 (1XLiNA-2) or a 2X concentration of LiNA-2 (2XLiNA-2). In another embodiment, the adjuvant formulation is 1XLiNA-2, wherein 1XLiNA-2 is homogeneous 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, wherein 2XLiNA-2 is homogeneous 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.

[0103] 5. Uses of adjuvant formulations containing MPLA-containing liposomes (L(MPLA)) and saponins. The adjuvant formulations of embodiments of the present invention can be mixed with immunogens to obtain immunogenic compositions, such as vaccines. Immunogenic compositions may contain physiologically acceptable mediators, such as any of the mediators described in U.S. Patent No. 5,888,519. Immunogenic compositions may contain naturally occurring or artificially produced proteins, recombinant proteins, glycoproteins, peptides, carbohydrates, nucleic acids, haptens, intact viruses, bacteria, protozoa, or virus-like particles or conjugates thereof as immunogens. Exemplary nucleic acids or polynucleotides of immunogenic compositions include, but are not limited to, ribonucleic acid (RNA) (including mRNA) and deoxyribonucleic acid (DNA). In some embodiments, the immunogenic composition includes DNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the immunogenic composition includes RNA encoding a polypeptide or fragment thereof described herein. In some embodiments, the immunogenic composition includes mRNA polynucleotides encoding a polypeptide or fragment thereof described herein. In some embodiments, the immunogenic composition includes modified RNA molecules (modRNA). In some embodiments, the immunogenic composition includes sugars. In some embodiments, the immunogenic composition includes capsular bacterial sugars.

[0104] The immunizing composition may be suitably used as a vaccine for the following: varicella or herpes zoster, human respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus, human parainfluenza virus type 1 or 3, Lyme disease, Streptococcus pneumoniae, Clostridium difficile, Escherichia coli or Klebsiella pneumoniae, influenza, HIV-1, hepatitis A, hepatitis B, human papillomavirus, meningococcal meningitis A, meningococcal meningitis B, meningococcal meningitis C, tetanus, diphtheria, pertussis, polio, Haemophilus influenzae type B, dengue fever, hand-foot-mouth disease, typhoid fever, pneumococcus, Japanese encephalitis virus, anthrax, herpes zoster, malaria, norovirus, or cancer. The immunizing composition may be suitably used in methods of treating or preventing disease or infection in an individual (preferably a human) caused by a pathogen associated with an infectious disease, wherein the pathogen is selected from Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcaneobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae. henselae), BK virus, Blastocystis hominis, Blastocystis dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, certain species of Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepaciaBurkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, certain species of Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prions, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, certain species of Clostridium spp, Clostridium tetanus The following are listed: * *Tetani*, certain species of *Coccidioides* spp., coronavirus, *Corynebacterium diphtheriae*, *Coxiella burnetii*, Crimean-Congohemorrhagic 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*, and *Ehrlichia* genus. Enterococcus genus, Enterovirus genus, Enterovirus (mainly Coxsackie A virus and Enterovirus 71)(EV71), certain species of Epidermophyton spp., Epstein-Barr Virus (EBV), Escherichia coli O157:H7, Escherichia coli O111 and O104:H4, Escherichia coli fimbrial antigen H, Fasciola hepatica and Fasciola gigantica, FFI prions, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, certain species of Gnathostoma spp., GSS prions, Guanarito virus Hepatitis A virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipa virus (Hendra virus Nipah 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. 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, Klebsiella pneumoniae, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenesMonocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, certain species of Malassezia spp., Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri fowleri), Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, certain species of Nocardia, Onchocerca volvulus, Orientiatsutsugamushi, Orthomyxoviridae family (influenza virus), Paracoccidioides brasiliensis, certain species of Paragonimus, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, poliovirus, rabies virus Respiratory syncytial virus (RSV), rhinovirus, rhinoviruses, RickettsiaRickettsia 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 Nombrevirus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus, Streptococcus agalactiae Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, certain species of Trichophyton spp., Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum Varicella-zoster virus (VZV), varicella-zoster virus (VZV), varicella major or varicella minor, vCJD prions, and Venezuelan equine encephalitis virus (Venezuelan equine virus).The bacteria include encephalitisvirus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0105] This invention provides immunogenic compositions comprising an immunogen and an adjuvant formulation as described herein. In another aspect, this invention provides a method for inducing an immune response in an individual, comprising administering the immunogenic composition. In some embodiments, the immunogenic composition increases the individual's neutralizing titer specific to the immunogen. In specific embodiments, after administration of the immunogenic composition, the neutralizing titer in the individual increases by at least about 1.01 times, about 1.1 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times, about 100 times, or more. In a particular embodiment, the immunogenic composition comprises LiNA-2 and has a neutralizing titer increased by at least 2-fold or more in an individual. In other particular embodiments, the immunogenic composition comprises LiNA-2 and has a neutralizing titer increased by at least 10-fold or more in an individual.

[0106] Example To better understand the present invention, the following embodiments are described. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. The following embodiments illustrate some implementations of the present invention.

[0107] In the following examples, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), and the synthetic monophosphatidyllipid A (MPLA) (3D-PHAD™) were available from Avanti Polar Lipids (Alabaster, Ala., USA). Purified QS-21 was available from Desert King International (San Diego, Calif., USA).

[0108] In the following examples, cholesterol content can be analyzed using established methods to confirm the cholesterol concentration of the liposome composition and indirectly confirm the phospholipid concentration. See, for example, Zlatkis et al., 1953. J. Lab. Clin. Med., 41: 486-492. The cholesterol concentration of the liposome composition can be determined from the cholesterol standard curve.

[0109] Example 1: Preparation of homogeneous adjuvant formulation: using a process that involves microfluidic mixing with saponin and aseptic filtration. Liposome adjuvant drug products with a size of 30 to 200 nm and capable of sterile filtration are prepared using microfluidic mixing. The liposomes are formed by mixing lipids (DMPG, DMPC, cholesterol, and MPLA) at a concentration of 0.8X to 2X in an organic solvent (ethanol, isopropanol, or other solvents) via sonication, heating, or a combination of both (organic phase), and then mixing the organic phase with an aqueous phase containing saponin QS-21. Mixing occurs in a microfluidic mixer. The mixer can be designed as a Y-type, T-type, or coaxial type (Y-type connector mixer from Precision Nanosystem, T-type connector and coaxial mixer from IDEX) or any other configuration that generates turbulence. A schematic diagram of this process is shown in [image / image / details]. Figure 1 middle.

[0110] The lipids (organic phase) in the organic solvent were heated to 65°C. The organic phase was pumped into a buffer (aqueous phase) maintained at RT (22°C to 25°C). The buffer (aqueous phase) contained 150 mM NaCl and 10 mM phosphate at pH 6.2 containing 0.07 to 0.35 mg / mL of QS-21. The lipids (organic phase) were introduced into the aqueous phase within a microfluidic chip with a Y-type mixing connector. The total flow rate was controlled at approximately 12 mL / min using a syringe pump, with an aqueous phase:organic phase volume ratio of 3:1. The concentration of QS-21 in the aqueous phase was controlled at 0.07 mg / mL, while maintaining a constant aqueous phase:organic phase volume ratio of 3:1 and a constant total flow rate of 12 mL / min, thereby resulting in a liposomal adjuvant drug product with a size of approximately 107 nm and a PDI of 0.16. Data from the formed liposomal adjuvant drug product are provided in Table 1 below. The organic solvent in the liposomes is removed by tangential flow filtration (TFF) using a hollow fiber membrane, a cartridge membrane, or a rotary centrifuge membrane. The concentration of the final product can be adjusted by controlling the ultrafiltration and percolation steps. The molecular weight cutoff (MWCO) of the membrane used for TFF is in the range of 100 to 500 kDa. After TFF, the liposome adjuvant product is resuspended in a buffer solution and passed through a bioload reduction filter (0.45 μm), and then sterile filtered (0.22 μm) before use. This process allows for sterile filtration of the final liposome adjuvant product. The above process can be a continuous or discontinuous process and allows for sterile filtration of the formed liposome adjuvant product. A "continuous process" means that all unit operations of forming liposomes by microfluidic mixing of the organic and aqueous phases, subsequent removal of the organic phase and concentration in TFF, and subsequent sterile filtration can be completed as a series of consecutive steps before filling is complete. In a “discontinuous process”, microfluidic mixing, TFF and aseptic filtration can be independent process steps before filling is completed.

[0111] Table 1

[0112] Example 2: Preparation of homogeneous adjuvant formulation: Using a microfluidic mixing process with saponin, which enables aseptic filtration, alternative methods Liposomes with sizes ranging from 30 to 200 nm are produced using a microfluidic mixing process. Liposomes are formed by dissolving lipids (DMPG, DMPC, cholesterol, and MPLA) at a concentration of 0.8X to 2X in an organic solvent (ethanol, isopropanol, or other solvents) (see Table 2) (organic phase) and mixing the organic phase with an aqueous buffer. Mixing leads to the formation of intermediate liposomes. Once the intermediate liposomes are formed, the final liposomal adjuvant drug product is formed by adding saponin QS-21 dissolved in the aqueous phase (i.e., the buffer system) on-line. Mixing occurs in a microfluidic mixer. The mixer can be designed as a Y-type, T-type, or coaxial type (Y-type connector mixers from Precision Nanosystems, T-type connector and coaxial mixers from IDEX) or any other configuration that generates turbulence. The lipids (organic phase) in the organic solvent are heated to 65°C. The organic phase is pumped into a buffer (aqueous phase) maintained at RT (22°C to 25°C). The buffer (aqueous phase) is a 10 mM phosphate buffer at pH 6.2 containing 150 mM NaCl. The lipid (organic phase) is introduced into the aqueous phase within a microfluidic chip with a Y-type, T-type, or coaxial hybrid connector. The total flow rate is controlled at approximately 12 mL / min to 240 mL / min using a syringe pump, peristaltic pump, or HPLC pump, with an aqueous phase:organic phase volume ratio of 3:1 to form intermediate liposomes. QS-21 dissolved in the aqueous phase or buffer is added to this intermediate liposome production line to form the final liposome adjuvant drug product, such as... Figure 2 As depicted in the diagram, the organic solvent in the liposomes is removed by tangential flow filtration (TFF) using a hollow fiber membrane, a cartridge membrane, or a rotary centrifuge membrane. The concentration of the final product can be adjusted by controlling the ultrafiltration and percolation steps. The molecular weight cutoff (MWCO) of the membrane used for TFF is in the range of 100 to 500 kDa. After TFF, the liposomal adjuvant product is resuspended in a buffer and passed through a bioload reduction filter (0.45 μm), and then aseptically filtered (0.22 μm) before use. This process allows for the aseptic filtration of the final liposomal adjuvant product. A schematic diagram of this process is shown in [the diagram / illustration]. Figure 2 middle.

[0113] The above process can be a continuous or discontinuous process and allows for aseptic filtration of the formed liposome adjuvant drug product. A "continuous process" means that all unit operations—microfluidic mixing of the organic and aqueous phases, subsequent removal of the organic phase and concentration in TFF, followed by aseptic filtration to form liposomes—can be completed as a series of consecutive steps before filling is complete. In a "discontinuous process," the unit operations of microfluidic mixing, TFF, and aseptic filtration can be independent process steps before filling is complete.

[0114] Example 3: Preparation of homogeneous adjuvant formulations using microfluidic mixing Preparation of intermediate liposomes (30 to 200 nm) Figure 3 A brief overview of the process for manufacturing these homogeneous LiNA-2 adjuvants is provided. The process steps include lipid preparation in an organic phase, aqueous phase preparation, microfluidic mixing of the two solutions to form liposomes, removal of the organic phase using tangential flow filtration, subsequent filtration of the liposomes and recombination (i.e., in a mixed form) with a sterile filtered QS-21 solution to form the final homogeneous adjuvant drug product. The final homogeneous adjuvant drug product is then filled into glass vials.

[0115] Lipid systems with sizes ranging from 50 to 200 nm were produced using a microfluidic mixing process. Liposomes were produced by dissolving lipids (DMPG, DMPC, cholesterol, and MPLA) at concentrations of 0.8X or 2X according to Table 2 in an organic solvent (ethanol) (organic phase). The lipids (organic phase) in the organic solvent were heated to 65°C. The organic phase was pumped into a buffer solution (aqueous phase) maintained at room temperature (22°C to 25°C). The buffer solution (aqueous phase) was a 10 mM phosphate buffer at pH 6.2 containing 150 mM NaCl or other suitable buffer system as described herein. The lipids (organic phase) were introduced into the aqueous phase using a Y-type or T-type mixing connector with a characteristic length range of 300 to 500 µm. The total flow rate was in the range of 12 mL / min to 240 mL / min or up to 3 L / min, and the aqueous phase:organic phase volume ratio varied from 1:1 to 3:1.

[0116] The formation of intermediate liposomes was screened across a design space that included the dimensions of the mixer geometry (Y-type, T-type, coaxial, or any other microfluidic mixer), the aqueous phase:organic phase ratio, the lipid concentration in the organic phase, and the flow rate. The results are summarized in Table 3.

[0117] Table 2: Lipid Preparation (in 200% ethanol) [Organic Phase]

[0118] Table 3: Test Conditions

[0119] Organic solvents in the liposome intermediates can be removed by tangential flow filtration using hollow fiber membranes, boxed membranes, or rotary centrifuge membranes. The concentration of the final product can be adjusted by controlling the ultrafiltration and percolation steps. The molecular weight cutoff (MWCO) of the membrane used for TFF is in the range of 100 to 500 kDa. After TFF, the intermediate liposomes are resuspended in buffer and passed through a bioload reduction filter (0.45 μm) and then aseptically filtered (0.22 μm).

[0120] Example 4: Adjuvant formulation This invention provides a method for manufacturing 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 homogeneous adjuvant formulations of this invention, which include a homogeneous adjuvant formulation called LiNA-2 (Novel Liposome Adjuvant-2), which may have a concentration of 0.5X LiNA-2 or 0.8X LiNA-2 or 1X LiNA-2 or 2X LiNA-2, or any other concentration variation of 1X LiNA-2. The composition of such adjuvant formulations is listed in Table 4 below: Table 4: Adjuvants in the final pharmaceutical product

[0121] Example 5: Immunogenicity of Clostridium difficile vaccine antigens formulated with different LiNA-2 adjuvants The relative immunogenicity of Clostridium difficile toxoid antigens prepared in rats with aluminum hydroxide (Al(OH)3) and different LiNA-2 adjuvant formulations (homogeneous and heterogeneous) was compared. Homogeneous and heterogeneous LiNA-2 adjuvants are those described in this paper and Table 5. The final rat LiNA-2 adjuvant dose was prepared by diluting 1X concentration of LiNA-2 at a 1:5 dilution with PBS buffer at pH 6.2.

[0122] Table 5: LiNA-2 adjuvant formulations

[0123] The toxin neutralization assay (TNA) described below is used to measure the functional cytotoxic activity of serum at multiple time points after immunization.

[0124] Wistar Han rats (n=10 per group, 8 to 10 weeks old, Charles River Laboratories) were immunized intramuscularly (IM) according to the study design in Table 6. Group 1 received Clostridium difficile vaccine antigen formulated with Al(OH)3. Groups 2 and 3 received Clostridium difficile vaccine antigen formulated with homogeneous and heterogeneous LiNA-2 adjuvants, respectively.

[0125] Serum was collected at multiple time points, and the ability to neutralize toxin cytotoxic activity was measured using TNA. Neutralizing titers of toxin B in serum from individual animals were shown. Figure 4 In this study, examples were given of toxoids formulated with homogeneous and heterogeneous LiNA-2 that elicited similar immune responses capable of neutralizing the toxicity of toxins on B cells.

[0126] Table 6: Rat Study Design Using LiNA-2 Adjuvant Formulation

[0127] Toxin neutralization assay (TNA) The immune response induced by the application of the compositions of the present invention can be determined using a toxin neutralization assay (TNA), ELISA, or preferably a cytotoxicity assay, as described in WIPO Patent Application WO / 2012 / 143902, U.S. Patent No. 9187536, and WIPO Patent Application WO / 2014 / 060898, the full text of which is incorporated herein by reference.

[0128] The toxin neutralization assay (TNA) can be used to quantify antibodies that neutralize Clostridium difficile toxins. In this assay, serially diluted serum is incubated with a fixed amount of Clostridium difficile toxin A or toxin B. Test cells (e.g., Vero cells) are then added, and the serum-toxin-cell mixture is incubated under appropriate conditions (e.g., 37°C for 6 days). The ability of serum to neutralize the cytotoxic effects of Clostridium difficile toxins can be determined by cell viability and is correlated with cell viability. The assay utilizes the accumulation of acid metabolites in closed culture wells as an indicator of normal cellular respiration. In cells exposed to the toxin, metabolism and CO2 production decrease; therefore, the pH rises (e.g., to 7.4 or higher), as indicated by phenol red pH indicator in cell culture media. The medium appears red at this pH. However, cell controls or cells exposed to toxins neutralized by antibodies metabolize and produce CO2 at normal levels; therefore, the pH is maintained (e.g., at 7.0 or lower), and the medium appears yellow at this pH. Therefore, Clostridium difficile toxin neutralizing antibodies are correlated with the ability of serum to neutralize the metabolic effects of Clostridium difficile toxin on cells, as demonstrated by its ability to maintain a specific pH (e.g., 7.0 or lower). Color changes in the culture medium can be measured using a disc reader (e.g., at 562 nm to 630 nm) to further calculate the antitoxin neutralizing antibody titer under 50% inhibition of Clostridium difficile toxin-mediated cytotoxicity. In one aspect, the composition induces a toxin neutralizing antibody titer that, when measured under the same conditions in a toxin neutralization assay, is at least 1.01 times greater in an individual after receiving a dose of the composition than in an individual before receiving that dose, for example, at least 1.01 times, 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 32 times, or more.

[0129] In summary, in this embodiment, 384-well microtiter discs were used to seed IMR-90 cells as targets for toxin-mediated cytotoxicity. The ability of each test serum sample to neutralize toxin A or toxin B was analyzed individually. Four serial dilutions of the test serum were mixed with fixed concentrations of toxin A (TcdA) or toxin B (TcdB) in a humidified incubator (37°C / 5% CO2) for 60 minutes to allow for toxin neutralization. All discs included reference standards and mass controls composed of antitoxin antibodies of known titers to monitor assay performance. After 60 minutes of incubation, the toxin-antiserum mixture was applied to an IMR-90 cell monolayer and the discs were incubated for another 72 hours. The viability of the IMR-90 cell monolayer was then tested using a luciferase-based CellTiter-Glo® reagent, which provides a measure of ATP levels in metabolically active cells and reports in relative luminescent 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 value of the test sample to a calibration curve from antitoxin A or B reference standards using a standard statistical analysis system (SAS®) procedure. Functional antibody concentrations are expressed as arbitrary units / mL (or neutralizing units / mL) of serum. The limits of quantitation (LLOQ) for TcdA and TcdB TNA assays are 75.9 and 249.7 neutralizing units / mL of serum, respectively.

[0130] Example 6: Preparation of intermediate liposomes using a coaxial microfluidic mixer intermediate liposomes are produced using a coaxial mixing device (such as...) Figure 5 The intermediate liposomes (described in the text) were manufactured using a range of total flow rates and aqueous-to-organic volume ratios. The experimental objectives were first to test the effect of mixing parameters on the size of the intermediate liposomes, and second to demonstrate that the intermediate liposomes could be manufactured using this coaxial mixing technique compared to a conventional jet mixer (i.e., a T-mixer).

[0131] In this experiment, the intermediate liposomes were prepared using a 14:0 ratio of cardiolipin (Avanti). ® Polar Lipids (CAS No. 63988-21-6) is used as a 3D PHAD (Avanti ® A 1:1 molar substitute for Polar Lipids (CAS No. 1699735-79-9) was used. To support the use of the 14:0 cardiolipin material, a low-capacity NanoAssembler was used in the control experiment. TM Ignite TMThe device operates to ensure that the size of intermediate liposomes generated using a 1:1 molar substitute (i.e., 14:0 cardiolipin) is comparable to that of intermediate liposomes manufactured using 3D-PHAD.

[0132] In short, intermediate liposomes are produced by dissolving lipids (DMPG, DMPC, cholesterol, and 3D-PHAD or cardiolipin) in an organic solvent (USP grade 200 pure ethanol) through sonication, heating, or a combination of both to form an organic phase. The lipids in the organic phase are heated to 65°C. The organic phase is then pumped into a buffer (aqueous phase) and maintained at room temperature (22°C to 25°C). The buffer (aqueous phase) is a 10 mM phosphate buffer at pH 6.2 containing 150 mM NaCl. The lipids (organic phase) are then injected using a NanoAssembler equipped with a syringe pump. TM Ignite TM The mixing device was introduced into the aqueous phase. The aqueous-to-organic phase ratio was kept constant at 3. Both 3D-PHAD and cardiolipin were tested at total flow rates of 6, 12, and 18 (mL / min).

[0133] As shown in Table 7 below, the particle size (Z mean, mean, and D90) and PDI of liposomes containing intermediates of 3D-PHAD or cardiolipin were measured.

[0134] Table 7: Controlled experiments comparing 3D-PHAD and 14:0 cardiolipin

[0135] This control experiment demonstrates that intermediate liposomes manufactured with a 1:1 molar substitute for 3D-PHAD (also 14:0 cardiolipin) are reasonably comparable in terms of particle size, although minor differences that may be attributable to assay variations were observed.

[0136] Based on these results, subsequent experiments were conducted to manufacture intermediate liposomes using a coaxial mixing device and 14:0 cardiolipin.

[0137] In short, intermediate liposomes are produced by dissolving lipids (DMPG, DMPC, cholesterol, and cardiolipin) according to Table 8 in an organic solvent (USP grade anhydrous ethanol (200 proof ethanol)) to form an organic phase. The lipids in the organic phase are heated to 65°C. The organic phase is then pumped into a buffer (aqueous phase) and maintained at room temperature (22°C to 25°C). The buffer (aqueous phase) is a 10 mM phosphate buffer at pH 6.2 containing 150 mM NaCl.

[0138] Table 8: Lipid Preparation (in anhydrous ethanol) [Organic Phase]

[0139] The lipids (organic phase) were introduced into the aqueous phase using a coaxial mixer equipped with a peristaltic pump. The coaxial mixer used is depicted in... Figure 5 The mixer is constructed of stainless steel and consists of an outer chamber in which an aqueous phase flows and a parallel, coaxial inner chamber in which an organic phase flows. The outer chamber has an inner diameter of approximately 9.47 mm and a length of 72.5 mm. The inner chamber has a diameter of approximately 489 micrometers and a length of 30.7 mm. The length difference between the outer and inner chambers represents the initial volume in which the nanoprecipitation reaction occurs.

[0140] The tested aqueous-to-organic phase ratios were 3, 4, and 5. These different ratios were achieved by varying the relative flow rates of the aqueous and organic phases entering the mixer via a peristaltic pump. Total flow rates of 300, 500, 700, and 900 mL / min were tested. The parameters measured, as shown in Table 9 below, are z-mean particle size, PDI, mean particle size, and D90.

[0141] Table 9: Parameters of intermediate liposomes prepared in a coaxial mixer

[0142] DLS reaction surface plotting is provided for Figure 6 The relationship between the z-mean liposome size and the total flow rate relative to the aqueous:organic phase ratio (v / v) when cardiolipin was used to replace 3D-PHAD at a constant lipid concentration of 2X with a 1:1 molar ratio, was depicted. The results demonstrate that the liposome size (z-mean) can be modulated during manufacturing by adjusting the aqueous:organic phase volume ratio and the total flow rate.

[0143] The following clauses describe additional aspects of this disclosure: C1. A method for preparing a homogeneous adjuvant formulation comprising a liposomal bilayer, the liposomal bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposomal composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposomal composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase into the aqueous phase from step (ii) to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0144] Method C2. C1, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21 or mixtures thereof.

[0145] Methods C3 and C2, wherein the saponin is QS-21.

[0146] C4. Any of C1 to C3, wherein the amount of saponin is about 0.07 mg / ml to about 0.35 mg / ml.

[0147] Methods C5 and C4, wherein the amount of saponin is approximately 0.07 mg / ml.

[0148] C6. The method of any one of C1 to C5, wherein the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0149] C7. The method of any one of C1 to C6, wherein in step (i), phospholipids, cholesterol and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating or a combination thereof.

[0150] Methods C8 and C7, wherein the organic solvent is ethanol or isopropanol.

[0151] Methods C9, C7, or C8, wherein the organic phase is heated to a temperature between 45°C and 65°C.

[0152] C10. The method of any one of C1 to C9, wherein the buffer solution in step (ii) contains 10 mM phosphate and 150 mM NaCl at pH 6.2.

[0153] C11. The method of any one of C1 to C10, wherein the aqueous phase in step (ii) is at a temperature between 20°C and 25°C.

[0154] C12. The method of any one of C1 to C11, wherein the flow rate of step (ii) is from 12 mL / min to 240 mL / min or up to 3 L / min.

[0155] C13. The method of any one of C1 to C12, wherein the mass ratio of water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:1.

[0156] C14. The method of any of C1 to C13, wherein the microfluidic mixer in step (iii) is injected using a pump or syringe.

[0157] The method of C15. C14, wherein the microfluidic mixer in step (iii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer.

[0158] The method of C16. C15, wherein the microfluidic mixer of step (iii) has an inner diameter ranging from 300 µm to 1,000 µm.

[0159] C17. The method of any one of C1 to C16, wherein the organic phase for removing saponin-containing liposomes in step (iv) is obtained by tangential flow filtration (TFF).

[0160] The method of C18. C17, wherein the TFF is TFF percolation.

[0161] The method of C19, C17 or C18, wherein the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa.

[0162] C20. The method of any of C1 to C19, wherein the concentration in step (v) is achieved by TFF, wherein the TFF comprises percolation, ultrafiltration, or both.

[0163] C21. The method of any of C1 to C20, wherein the filtration in step (vi) includes a bioburden reduction filter and a sterile filter.

[0164] The methods of C22 and C21, wherein the bioburden reduction filter is up to 0.45 microns.

[0165] The method of C23, C21 or C22, wherein the sterile filter is 0.22 microns.

[0166] C24. The method of any one of C1 to C23, wherein the concentration in step (v) and the filtration in step (vi) occur at room temperature.

[0167] C25. A method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, the liposome bilayer comprising (a) a monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) The intermediate liposomes from step (ii) are mixed with saponin, wherein the saponin is first dissolved in a buffer solution or water to form saponin-containing liposomes. (iv) Remove the organic phase containing saponin liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); and (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.30. Homogeneous adjuvant formulations were thus prepared.

[0168] The method of C26. C25, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21 or mixtures thereof.

[0169] Methods C27 and C26, wherein the saponin is QS-21.

[0170] The method of any one of C25 to C27, wherein the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

[0171] C29. The method of any one of C25 to C28, wherein in step (i), phospholipids, cholesterol and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating or a combination thereof.

[0172] Methods C30 and C29, wherein the organic solvent is ethanol or isopropanol.

[0173] Methods C31, C29, or C30, wherein the organic phase is heated to a temperature between 45°C and 65°C.

[0174] The method of any one of C32, C25 to C31, wherein the buffer solution contains 10 mM phosphate and 150 mM NaCl at pH 6.2.

[0175] The method of any one of C33, C25 to C32, wherein the aqueous phase in step (ii) is at a temperature between 20°C and 25°C.

[0176] The method of any one of C34, C25 to C33, wherein the flow rate of step (ii) is from 12 mL / min to 240 mL / min or up to 3 L / min.

[0177] C35. The method of any one of C25 to C34, wherein the mass ratio of water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:1.

[0178] C36. The method of any of C25 to C35, wherein the microfluidic mixer in step (ii) is injected using a pump or syringe.

[0179] The method of C37. C36, wherein the microfluidic mixer in step (ii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer.

[0180] C38. The method of any of C25 to C37, wherein the microfluidic mixer of step (ii) has a length ranging from 300 µm to 1,000 µm.

[0181] C39. The method of any of C25 to C38, wherein the organic phase for removing saponin-containing liposomes in step (iv) is obtained by tangential flow filtration (TFF).

[0182] The methods of C40 and C39, wherein the TFF is TFF percolation.

[0183] The method of C41, C39 or C40, wherein the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa.

[0184] C42. The method of any of C25 to C41, wherein the concentration in step (v) is achieved by TFF, wherein the TFF comprises percolation, ultrafiltration or both.

[0185] The method of any one of C43, C25 to C42, wherein the filtration in step (vi) includes a bioburden reduction filter and a sterile filter.

[0186] The methods of C44 and C43, wherein the bioburden reduction filter is up to 0.45 microns.

[0187] Methods C45, C43, or C44, wherein the sterile filter reaches 0.22 microns.

[0188] C46. The method of any one of C25 to C45, wherein the concentration in step (v) and the filtration in step (vi) occur at room temperature.

[0189] C47. A method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, the liposome bilayer comprising (a) a monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein the phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearatel phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearatel phosphatidylglycerol (DSPG), and combinations thereof, wherein the molar percentage concentration of cholesterol in the liposome composition is greater than 50% (mol / mol), the method comprising the following steps: (i) Dissolving phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of organic phase to aqueous phase to form intermediate liposomes, wherein the aqueous phase contains buffer or water. (iii) Remove the organic phase of the intermediate liposome from step (ii); (iv) Concentrate the intermediate liposome from step (iii); (v) The intermediate liposome from step (iv) is sterile filtered; (vi) Sterilely filter the saponin; and (vii) The intermediate liposomes from step (v) are sterilized and mixed with the saponin from step (vi) to form a final adjuvant formulation having a size range of about 30 to 400 nm and a polydispersity index of 0.05 to 0.50. Homogeneous adjuvant formulations were thus prepared.

[0190] Methods C48 and C47, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21 or mixtures thereof.

[0191] Methods C49 and C48, wherein the saponin is QS-21.

[0192] The method of any one of C50, C47 to C49, wherein the liposome composition comprises dimyristic phosphatidylcholine (DMPC) and dimyristic phosphatidylglycerol (DMPG).

[0193] The method of any one of C51, C47 to C50, wherein in step (i), phospholipids, cholesterol and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating or a combination thereof.

[0194] Methods C52 and C51, wherein the organic solvent is ethanol or isopropanol.

[0195] Methods C53, C51, or C52, wherein the organic phase is heated to a temperature between 45°C and 65°C.

[0196] The method of any one of C54, C47 to C53, wherein the buffer solution in step (ii) contains 10 mM phosphate and 150 mM NaCl at pH 6.2.

[0197] The method of any one of C55, C47 to C54, wherein the aqueous phase in step (ii) is at a temperature between 20°C and 25°C.

[0198] The method of any of C56, C47 to C55, wherein the microfluidic mixer in step (ii) is injected using a pump or syringe.

[0199] The method of C57. C56, wherein the microfluidic mixer in step (ii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer.

[0200] The method of C58, C56 or C57, wherein the microfluidic mixer of step (ii) has a length ranging from 300 µm to 1,000 µm.

[0201] C59. The method of any one of C47 to C58, wherein the flow rate of step (ii) is from 12 mL / min to 240 mL / min or up to 3 L / min.

[0202] The method of any one of C60, C47 to C59, wherein the mass ratio of water in step (i) to the organic phase in step (ii) is in the range of 8:1 to 3:1, or 5:1 to 3:1.

[0203] C61. The method of any one of C47 to C60, wherein the organic phase of the removal of intermediate liposomes in step (iii) and the concentration in step (iv) are performed by tangential flow filtration (TFF).

[0204] The method of C62. C61, wherein the TFF is TFF percolation, ultrafiltration or both.

[0205] The method of C63, C61 or C62, wherein the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa.

[0206] C64. The method of any of C47 to C63, wherein the aseptic filtration in steps (v) and (vi) comprises a bioburden reduction filter and a aseptic filter.

[0207] The methods of C65 and C64, wherein the bioburden reduction filter is up to 0.45 microns.

[0208] Methods C66, C64, or C65, wherein the sterile filter reaches 0.22 microns.

[0209] C67. A homogeneous adjuvant formulation prepared by any one of C1 to C66.

[0210] C68. An adjuvant formulation obtained by any of the methods of C1 to C24.

[0211] C69. An adjuvant formulation obtainable by any of the methods from C1 to C24.

[0212] C70. An adjuvant formulation obtained by any one of C25 to C46.

[0213] C71. An adjuvant formulation obtainable by any of the methods from C25 to C46.

[0214] C72. An adjuvant formulation obtained by any of the methods of C47 to C67.

[0215] C73. An adjuvant formulation obtainable by any of the methods from C47 to C67.

[0216] C74. An adjuvant formulation comprising a monophosphoryl 3-deacyl lipid A phosphorylated hexaacyl disaccharide (3D-PHAD).

[0217] C75. An adjuvant formulation comprising 3D-PHAD, QS-21, 1,2-dimyristico-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristico-sn-glycerol-3-phospho-(1'-rac-glycerol) (DMPG) and cholesterol.

[0218] C76. An adjuvant formulation which is essentially composed of 3D-PHAD, QS-21, DMPC, DMPG and cholesterol.

[0219] C77. An adjuvant formulation comprising 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

[0220] C78. An adjuvant formulation comprising essentially 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

[0221] C79. An adjuvant formulation comprising 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

[0222] C80. An adjuvant formulation comprising 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, a phosphate buffer solution at a concentration between about 1 mM and 50 mM, and sodium chloride at a concentration between about 50 mM and 250 mM.

[0223] C81. An adjuvant formulation comprising essentially 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, a phosphate buffer solution at a concentration between about 1 mM and 50 mM, and sodium chloride at a concentration between about 50 mM and 250 mM.

[0224] C82. An adjuvant formulation comprising 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, phosphate buffer at a concentration between about 1 mM and 50 mM, and sodium chloride at a concentration between about 50 mM and 250 mM.

[0225] C83. An adjuvant formulation comprising 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, 10 mM phosphate buffer, and 150 mM sodium chloride.

[0226] C84. An adjuvant formulation comprising essentially 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, 10 mM phosphate buffer and 150 mM sodium chloride.

[0227] C85. An adjuvant formulation comprising 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, 10 mM phosphate buffer and 150 mM sodium chloride.

[0228] An adjuvant formulation of any one of C86, C68 to C85, comprising 3D-PHAD at a concentration between about 0.2 mg / ml and about 0.6 mg / ml.

[0229] An adjuvant formulation of any one of C87, C68 to C85, comprising 3D-PHAD at a concentration of about 0.4 mg / ml.

[0230] An adjuvant formulation of any one of C88, C68 to C85, comprising 3D-PHAD at a concentration between about 0.4 mg / ml and about 1.2 mg / ml.

[0231] An adjuvant formulation of any one of C89, C68 to C85, comprising 3D-PHAD at a concentration of about 0.8 mg / ml.

[0232] An adjuvant formulation of any one of C90, C68 to C89, comprising DMPC at a concentration between about 7 mg / ml and about 21 mg / ml.

[0233] An adjuvant formulation of any one of C91, C68 to C89, comprising DMPC at a concentration of about 14 mg / ml.

[0234] C92. An adjuvant formulation of any of C68 to C89, comprising DMPC at a concentration between about 14 mg / ml and about 42 mg / ml.

[0235] An adjuvant formulation of any one of C93, C68 to C89, comprising DMPC at a concentration of approximately 28 mg / ml.

[0236] An adjuvant formulation of any one of C94, C68 to C93, comprising DMPG at a concentration between about 0.8 mg / ml and about 2.4 mg / ml.

[0237] An adjuvant formulation of any one of C95, C68 to C93, comprising DMPG at a concentration of approximately 1.6 mg / ml.

[0238] An adjuvant formulation of any one of C96, C68 to C93, comprising DMPG at a concentration between about 1.6 mg / ml and about 4.8 mg / ml.

[0239] An adjuvant formulation of any one of C97, C68 to C93, comprising DMPG at a concentration of approximately 3.2 mg / ml.

[0240] An adjuvant formulation of any one of C98, C68 to C97, containing cholesterol at a concentration between about 5 mg / ml and about 17 mg / ml.

[0241] An adjuvant formulation of any one of C99, C68 to C97, containing cholesterol at a concentration of about 11 mg / ml.

[0242] An adjuvant formulation of any one of C100, C68 to C97, containing cholesterol at a concentration between about 10 mg / ml and about 34 mg / ml.

[0243] An adjuvant formulation of any one of C101, C68 to C97, containing cholesterol at a concentration of approximately 22 mg / ml.

[0244] An adjuvant formulation of any one of C102, C68 to C101, comprising QS-21 at a concentration between about 0.1 mg / ml and about 0.3 mg / ml.

[0245] An adjuvant formulation of any one of C103, C68 to C101, comprising QS-21 at a concentration of about 0.2 mg / ml.

[0246] An adjuvant formulation of any one of C104, C68 to C101, comprising QS-21 at a concentration between about 0.2 mg / ml and about 0.6 mg / ml.

[0247] An adjuvant formulation of any one of C105, C68 to C101, comprising QS-21 at a concentration of about 0.4 mg / ml.

[0248] An adjuvant formulation of any one of C106, C68 to C105, comprising DMPC and DMPG having a DMPC to DMPG molar ratio (mol / mol) between about 10:1 and about 8:1.

[0249] An adjuvant formulation of any one of C107, C68 to C105, comprising DMPC and DMPG having a DMPC to DMPG molar ratio (mol / mol) of approximately 9:1.

[0250] An adjuvant formulation of any one of C108, C68 to C107, comprising DMPC, DMPG and 3D-PHAD, wherein the molar ratio of 3D-PHAD to phospholipid is between about 1:80 and about 1:95.

[0251] An adjuvant formulation of any one of C109, C68 to C107, comprising DMPC, DMPG and 3D-PHAD, wherein the molar ratio of 3D-PHAD to phospholipid is approximately 1:88.

[0252] An adjuvant formulation of any one of C110, C68 to C109, wherein the adjuvant formulation has a cholesterol:phospholipid molar ratio greater than 1.

[0253] C111. An adjuvant formulation of any one of C68 to C109, wherein the adjuvant formulation has a cholesterol:phospholipid molar ratio between about 55:50 and about 55:40.

[0254] An adjuvant formulation of any one of C112, C68 to C109, wherein the adjuvant formulation has a cholesterol:phospholipid molar ratio of approximately 55:45.

[0255] An adjuvant formulation of any one of C113, C68 to C112, wherein the liposomes in the adjuvant formulation have a size range between about 30 nm and about 400 nm.

[0256] An adjuvant formulation of any one of C114, C68 to C112, wherein the liposomes in the adjuvant formulation have a size range between about 30 nm and about 200 nm.

[0257] An adjuvant formulation of any one of C115, C68 to C112, wherein the liposomes in the adjuvant formulation have a size of less than about 200 nm.

[0258] An adjuvant formulation of any one of C116, C68 to C115, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) between about 0.05 and about 0.5.

[0259] An adjuvant formulation of any one of C117, C68 to C115, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) between about 0.05 and about 0.3.

[0260] An adjuvant formulation of any one of C118, C68 to C115, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) of less than about 0.3.

[0261] An adjuvant formulation of any one of C119, C68 to C112, wherein the liposomes in the adjuvant formulation have a size range between about 30 nm and about 1400 nm.

[0262] An adjuvant formulation of any one of C120, C68 to C112, wherein the liposomes in the adjuvant formulation have a size range between about 300 nm and about 1000 nm.

[0263] An adjuvant formulation of any one of C121, C68 to C112, wherein the liposomes in the adjuvant formulation have a size greater than about 300 nm.

[0264] An adjuvant formulation of any one of C122, C68 to C112, or C119 to C121, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) between about 0.4 and about 1.

[0265] An adjuvant formulation of any one of C123, C68 to C112, or C119 to C121, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) greater than about 0.5.

[0266] An adjuvant formulation of any one of C124, C68 to C112, or C119 to C121, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) greater than about 0.4.

[0267] C125. An immunogenic composition comprising an adjuvant formulation of any one of C68 to C124 and an immunogen.

[0268] C126. A method of inducing an immune response in an individual, comprising administering an immunizing composition of C125 to the individual.

[0269] The methods of C127 and C126, wherein, after administration, the titer of neutralizing antibodies specific to the immunogen increases in individuals.

[0270] The methods of C128 and C127 involve an increase in neutralizing antibody titer of at least approximately 10-fold or more in an individual.

[0271] The methods of C129 and C127, wherein the neutralizing antibody titer increases by at least about 100 times or more in an individual.

[0272] The method of any one of C130, C125 to C129, wherein the immunogen is specific to Clostridium difficile.

Claims

1. A method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, said liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition, said liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein said 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, wherein the molar percentage concentration of said cholesterol in said liposome composition is greater than 50% (mol / mol), said method comprising the following steps: (i) Dissolving the phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) Mix the buffer solution or water with the saponin to form an aqueous phase; (iii) The organic phase of step (i) is mixed in a microfluidic mixer at a specific flow rate and at a specific ratio of the organic phase to the aqueous phase to form saponin-containing liposomes; (iv) Remove the organic phase of the saponin-containing liposome from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.

30. The homogeneous adjuvant formulation is thus prepared.

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

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

4. The method according to any one of claims 1 to 3, wherein the amount of saponin is from about 0.07 mg / ml to about 0.35 mg / ml.

5. The method according to claim 4, wherein the amount of saponin is about 0.07 mg / ml.

6. The method according to any one of claims 1 to 5, wherein the liposome composition comprises dimyristic phosphatidylcholine (DMPC) and dimyristic phosphatidylglycerol (DMPG).

7. The method according to any one of claims 1 to 6, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating or a combination thereof.

8. The method according to claim 7, wherein the organic solvent is ethanol or isopropanol.

9. The method according to claim 7 or 8, wherein the organic phase is heated to a temperature between 45°C and 65°C.

10. The method according to any one of claims 1 to 9, wherein the buffer solution in step (ii) comprises 10 mM phosphate and 150 mM NaCl at pH 6.

2.

11. The method according to any one of claims 1 to 10, wherein the aqueous phase in step (ii) is at a temperature between 20°C and 25°C.

12. The method according to any one of claims 1 to 11, wherein the flow rate in step (ii) is from 12 mL / min to 240 mL / min or up to 3 L / min.

13. The method according to any one of claims 1 to 12, wherein the mass ratio of water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:

1.

14. The method according to any one of claims 1 to 13, wherein the microfluidic mixer in step (iii) is injected using a pump or syringe.

15. The method according to claim 14, wherein the microfluidic mixer in step (iii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer.

16. The method according to claim 14 or 15, wherein the microfluidic mixer in step (iii) has an inner diameter ranging from 300 µm to 1,000 µm.

17. The method according to any one of claims 1 to 16, wherein the organic phase for removing the saponin-containing liposomes in step (iv) is removed by tangential flow filtration (TFF).

18. The method of claim 17, wherein the TFF is a TFF percolation filter.

19. The method of claim 17 or 18, wherein the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa.

20. The method according to any one of claims 1 to 19, wherein the concentration in step (v) is achieved by TFF, wherein TFF comprises percolation, ultrafiltration, or both.

21. The method according to any one of claims 1 to 20, wherein the filtration in step (vi) comprises a bioburden reduction filter and a sterile filter.

22. The method of claim 21, wherein the bioburden reduction filter has a diameter of 0.45 microns.

23. The method of claim 21 or 22, wherein the sterile filter has a diameter of 0.22 micrometers.

24. The method according to any one of claims 1 to 23, wherein the concentration in step (v) and the filtration in step (vi) occur at room temperature.

25. A method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, said liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition, said liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein said 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, wherein the molar percentage concentration of said cholesterol in said liposome composition is greater than 50% (mol / mol), said method comprising the following steps: (i) Dissolving the phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of the organic phase to the aqueous phase to form intermediate liposomes, wherein the aqueous phase comprises a buffer or water; (iii) The intermediate liposomes from step (ii) are mixed with saponin, wherein the saponin is first dissolved in a buffer solution or water to form saponin-containing liposomes. (iv) Remove the organic phase of the saponin-containing liposomes from step (iii); (v) Concentrate the saponin-containing liposomes from step (iv); (vi) The saponin-containing liposomes from step (v) are aseptically filtered to form a final adjuvant formulation having a size range of about 30 to 200 nm and a polydispersity index of 0.05 to 0.

30. The homogeneous adjuvant formulation is thus prepared.

26. The method according to claim 25, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21 or mixtures thereof.

27. The method according to claim 26, wherein the saponin is QS-21.

28. The method according to any one of claims 25 to 27, wherein the liposome composition comprises dimyristic phosphatidylcholine (DMPC) and dimyristic phosphatidylglycerol (DMPG).

29. The method according to any one of claims 25 to 28, wherein in step (i), the phospholipids, cholesterol and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating or a combination thereof.

30. The method of claim 29, wherein the organic solvent is ethanol or isopropanol.

31. The method according to claim 29 or 30, wherein the organic phase is heated to a temperature between 45°C and 65°C.

32. The method according to any one of claims 25 to 31, wherein the buffer solution comprises 10 mM phosphate and 150 mM NaCl at pH 6.

2.

33. The method according to any one of claims 25 to 32, wherein the aqueous phase in step (ii) is at a temperature between 20°C and 25°C.

34. The method according to any one of claims 25 to 33, wherein the flow rate in step (ii) is from 12 mL / min to 240 mL / min or up to 3 L / min.

35. The method according to any one of claims 25 to 34, wherein the mass ratio of the water in step (ii) to the organic phase in step (i) is in the range of 8:1 to 3:1, or 5:1 to 3:

1.

36. The method according to any one of claims 25 to 35, wherein the microfluidic mixer in step (ii) is injected using a pump or syringe.

37. The method of claim 36, wherein the microfluidic mixer in step (ii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer.

38. The method according to any one of claims 25 to 37, wherein the microfluidic mixer in step (ii) has a length ranging from 300 µm to 1,000 µm.

39. The method according to any one of claims 25 to 38, wherein the organic phase for removing the saponin-containing liposomes in step (iv) is obtained by tangential flow filtration (TFF).

40. The method of claim 39, wherein the TFF is a TFF percolation filter.

41. The method of claim 39 or 40, wherein the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa.

42. The method according to any one of claims 25 to 41, wherein the concentration in step (v) is achieved by TFF, wherein the TFF comprises percolation, ultrafiltration, or both.

43. The method according to any one of claims 25 to 42, wherein the filtration in step (vi) comprises a bioburden reduction filter and a sterile filter.

44. The method of claim 43, wherein the bioburden reduction filter has a diameter of 0.45 microns.

45. The method of claim 43 or 44, wherein the sterile filter has a diameter of 0.22 microns.

46. ​​The method according to any one of claims 25 to 45, wherein the concentration in step (v) and the filtration in step (vi) occur at room temperature.

47. A method for preparing a homogeneous adjuvant formulation comprising a liposome bilayer, said liposome bilayer comprising (a) monophosphatidyllipid A (MPLA), (b) a saponin, and (c) a liposome composition, said liposome composition comprising (i) at least one phospholipid selected from phosphatidylcholine (PC) and / or phosphatidylglycerol (PG) and (ii) cholesterol, wherein said 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, wherein the molar percentage concentration of said cholesterol in said liposome composition is greater than 50% (mol / mol), said method comprising the following steps: (i) Dissolving the phospholipids, cholesterol, and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase; (ii) The organic phase from step (i) is mixed in a microfluidic mixer at a specific flow rate and a specific ratio of the organic phase to the aqueous phase to form intermediate liposomes, wherein the aqueous phase comprises a buffer or water; (iii) Remove the organic phase of the intermediate liposomes described in step (ii); (iv) Concentrate the intermediate liposomes described in step (iii); (v) The intermediate liposomes from step (iv) are aseptically filtered; (vi) Sterilely filter the saponin; and (vii) The intermediate liposomes from step (v) and the saponin from step (vi) are sterilized and mixed to form a final adjuvant formulation having a size range of about 30 to 400 nm and a polydispersity index of 0.05 to 0.

50. The homogeneous adjuvant formulation is thus prepared.

48. The method according to claim 47, wherein the saponin is selected from the group consisting of QS-7, QS-18, QS-21 or mixtures thereof.

49. The method according to claim 48, wherein the saponin is QS-21.

50. The method according to any one of claims 47 to 49, wherein the liposome composition comprises dimyristic phosphatidylcholine (DMPC) and dimyristic phosphatidylglycerol (DMPG).

51. The method according to any one of claims 47 to 50, wherein in step (i), phospholipids, cholesterol and MPLA are dissolved in an organic solvent by ultrasonic treatment, heating or a combination thereof.

52. The method according to claim 51, wherein the organic solvent is ethanol or isopropanol.

53. The method according to claim 51 or 52, wherein the organic phase is heated to a temperature between 45°C and 65°C.

54. The method according to any one of claims 47 to 53, wherein the buffer solution in step (ii) comprises 10 mM phosphate and 150 mM NaCl at pH 6.

2.

55. The method according to any one of claims 47 to 54, wherein the aqueous phase in step (ii) is at a temperature between 20°C and 25°C.

56. The method according to any one of claims 47 to 55, wherein the microfluidic mixer in step (ii) is injected using a pump or syringe.

57. The method according to claim 56, wherein the microfluidic mixer in step (ii) is a Y-connector, a T-connector, or a coaxial microfluidic mixer.

58. The method according to claim 56 or 57, wherein the microfluidic mixer in step (ii) has a length ranging from 300 µm to 1,000 µm.

59. The method according to any one of claims 47 to 58, wherein the flow rate in step (ii) is from 12 mL / min to 240 mL / min or up to 3 L / min.

60. The method according to any one of claims 47 to 59, wherein the mass ratio of the water in step (i) to the organic phase in step (ii) is in the range of 8:1 to 3:1, or 5:1 to 3:

1.

61. The method according to any one of claims 47 to 60, wherein the removal of the organic phase of the intermediate liposome in step (iii) and the concentration in step (iv) are performed by tangential flow filtration (TFF).

62. The method of claim 61, wherein the TFF is TFF percolation, ultrafiltration, or both.

63. The method according to claim 61 or 62, wherein the TFF comprises a membrane having a molecular weight cutoff (MWCO) in the range of 100 to 500 kDa.

64. The method according to any one of claims 47 to 63, wherein the aseptic filtration in steps (v) and (vi) comprises a bioburden reduction filter and a aseptic filter.

65. The method of claim 64, wherein the bioburden reduction filter has a diameter of 0.45 microns.

66. The method of claim 64 or 65, wherein the sterile filter has a diameter of 0.22 micrometers.

67. A homogeneous adjuvant formulation prepared by the method according to any one of claims 1 to 66, wherein the adjuvant formulation has a size range between about 30 nm and about 200 nm and a polydispersity index between about 0.05 and about 0.

30.

68. The adjuvant formulation of claim 67, wherein the adjuvant formulation has a size range between about 100 nm and about 150 nm.

69. The adjuvant formulation according to claim 67 or 68, wherein the adjuvant formulation has a polydispersity index between about 0.05 and about 0.

2.

70. The adjuvant formulation according to any one of claims 67 to 69, wherein the adjuvant formulation has a polydispersity index of about 0.

1.

71. The adjuvant formulation according to any one of claims 67 to 70, wherein the adjuvant formulation comprises monophosphoryl 3-deacyl lipid A phosphorylated hexaacyl disaccharide (3D-PHAD), QS-21, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycerol-3-phospho-(1'-rac-glycerol) (DMPG) and cholesterol.

72. The adjuvant formulation according to any one of claims 67 to 71, wherein the adjuvant formulation comprises 3D-PHAD, QS-21, DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

73. The adjuvant formulation according to any one of claims 67 to 72, wherein the adjuvant formulation comprises 3D-PHAD at a concentration of about 0.4 mg / ml or about 0.8 mg / ml.

74. The adjuvant formulation according to any one of claims 67 to 73, wherein the adjuvant formulation comprises DMPC at a concentration of about 14 mg / ml or about 28 mg / ml.

75. The adjuvant formulation according to any one of claims 67 to 74, wherein the adjuvant formulation comprises DMPG at a concentration of about 1.6 mg / ml or about 3.2 mg / ml.

76. The adjuvant formulation according to any one of claims 67 to 75, wherein the adjuvant formulation comprises cholesterol at a concentration of about 11 mg / ml or about 22 mg / ml.

77. The adjuvant formulation according to any one of claims 67 to 76, wherein the adjuvant formulation comprises QS-21 at a concentration of about 0.2 mg / ml or about 0.4 mg / ml.

78. The adjuvant formulation according to any one of claims 67 to 77, wherein the adjuvant formulation comprises DMPC and DMPG, and the molar ratio of DMPC to DMPG (mol / mol) is between about 10:1 and about 8:

1.

79. The adjuvant formulation according to any one of claims 71 to 78, wherein the adjuvant formulation has a cholesterol:phospholipid molar ratio between about 55:50 and about 55:

40.

80. The adjuvant formulation according to any one of claims 71 to 79, wherein the adjuvant formulation has a 3D-PHAD:phospholipid molar ratio between about 1:80 and about 1:

95.

81. An immunogenic composition comprising an adjuvant formulation and an immunogen as described in any one of claims 67 to 80.

82. A method of inducing an immune response in an individual, comprising administering to the individual the immunogenic composition as described in claim 81.

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