WO2025057058A1

WO2025057058A1 - Methods for producing an adjuvant

Info

Publication number: WO2025057058A1
Application number: PCT/IB2024/058787
Authority: WO
Inventors: Naveen PALATH; Kirk ROFFI
Original assignee: Pfizer Inc.
Priority date: 2023-09-13
Filing date: 2024-09-10
Publication date: 2025-03-20

Abstract

This invention provides methods for producing homogeneous adjuvant formulations comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome composition comprising (i) at least one phospholipid selected from 5 phosphatidylcholine (PC) and/or phosphatidylglycerol (PG), 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 a combination thereof, and (ii) cholesterol wherein the mole 0 percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), and further provides the homogeneous adjuvant formulations produced from said methods.

Description

METHODS FOR PRODUCING AN ADJUVANT

FIELD OF THE INVENTION

This invention provides methods for producing homogeneous adjuvant formulations comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol, wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), and further provides the homogeneous adjuvant formulations produced from said methods.

BACKGROUND OF THE INVENTION

A commercial liposomal adjuvant formulation, known as AS01 , containing both monophosphoryl lipid A (MPLA) and QS-21 saponin, was used as the adjuvant for a Shingles vaccine in 2017 in the United States for adults >50 years of age. The US Army has developed another liposome-based formulation containing both MPLA and QS-21 saponin, which is known as ALFQ (Army Liposome Formulation Q). ALFQ has been shown to be potent as a liposomal vaccine adjuvant in rodents and non-human primate studies, and it has also been shown to be non-pyrogenic and nontoxic in preclinical studies. The improved safety profile of ALFQ can be attributed to the irreversible binding by liposomal cholesterol to free QS-21 to form a complex that prevents hemolysis resulting from the binding of QS-21 to erythrocytes. The size of ALFQ increases from 50 to 100 nm to as large as approximately 30,000 nm during the manufacture of ALFQ when soluble QS-21 is added to a suspension of the liposome intermediate. As described in the literature, the ALFQ liposome intermediate is prepared by a rehydration process (Beck et al., Biochimica et Biophysica Acta 1848 (2015) 775-780; Singh et al., Biochemical and Biophysical Research Communications 529 (2020) 362-365; Matyas et al., Methods in Enzymology 373 (2003) 34-50). In that process, lipids are mixed and dissolved in organic solvent, dried under vacuum, and then liposomes are formed in PBS and downsized by microfluidizer to 30-100 nm. In the most widely used of these methods, a thin lipid film (from an organic solvent) is deposited on the walls of a container, an aqueous solution of the material to be encapsulated is added, and the container is agitated (Bangham et al., J. Mol. Biol. 13 (1965) 238-252). Under the right conditions, this process results in the formation of multilamellar vesicles of liposomes. However, this method is not generally scalable for manufacturing, due to size limitations of the equipment used, i.e. a rotary evaporator (e.g. Rotavap).

In another process, an organic solution of lipid is freeze-dried, resulting in a lyophilized product with physical properties for easy hydration by an aqueous solution of the material to be encapsulated. This method of production is limited due to the variability of drug encapsulation in the liposome between batches. For example, Conrad et al. (Biochim. Biophys. Acta 332 (1974) 36-46) shows that a standard deviation in encapsulation efficiency of 12-13% was found between independently prepared liposome preparations using this method.

Therefore, there is a need for reproducible processes that can control the size and polydispersity of liposomal adjuvant formulations containing MPLA and a saponin including, but not limited to, QS-21 . Additionally, there is a need for processes that are sufficiently robust to be used at large scale, i.e. scalable for clinical and commercial manufacturing.

SUMMARY OF THE INVENTION

This invention provides methods for producing a homogeneous adjuvant formulation as described herein that are 1) more reproducible and well-controlled enabling the manufacture of a homogeneous liposomal adjuvant formulation having a defined size and polydispersity, and 2) more scalable than traditional batch methods such as those described above (e.g. rehydration process) or bulk mixing. The invention leverages the advantages of semi-continuous flow chemistry, that is, by forming liposomes in a microfluidic mixer, wherein process parameters such as the chemical composition, flow regime, flowrate, and temperature can be precisely controlled resulting in a reproducible product (i.e. homogeneous). A semi-continuous flow process is more scalable because it’s not limited by the size of the vessel (as is the case with a rotary evaporator), rather more microfluidic mixers can be added in-parallel and the volumetric flowrate increased as needed to achieve the desired throughput for clinical or commercial scale. These advantages enable the establishment of a Process Control Strategy in compliance with FDA guidance for good manufacturing practice (CGMP) which includes both examination of material quality and equipment monitoring in order to assure that in-process materials and the finished product meet predetermined quality requirements and do so consistently and reliably (see https://www.fda.gov/files/drugs/published/Process-Validation-General-Principles-and- Practices.pdf, pages 5 and 9). Another advantage of the invention is that the liposomal adjuvant formulations formed according to the methods described herein do not require the additional steps of downsizing unlike those described in PCT International Application No. PCT/IB2023/052255. Accordingly, this invention provides reproducible processes that can control the size and polydispersity of homogeneous liposomal adjuvant formulations containing MPLA and a saponin for large scale manufacturing (i.e. scalable manufacturing) in amounts sufficient for clinical and commercial use.

This invention provides a first method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(i) dissolving the phospholipids, cholesterol and MPLA in an organic solvent or a mixture of organic solvents to form an organic phase;

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(iii) mixing the organic phase of step (i) into an aqueous phase of step (ii) in a microfluidic mixer at a specific flowrate and at a specific ratio of the organic phase to the aqueous phase to form a saponin-containing liposome;

(iv) removing the organic phase of the saponin-containing liposome of step (iii);

(v) concentrating the saponin-containing liposome of step (iv); and

(vi) sterile filtering the saponin-containing liposome of step (v), to form a final adjuvant formulation having a size range of about 30-200 nm with a polydispersity index of 0.05 to 0.30, thereby producing the homogeneous adjuvant formulation.

In an embodiment, said first method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1 , said method comprising the steps of:

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(v) concentrating the saponin-containing liposome of step (iv); and

This invention also provides a second method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(ii) mixing the organic phase of step (i) into an aqueous phase, wherein the aqueous phase comprises a buffer or water, in a microfluidic mixer at a specific flowrate and at a specific ratio of the organic phase to the aqueous phase to form an intermediate liposome;

(iii) mixing the intermediate liposome of step (ii) with a saponin, wherein the saponin is first dissolved in a buffer or water, to form a saponin-containing liposome;

(iv) removing the organic phase of the saponin-containing liposome of step (iii); (v) concentrating the saponin-containing liposome of step (iv); and

In an embodiment, said second method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1 , said method comprising the steps of:

(v) concentrating the saponin-containing liposome of step (iv); and

This invention further provides a third method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(iii) removing the organic phase of the intermediate liposome of step (ii);

(iv) concentrating the intermediate liposome of step (iii);

(v) sterile filtering the intermediate liposome of step (iv);

(vi) sterile filtering the saponin; and

(vii) aseptically mixing the intermediate liposome of step (v) and the saponin of step (vi) to form a final adjuvant formulation having a size range of about 30-400 nm with a polydispersity index of 0.05 to 0.50, thereby producing the homogeneous adjuvant formulation.

In an embodiment, said third method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the molar ratio of cholesterol to phospholipid in the liposome composition is greater than 1 , said method comprising the steps of:

(ii) mixing the organic phase of step (i) into an aqueous phase, wherein the aqueous phase comprises a buffer or water, in a microfluidic mixer at a specific flowrate and at a specific ratio of the organic phase to the aqueous phase to form an intermediate liposome; (iii) removing the organic phase of the intermediate liposome of step (ii);

(iv) concentrating the intermediate liposome of step (iii);

(v) sterile filtering the intermediate liposome of step (iv);

(vi) sterile filtering the saponin; and

This invention also provides an adjuvant formulation produced by any one of the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the process to form homogeneous liposomal adjuvants wherein saponin is added to the aqueous phase.

FIG. 2 shows a schematic representation of the process to form homogeneous liposomal adjuvants wherein saponin is added inline to intermediate liposomes.

FIG. 3 shows an overview of the process for manufacturing homogeneous liposomal adjuvants as described in Example 3.

FIG. 4 depicts the neutralization titers of individual animals (rats) immunized with C. difficile toxoid antigens formulated with different LiNA-2 adjuvants (homogeneous and heterogeneous), as described in Example 5.

FIG. 5 depicts a schematic cross-sectional view, in an axial plane, of a coaxial mixer.

FIG. 6 depicts a Dynamic light scattering (DLS) response surface plot of the data obtained with intermediate liposomes produced by a coaxial mixer, as described in Example 6. In this figure, the z-average particle size vs. total flowrate vs. aqueous phase:organic phase ratio (v/v) relationship is depicted.

DETAILED DESCRIPTION OF THE INVENTION

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

Multiple processes are described herein to prepare homogeneous liposomal adjuvant formulations scalable for manufacturing. The homogeneous processes are controlled and robust processes generating size-controlled liposomes.

Examples 1 -3 describe processes that are scalable for manufacturing homogeneous liposomal adjuvant formulations. The processes described herein create a homogeneous formulation having liposomes with a size range less than 1 micrometer and a controlled polydispersity index (PDI) ranging from 0.05 to 0.5. The processes described herein are reproducible and well-controlled which enables the manufacture of a homogeneous liposomal adjuvant formulation having a defined size.

Also described herein are multiple solvent injection methods via microfluidic approach. As used herein, “microfluidic mixing” shall include mixing to achieve a thorough and rapid mixing of multiple streams in microscale devices like s microchannels within microfluidic mixers. In such devices, sample mixing is essentially achieved by enhancing the diffusion effect between the flow of the different species (e.g. organic phase and aqueous phase) to prepare the intermediate liposomes or final liposomal adjuvant drug product. This method involves mixing of lipid solution in ethanol or other organic solvents (eg. isopropyl alcohol) with aqueous buffer. In Example 1 , the saponin is dissolved in the aqueous phase and forms the final liposomal adjuvant as one continuous process. In Example 2, the saponin in buffer is added in-line with the intermediate liposomes formed from mixing of organic and aqueous phase and forms the final liposomal adjuvant as one continuous process. In Example 3 the intermediate liposome is aseptically compounded (i.e. mixed) with saponin QS-21 in buffer to form the final adjuvant drug product. The processes are performed at room temperature. The size of intermediate liposomes can be generated from 30 nm to 400 nm with a PDI <0.5. The size and PDI of the intermediate and the final adjuvant drug product can be controlled by adjusting the parameters of solvent injection such as: temperature, flow rate, or flow rate ratio of organic to aqueous phases and type of mixer.

Overall, provided herein are novel processes to prepare homogeneous adjuvant formulations comprising a MPLA-containing liposome composition comprising a saponin (e.g., QS-21). These processes can control the size and polydispersity of the liposomal adjuvant formulations and are easily scalable for clinical and commercial manufacturing and reproducible compared to conventional processes. In addition, provided herein is a novel homogeneous MPLA-containing liposome composition comprising a saponin (e.g. QS-21).

Accordingly, this invention provides a first method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(v) concentrating the saponin-containing liposome of step (iv); and

(vi) sterile filtering the saponin-containing liposome of step (v), to form a final adjuvant formulation having a size range of about 30-200 nm with a polydispersity index of 0.05 to 0.30, thereby producing the homogeneous adjuvant formulation. In one embodiment of the first method for producing a homogeneous adjuvant composition, the saponin is selected from the group consisting of QS-7, QS- 18, QS-21 , or a mixture thereof. In a preferred embodiment the saponin is QS-21 .

In another embodiment of the first method for producing a homogeneous adjuvant composition, wherein the saponin is in an amount from about 0.07 mg/ml to about 0.35 mg/ml. In one aspect, the amount of the saponin is about 0.07 mg/ml. In a preferred aspect, the amount of the saponin is 0.07 mg/ml.

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

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

In one embodiment of the first method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises a chelating agent, for example, EDTA. In one embodiment of the first method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises phosphate-buffered saline (PBS). In one embodiment of the first method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises phosphate-buffered saline (PBS) and EDTA. In another embodiment of the first method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2. In another embodiment of the first method for producing a homogeneous adjuvant composition, the buffer of step (ii) has a pH of 6.2 and comprises 10 mM phosphate, 150 mM NaCI, and ETDA.

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

In another embodiment of the first method for producing a homogeneous adjuvant composition, the mass ratio of aqueous phase of step (ii) to organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1 . In a preferred embodiment, the ratio is 3:1 .

In another embodiment of the first method for producing a homogeneous adjuvant composition, the microfluidic mixer of step (iii) uses a pump or syringe injection. In one aspect, the microfluidic mixer of step (iii) is a Y-junction, T-junction or coaxial microfluidic mixer. In another aspect, the microfluidic mixer of step (iii) has an internal diameter size ranging from 300 pm to 1 ,000 pm. In another embodiment of the first method for producing a homogeneous adjuvant composition, wherein removing the organic phase of the saponin-containing liposome of step (iv) is by Tangential Flow Filtration (TFF). In another aspect, the TFF is TFF diafiltration. In another embodiment of the first method for producing a homogeneous adjuvant composition, the concentrating of step (v) is by TFF, wherein TFF comprises diafiltration, ultrafiltration or both.

In another aspect, the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa. In one aspect, the membrane is a hollow fiber membrane, a cassette membrane or a spin centrifugation membrane.

In another embodiment of the first method for producing a homogeneous adjuvant composition, the filtering of step (vi) comprises a bioburden reduction filter and a sterile filter. In one aspect, the bioburden reduction filter is up to 0.45 microns. In another aspect, the sterile filter is up to 0.22 microns.

In another embodiment of the first method for producing a homogeneous adjuvant composition, wherein the concentrating of step (v) and the filtering of step (vi) occur at room temperature.

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(iv) removing the organic phase of the saponin-containing liposome of step (iii); (v) concentrating the saponin-containing liposome of step (iv) to form a final adjuvant formulation having a size range of about 30-200 nm with a polydispersity index of 0.05 to 0.30, thereby producing the homogeneous adjuvant formulation.

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(v) concentrating the saponin-containing liposome of step (iv); and

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

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

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

In one embodiment of the second method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises a chelating agent, for example, EDTA. In one embodiment of the second method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises phosphate-buffered saline (PBS). In one embodiment of the second method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises phosphate-buffered saline (PBS) and EDTA. In another embodiment of the second method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2. In another embodiment of the second method for producing a homogeneous adjuvant composition, the buffer of step (ii) has a pH of 6.2 and comprises 10 mM phosphate, 150 mM NaCI, and ETDA.

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

In another embodiment of the second method for producing a homogeneous adjuvant composition, the mass ratio of aqueous phase of step (ii) to organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1 . In a preferred embodiment, the ratio is 3:1 .

In another embodiment of the second method for producing a homogeneous adjuvant composition, the microfluidic mixer of step (ii) uses a pump or syringe injection. In one aspect, the microfluidic mixer of step (ii) is a Y-junction, T-junction or coaxial microfluidic mixer. In another aspect, the microfluidic mixer of step (ii) has an internal diameter size ranging from 300 pm to 1 ,000 pm.

In another embodiment of the second method for producing a homogeneous adjuvant composition, wherein removing the organic phase of the saponin-containing liposome of step (iv) is by Tangential Flow Filtration (TFF). In another aspect, the TFF is TFF diafiltration. In another embodiment of the second method for producing a homogeneous adjuvant composition, wherein the concentrating of step (v) is by TFF, wherein TFF comprises diafiltration, ultrafiltration or both.

In another embodiment of the second method for producing a homogeneous adjuvant composition, wherein the filtering of step (vi) comprises a bioburden reduction filter and a sterile filter. In one aspect, the bioburden reduction filter is up to 0.45 microns. In another aspect, the sterile filter is up to 0.22 microns.

In another embodiment of the second method for producing a homogeneous adjuvant composition, wherein the concentrating of step (v) and the filtering of step (vi) occur at room temperature.

(v) concentrating the saponin-containing liposome of step (iv);

(iv) concentrating the intermediate liposome of step (iii);

(v) sterile filtering the intermediate liposome of step (iv);

(vi) sterile filtering the saponin; and

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

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

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

In one embodiment of the third method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises a chelating agent, for example, EDTA. In one embodiment of the third method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises phosphate-buffered saline (PBS). In one embodiment of the third method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises phosphate- buffered saline (PBS) and EDTA. In another embodiment of the third method for producing a homogeneous adjuvant composition, the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2. In another embodiment of the third method for producing a homogeneous adjuvant composition, the buffer of step (ii) has a pH of 6.2 and comprises 10 mM phosphate, 150 mM NaCI, and ETDA.

In another aspect, the aqueous phase of step (ii) is at a temperature between 20°C to 25°C. In another aspect, the flowrate of step (ii) is 12 mL/min to 240 mL/min or up to 3L/min. In another embodiment of the third method for producing a homogeneous adjuvant composition, the mass ratio of aqueous phase of step (ii) to organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1 . In a preferred embodiment, the ratio is 3:1 .

In another embodiment of the third method for producing a homogeneous adjuvant composition, the microfluidic mixer of step (ii) uses a pump or syringe injection. In one aspect, the microfluidic mixer of step (ii) is a Y-junction, T-junction or coaxial microfluidic mixer. In another aspect, the microfluidic mixer of step (ii) has an internal diameter size ranging from 300 pm to 1 ,000 pm.

In another embodiment of the third method for producing a homogeneous adjuvant composition, wherein removing the organic phase of the intermediate liposome of step (iii) is by Tangential Flow Filtration (TFF). In another aspect, the TFF is TFF diafiltration, ultrafiltration or both. In another aspect, the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa. In one aspect, the membrane is a hollow fiber membrane, a cassette membrane or a spin centrifugation membrane.

In another embodiment of the third method for producing a homogeneous adjuvant composition, wherein the sterile filtering of step (v) and step (vi) comprises a bioburden reduction filter and a sterile filter. In one aspect, the bioburden reduction filter is up to 0.45 microns. In another aspect, the sterile filter is up to 0.22 microns.

(iii) removing the organic phase of the intermediate liposome of step (ii); (iv) concentrating the intermediate liposome of step (iii);

(v) sterile filtering the intermediate liposome of step (iv);

(vi) sterile filtering the saponin; and

This invention also provides a homogeneous adjuvant formulation produced by any one of the methods described herein.

In one aspect, the liposome composition of the adjuvant formulation may comprise cholesterol at a mole percent concentration of over 50% (mol/mol), of about 55% to about 71 % (mol/mol), or preferably about 55% (mol/mol). In one aspect, the liposome composition of the adjuvant formulation may comprise a phosphatidylcholine phospholipid (PC) selected from the group consisting of: dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearyl phosphatidylcholine (DSPC). In another aspect, the liposome composition of the adjuvant formulation may comprise a phosphatidylglycerol phospholipid (PG) selected from the group consisting of: dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). In a further aspect, the liposome composition of the adjuvant formulation may comprise a combination of (i) a phosphatidylcholine phospholipid (PC) selected from the group consisting of: dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and distearyl phosphatidylcholine (DSPC), and (ii) a phosphatidylglycerol phospholipid (PG) selected from the group consisting of: dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). The liposome composition of the adjuvant formulation may have a ratio of the PC to the PG (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 1 1 :1 , about 12:1 , about 13:1 , about 14:1 , or about 15:1 . The liposome composition of the adjuvant formulation may comprise multi-lamellar vesicles (MLV) or small uni-lamellar vesicles (SUV), wherein small uni-lamellar vesicles are about 50 to about 100 nm in diameter, and wherein multi- lamellar vesicles are about 30 nm to about 400 nm in diameter. In a preferred embodiment, the liposome composition of the adjuvant formulation comprises a PC and a PG, wherein the PC is dimyristoyl phosphatidylcholine (DMPC) and the PG is dimyristoyl phosphatidylglycerol (DMPG), having a mole ratio of PC to PG (mol/mol) of about 9:1 . In another aspect, the liposome composition of the adjuvant formulation may comprise about 5 mg or less, about 4 mg or less, about 3 mg or less, about 2 mg or less, about 1 mg or less, about 0.9 mg or less, about 0.8 mg or less, about 0.7 mg or less, about 0.6 mg or less, about 0.5 mg or less, about 0.4 mg or less, about 0.3 mg or less, about 0.2 mg or less, about 0.1 mg or less, about 0.09 mg or less, about 0.08 mg or less, about 0.07 mg or less, about 0.06 mg or less, about 0.05 mg or less, about 0.04 mg or less, about 0.03 mg or less, about 0.02 mg or less, or about 0.01 mg or less of MPLA (total weight per ml liposome suspension). The liposome composition of the adjuvant formulation may have a MPLA:phospholipid mole ratio of about 1 :5.6 to about 1 :880, or about 1 :88 to about 1 :220. In a preferred embodiment, the liposome composition of the adjuvant formulation comprises a PC and a PG, wherein the PC is dimyristoyl phosphatidylcholine (DMPC) and the PG is dimyristoyl phosphatidylglycerol (DMPG), having a MPLA:phospholipid mole ratio of about 1 :220, about 1 :88 or about 1 :5.6, preferably 1 :88.

In a further aspect, the adjuvant formulation may have a content of saponin (total weight per ml 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 comprises a content of saponin of about 0.15 to 0.4 mg/ml.

In another aspect, the adjuvant formulation is a homogeneous adjuvant composition comprising liposomes that range in size from between about 1 nm and about 500 nm. In some embodiments, the liposomes within the adjuvant formulation range in size from 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 range in size from 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 range in size from 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 of 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.

In a further aspect, the adjuvant formulation is a homogeneous adjuvant composition comprising liposomes that have 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 that have a PDI 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.

In yet another aspect, the adjuvant formulation is a heterogeneous adjuvant composition comprising liposomes that range in size from between about 1 nm and about 10 pM. In some embodiments, the liposomes within the adjuvant formulation range in size from between about 30 nm and about 4 pM. In other embodiments, the liposomes within the adjuvant formulation range in size from between about 30 nm and about 1400nm. In still other embodiments, the liposomes within the adjuvant formulation range in size from between about 30 nm and about 1000 nm. In some embodiments, the liposomes within the adjuvant formulation range in size from 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 range in size from between about 300 nm and about 1000 nm. In other embodiments, the liposomes within the adjuvant formulation have a size of 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 of greater than 300 nm.

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

Also provided is an immunogenic composition comprising an immunogen and the adjuvant formulation. The immunogenic composition may typically comprise a physiologically acceptable vehicle. The immunogen of the immunogenic composition can be selected from the group consisting of a naturally-occurring or artificially-created protein, a recombinant protein, a glycoprotein, a peptide, a carbohydrate, a hapten, a whole virus, a bacterium, a protozoan, and a virus-like particle. A method of immunizing an animal comprising administering the immunogenic composition is also provided.

Further provided is a method of reducing toxicity of a saponin as an adjuvant or preparing an adjuvant formulation comprising adding a monophosphoryl lipid A (MPLA)-containing liposome composition to the saponin, wherein the liposome composition comprises i) a lipid bilayer comprising phospholipids in which the hydrocarbon chains have a melting temperature in water of >23° C and ii) cholesterol at a mole percent concentration of greater than about 50% (mol/mol). The saponin may be selected from the group consisting of QS-7, QS-18, QS-21 , and a mixture thereof. Preferably, the saponin is QS-21. The liposome composition may comprise cholesterol at a mole percent concentration of about 55% to about 71 % (mol/mol), preferably about 55% (mol/mol).

1 . Definitions

An “immunogen” is an agent capable of inducing humoral and/or cell-mediated immune response. The immunogen as described herein can be an antigen, a hapten, or an inactivated pathogen. An immunogenic composition as described herein can be, for example, a vaccine formulation.

As used herein, the term “homogeneous” shall mean a final adjuvant formulation comprising liposomes having a size range of about 30 nm - 400 nm as determined by methods known in the art including, but not limited to, Dynamic light scattering (DLS), Transmission electron microscopy or Cryogenic electron microscopy (e.g. cryo-TEM or cryo-EM), Nanoparticle Tracking Analysis (NTA, e.g. ViewSizer). A “homogeneous” adjuvant formulation may also mean a final adjuvant formulation comprising liposomes having a polydispersity index (PDI) of between about 0.05 to 0.5 or between about 0.05 to about 0.3, preferably about 0.3. Polydispersity is measured by a polydispersity index (PDI). Calculations used for the determination of size and PDI parameters may be found in the ISO standard documents 13321 :1996 E and ISO 22412:2008 (Worldwide M.l. Dynamic Light Scattering, Common Terms Defined. Malvern Instruments Limited; Malvern, UK: 2011. Pp. 1-6. Inform White Paper). As used herein a “homogeneous” adjuvant formulation shall also mean a “monodisperse” adjuvant formulation.

As used herein, the following terms have the same meaning and are used interchangeably: “homogeneous adjuvant formulation”, “homogeneous liposomal adjuvant formulation”, “homogeneous liposomal adjuvant”, “homogeneous LiNA-2", “liposomal adjuvant drug product”, “final adjuvant drug product”, “final liposomal adjuvant drug product”, “final homogeneous adjuvant drug product”, “final adjuvant formulation”, and the like.

As used herein, the term “buffer” shall mean any solution that resists changes in pH when acid or alkali is added to it. In some embodiments, the buffers disclosed herein comprise a chelating agent, for example, EDTA.

As used herein, the term “size” shall refer to the diameter size of a particle or a population of particles. The diameter size 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 size is provided as the z-average of a population of particles. In some embodiments, the diameter size is provided as the mean of a population of particles. In some embodiments, the diameter size is provided as the D10, D50, D90, etc. of a population of particles. For example, in some embodiments, the D90 value of a population of particles is the value at which 90% of the particles in the population have a smaller diameter.

“Liposomes” as used herein refer to closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be uni-lamellar vesicles possessing a single membrane bilayer or multi-lamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase. Liposomes, as they are ordinarily used, consist of smectic mesophases, and can consist of either phospholipid or nonphospholipid smectic mesophases. Smectic mesophase is most accurately described by Small, HANDBOOK OF LIPID RESEARCH, Vol. 4, Plenum, N.Y., 1986, pp. 49-50. According to Small, “[w]hen a given molecule is heated, instead of melting directly into an isotropic liquid, it may instead pass through intermediate states called mesophases or liquid crystals, characterized by residual order in some directions but by lack of order in others. In general, the molecules of liquid crystals are somewhat longer than they are wide and have a polar or aromatic part somewhere along the length of the molecule. The molecular shape and the polar-polar, or aromatic, interaction permit the molecules to align in partially ordered arrays. These structures characteristically occur in molecules that possess a polar group at one end. Liquid crystals with long-range order in the direction of the long axis of the molecule are called smectic, layered, or lamellar liquid crystals. In the smectic states the molecules may be in single or double layers, normal or tilted to the plane of the layer, and with frozen or melted aliphatic chains.”

Lipid A is a set of complex, heavily acylated and amidated diglucosamine diphosphate molecules and is the lipid moiety common to all lipopolysaccharides (LPS; also known as endotoxin) from Gram-negative bacteria. LPS covers virtually the entire outer surface of all Gramnegative bacteria, and lipid A anchors the LPS into the outer lipid surface of the bacterium. The O-polysaccharide portion of LPS in wild-type smooth bacteria is linked to a relatively conserved core oligosaccharide that is expressed in rough mutants, and this in turn is linked to lipid A through highly conserved 2-keto-3-deoxyoctanoic acid sugars that are unique chemical structures sometimes required for bacterial viability and found only in LPS. See, e.g., Alving et al., 2012, Expert Rev. Vaccines 11 : 733-44. “Monophosphoryl lipid A” is a lipid A congener in which the glucosamine-1 -phosphate group on the polar head group has been removed. Numerous congeners of MPLA also exist.

“Microfluidic mixer” as used herein is a Y-junction, T-junction or coaxial microfluidic mixer. In one aspect, the microfluidic mixer of step has an internal diameter size ranging from 300 pm to 1 ,000 pm. The microfluidic mixer forms liposomes by a semi-continuous flow process. Examples of microfluidic mixers include, but are not limited to, Y-mixer from Precision Nanosystem or T-mixer from IDEX or a coaxial mixer or any other configuration where turbulent flow is generated. “Turbulent flow” shall mean a flow in which the fluid undergoes irregular fluctuations or mixing, which is in contrast to laminar flow in which the fluid moves in smooth paths or layers. In turbulent flow the speed of the fluid at a point is continuously undergoing changes in both magnitude and direction.

In one aspect, the microfluidic mixer as used herein is a coaxial mixer described in international publication number WO 2024/057209 (Darvari et. al.). In one embodiment, the microfluidic mixer as used herein is the coaxial mixer depicted in FIG. 5. which represents a coaxial flow device (1 ) extending along a main longitudinal axis X. The coaxial mixer depicted in FIG. 5 comprises a first (outer) tube (3) having an inlet (4) for a controlled flow of the organic phase or the aqueous phase and a second (inner) tube (5) having an inlet (6) for a controlled flow of the other of the organic phase or the aqueous phase. The first tube (3) has a mixing portion (7) for the continuous mixing of the organic phase and the aqueous phase and an outlet (9) for a resulting flow of a mixed solution comprising the organic phase and the aqueous phase. The second tube (5) is coaxially arranged, along the longitudinal axis X, within the first tube (3) and has an outlet (10) axially opening into said mixing portion (7) of the first tube (3). The mixing portion (7) further includes, between the outlet (10) of the second tube (5) and the turbulent mixing portion (11), a controlled micro-mixing environment portion (15) free of obstacle for the combined flow. A disrupting physical element extends over a certain length of the mixing portion (7), in this case in the turbulent mixing portion (11), from the downstream end of the controlled micro-mixing environment portion (15) to the outlet (9) of the first tube (3) and includes an alternating helical flow path (21) in the form of a helical groove, arranged on an inner surface of the first tube (3).

The “mole percent concentration of cholesterol” of a liposome composition as used herein refers to the ratio of Cholesterol:total phospholipid (i.e., phosphatidylcholine and phosphatidylglycerol) originally used in the preparation of the liposome composition.

A “physiologically acceptable vehicle” as used herein refers to a vehicle that is suitable for in vivo administration (e.g., oral, transdermal or parenteral administration) or in vitro use, i.e., cell culture. Exemplary physiologically acceptable vehicles can be those physiologically acceptable constituents of liposomes as disclosed in U.S. Pat. Nos. 4,186,183 and 4,302,459.

“Preferred” and “preferably” as used herein are to be construed for purposes of claim construction in Europe only. The terms should be read out of or omitted from the construction of the sentences and paragraphs in which they appear for purposes of U.S. claim construction.

The term “about” as used herein refers to ± 5% of the referenced value. Unless defined otherwise, 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 as used herein, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies and reference to “the dosage” includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

2. Saponin

For the present embodiments, a suitable saponin is Quil A, its derivatives thereof, or any purified component thereof (for example, QS-7, QS-18, QS-21 , or a mixture thereof). Quil A is a saponin preparation isolated from the South American tree Quillaja Saponaria Molina and was first found to have adjuvant activity. Dalsgaard et al., 1974, Archiv. fur die gesanite Virusforschung, 44: 243-254. Purified fragments of Quil A have been isolated by HPLC (EP 0362 278), including, for example, QS-7 and QS-21 (also known as QA7 and QA21 , respectively). QS- 21 is the 21st fraction purified from the sap of Quillaja Saponaria tree. QS-21 has been shown to induce CD8+ cytotoxic T cells (CTLs), Th1 cells, and a predominant lgG2a antibody response.

3. Monophosphoryl Lipid A (MPLA)-Containinq Liposomes (L(MPLA))

Liposomes are closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be uni-lamellar vesicles possessing a single membrane bilayer or multi- lamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences as described in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; and 6,043,094. Liposomes can be made without hydrophilic polymers. Therefore, liposome formulations may or may not contain hydrophilic polymers.

Liposomes may be comprised of any lipid or lipid combination known in the art. For example, the vesicle-forming lipids may be naturally-occurring or synthetic lipids, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also be glycolipids, cerebrosides, or cationic lipids, such as 1 ,2-dioleyloxy-3-(trimethylamino)propane (DOTAP); N-[1 -(2,3,-ditetradecyloxy)propyl]-N,N- dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1 (2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethylammonium bromide (DORIE); N-[1 -(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA); 3 [N — (N',N'-dimethylaminoethane) carbamoly]cholesterol (DCChol); or dimethyldioctadecylammonium (DDAB) also as disclosed in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the proper range to impart stability to the liposome vesicle, as disclosed in U.S. Pat. Nos. 5,916,588 and 5,874,104. Additional liposomal technologies are described in U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191 ; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 4,684,479. These described liposomes and lipid-coated microbubbles, and methods for their manufacture. Thus, one skilled in the art, considering both the present disclosure and the disclosures of these other patents could produce a liposome for the purposes of the present embodiments. For the present embodiments, the liposome compositions typically contain about 1 mM to about 150 mM phospholipids.

Any of the above exemplary liposomes includes monophosphoryl lipid A (MPLA), or could be combined with other liposomes and lipid A (MPLA). MPLA alone can be toxic to humans and animals. However, when present in liposomes, the toxicity is not detected. See, e.g., Alving et al., 2012. MPLA serves as a potent adjuvant and serves to raise the immunogenicity of the liposome and peptides, proteins, or haptens associated with the liposome.

For the present embodiments, a monophosphoryl lipid A (MPLA)-containing liposome (L(MPLA)) comprises (1) a lipid bilayer comprising phospholipids in which the hydrocarbon chains have a melting temperature in water of >23° C., usually dimyristoyl phosphatidylcholine (DMPC, e.g. 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine) and dimyristoyl phosphatidylglycerol (DMPG, e.g. 1 ,2-dimyristoyl-sn-glycero-3-phospho-(1 '-rac- glycerol)); (2) cholesterol (Choi) as a stabilizer: and (3) monophosphoryl lipid A (MPLA) as an immunostimulator. The particular composition comprises cholesterol at a mole percent concentration of greater than about 50% (mol/mol), preferably about 55% to about 71 % (mol/mol), or more preferably about 55% (mol/mol). Additionally in the present embodiments, monophosphoryl lipid A (MPLA)-containing liposome (L(MPLA)) comprising a saponin (e.g. QS-21) may be a homogeneous LiNA-2 adjuvant formulation, as described herein.

For the present embodiments, an L(MPLA) may comprise a phosphatidylcholine (PC) selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and disteaiyl phosphatidylcholine (DSPC). The L(MPLA) may also comprise a phosphatidylglycerol (PG) selected from dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG). The PC to PG ratio (mol/mol) of the liposome may be about 0.5:1 , about 1 :1 , about 2:1 , about 3:1 , about 4:1 , about 5:1 , about 6:1 , about 7:1 , about 8:1 , about 9:1 , about 10:1 , about 11 :1 , about 12:1 , about 13:1 , about 14:1 , or about 15:1 . The liposome may have a content of MPLA (total weight per ml 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 liposome may have a MPLA:phospholipid mole ratio of about 1 :5.6 to about 1 :880, preferably about 1 :88 to about 1 :220. Prior to the addition of a saponin, the liposome may comprise small uni-lamellar vesicles (SUV) or bi-lamellar vesicles. The small unilamellar or bilamellar vesicles may be about 50 to about 100 nm in diameter.

4. Adjuvant Formulations Comprising MPLA-Containinq Liposomes (L(MPLA) and Saponin

An adjuvant formulation known as AS01 (also known as AS01 B or AS01 E) was previously introduced by GlaxoSmithKline. In AS01 , the lipid bilayer was comprised of a neutral lipid that is “non-crystalline” at room temperature, such as dioleoyl phosphatidylcholine, cholesterol, MPLA, and QS-21 . See U.S. Patent No. 10,039,823. During manufacture of AS01 small unilamellar liposomal vesicles (SUV) are first created and purified QS-21 is then added to the SUV. The QS- 21 imparts unique properties in that it binds to the liposomal cholesterol where it causes perforations (holes) or other permanent structural changes in the liposomes. See, e.g., Paepenmuller et al., 2014, Int. J. Pharm., 475: 138-46. A reduced amount of free QS-21 presumably resulted in reduced local injection pain often caused by free QS-21. See, e.g., Waite et al., 2001 , Vaccine, 19: 3957-67; Mbawuike et al., 2007, Vaccine, 25: 3263-69. The AS01 B formulation was created for vaccines where the induction of a yet stronger T-cell-mediated immune response is required.” See Gargon et al., 2007, Expert. Rev. Vaccines, 6: 723-39. The AS01 formulation is being developed as an adjuvant for a variety of vaccines. See Garcon & Mechelen, 2011 , Expert. Rev. Vaccines, 10: 471 -86. The ASO1 formulation, as described in U.S. Patent No. 10,039,823, may contain cholesterol (sterol) at a mole percent concentration of 1 -50% (mol/mol), preferably 20-25% (mol/mol).

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

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

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

In a further aspect, the liposome composition of the adjuvant formulation may have a MPLA:phospholipid mole ratio of about 1 :5.6 to about 1 :880, or about 1 :88 to about 1 :220. In a preferred embodiment, the liposome composition of the adjuvant formulation comprises a PC and a PG, wherein the PC is dimyristoyl phosphatidylcholine (DMPC) and the PG is dimyristoyl phosphatidylglycerol (DMPG), having a MPLA:phospholipid mole ratio 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:phospholipid mole 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 mole 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 mole ratio between about 1 :80 and about 1 :95. In another particular embodiment, the liposome composition of the adjuvant formulation comprises DMPC, DMPG, and 3D-PHAD and has a 3D-PHAD:phospholipid mole ratio of about 1 :88.

In another embodiment, the invention provides an adjuvant formulation produced by the methods described herein wherein the adjuvant formulation comprises unilamellar liposomes having a liposome bilayer that consists of: (a) at least one phosphatidylcholine (PC) and/or phosphatidylglycerol (PG), as phospholipids, selected from the group consisting of: dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and distearyl phosphatidylglycerol (DSPG); (b) cholesterol; (c) monophosphoryl lipid A (MPLA); and (d) a saponin; and wherein the mole ratio of the cholesterol (b) to the phospholipids (a) is greater than about 50:50; and wherein the unilamellar liposomes have a median diameter size in 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 mole ratio of the cholesterol (b) to the phospholipids (a) is about 55:45 to about 71 :29. In one aspect, the mole ratio of the cholesterol (b) to the phospholipids (a) is about 55:50, about 55:45, about 55:40, about 55:35, or about 55:30. In another aspect, the mole ratio of the cholesterol (b) to the phospholipids (a) is about 55:45. In another aspect, dimyristoyl phosphatidylcholine (DMPC) is selected as a phospholipid, wherein additionally dimyristoyl phosphatidylglycerol (DMPG) is selected as a phospholipid. In another aspect, both a PC and a PG are selected as phospholipids, and wherein the ratio of the PC to the PG (mol/mol) is about 0.5:1 , about 1 :1 , about 2:1 , about 3:1 , about 4:1 , about 5:1 , about 6:1 , about 7:1 , about 8:1 , about 9:1 , about 10:1 , about 11 :1 , about 12:1 , about 13:1 , about 14:1 , or about 15:1. In another aspect, the 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 the phospholipids (a), and (ii) about 5 mg/ml or less of the MPLA (c).ln another aspect, the mole ratio of the MPLA (c) to the phospholipids (a) is about 1 :5.6 to about 1 :880. In a further aspect, the 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 the phospholipids (a), and (ii) about 1 mg/ml or less of the saponin (d). In another aspect, the mole ratio of the MPLA (c) to the phospholipids (a) is about 1 :88 to about 1 :220.

Examples of adjuvant formulations comprising an MPLA-containing liposome composition and at least one saponin (e.g. QS-21) made by the methods described herein include the homogeneous adjuvant formulations described herein, namely a Liposomal Novel Adjuvant-2 (LiNA-2) homogeneous adjuvant formulation. In one aspect the LiNA-2 homogeneous adjuvant comprises a synthetic TLR4 agonist, monophosphoryl lipid A (MPLA), a triterpenoid glycoside saponin (QS-21), 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine also known as dimyristoyl phosphatidylcholine (DMPC), 1 ,2-dimyristoyl-sn-glycero-3-phospho-(1 '-rac- glycerol) also known as dimyristoyl phosphatidylglycerol (DMPG), and cholesterol. In a preferred aspect, LiNA-2 homogeneous adjuvant comprises MPLA (e.g. 3D-PHAD®), 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 between about 1 mM and 10 mM. In some embodiments, the LiNA-2 adjuvant comprises a phosphate buffer 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 a phosphate buffer of about 10 mM. In another preferred aspect, LiNA-2 homogeneous adjuvant comprises MPLA (e.g. 3D-PHAD®), QS-21 , DMPC, DMPG and cholesterol in a phosphate buffer comprising sodium chloride (NaCI). In a further particular embodiment, the LiNA-2 adjuvant comprises 3D-PHAD®, QS-21 , DMPC, DMPG, cholesterol, and 10 mM phosphate buffer.

In some embodiments, the LiNA-2 adjuvant comprises sodium chloride. In some embodiments, the LiNA-2 adjuvant comprises between about 50 mM and about 500 mM sodium chloride. 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 a phosphate buffer. In a further particular embodiment, the LiNA-2 adjuvant comprises 3D-PHAD®, QS-21 , DMPC, DMPG, cholesterol, 150 mM sodium chloride, and a 10 mM phosphate buffer.

In one aspect, the LiNA-2 homogeneous adjuvant is designed to reconstitute lyophilized powder formulation for administration. In another aspect, the LiNA-2 homogeneous adjuvant is designed to be mixed with a liquid formulation for administration.

In a further aspect, LiNA-2 homogeneous adjuvant formulation may be LiNA-2 at 1X concentration (1XLiNA-2) or LiNA-2 at 2X concentration (2XLiNA-2) as compared to ALFQ concentration (ALFQ comprising (i) 7.0 mg/mL DMPC, (ii) 0.78 mg/ml DMPG, (iii) 5.4 mg/ml cholesterol, (iv) 0.2 mg/mL MPLA (3D-PHAD), and (v) 0.1 mg/ml QS-21). In another embodiment, the adjuvant formulation is 1XLiNA-2, wherein the 1XLiNA-2 is homogeneous comprising (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 the 2XLiNA-2 is homogeneous comprising (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 . 5. Uses of Adjuvant Formulations Comprising MPLA-Containinq Liposomes (L(MPLA) and Saponin

The adjuvant formulations of the present embodiments may be used to mix with an immunogen to obtain an immunogenic composition, for example, a vaccine. The immunogenic composition may comprise a physiologically acceptable vehicle, for example, any one of those described in U.S. Pat. No. 5,888,519. The immunogenic composition may comprise naturally- occurring or artificially-created proteins, recombinant proteins, glycoproteins, peptides, carbohydrates, nucleic acids, haptens, whole viruses, bacteria, protozoa, or virus-like particles, or conjugates thereof as the immunogen. Exemplary nucleic acids or polynucleotides of the immunogenic composition include, but are not limited to, ribonucleic acids (RNAs), including mRNA, and deoxyribonucleic acids (DNAs). 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 an mRNA polynucleotide encoding a polypeptide or fragment thereof described herein. In some embodiments, the immunogenic composition comprises a modified RNA molecule (modRNA). In some embodiments, the immunogenic composition comprising a saccharide. In some embodiments, the immunogenic composition comprising a capsular bacterial saccharide.

The immunogenic composition may be suitably used as a vaccine for chickenpox or shingles, human respiratory syncytial virus infection (RSV), Cytomegalovirus infection (CMV), Human metapneumovirus, Human parainfluenza viruses type 1 or type 3, Lyme disease, Streptococcus pneumonia, Clostridioides difficile, Escherichia coli or Klebsiella pneumoniae, influenza, HIV-1 , Hepatitis A, Hepatitis B, Human Papilloma virus, Meningococcal type A meningitis, Meningococcal type B meningitis, Meningococcal type C meningitis, Tetanus, Diphtheria, Pertussis, Polio, Haemophilus influenza type B, Dengue, Hand Foot and Mouth Disease, Typhoid, Pneumococcus, Japanese encephalitis virus, Anthrax, Shingles, Malaria, Norovirus, or cancer. The immunogenic composition may be suitably used in methods for treating or preventing a disease or infection in a subject, preferably wherein the subject is 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, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1 , DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coll O157:H7, 0111 and 0104:H4, Escherichia coll Fimbrial antigen H, Fasciola he patica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (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 monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowled, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, 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, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella-zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

The present invention provides an immunogenic composition comprising an immunogen and an adjuvant formulation as described herein. In another aspect, a method for inducing an immune response in a subject comprising administering the immunogenic composition is provided. In some embodiments, the immunogenic composition increases neutralizing titers specific for the immunogen in the subject. In certain embodiments, the neutralizing titers in the subject increase by at least about 1.01 -fold, about 1.1 -fold, about 1.5-fold, about 2-fold, about 3- fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10- fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, or more after administration of the immunogenic composition. In particular embodiments, the immunogenic composition comprises LiNA-2 and the neutralizing titers in the subject increase by at least 2-fold or more. In other particular embodiments, the immunogenic composition comprises LiNA-2 and the neutralizing titers in the subject increase by at least 10-fold or more.

EXAMPLES

In orderthat this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. The following Examples illustrate some embodiments of the invention.

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

Also, in the following Examples, cholesterol content may be analyzed to confirm the cholesterol, and indirectly, the phospholipid concentration of the liposome composition by the established methods. See, e.g., Zlatkis et al., 1953, J. Lab. Clin. Med., 4V. 486-492. The cholesterol concentration of the liposome composition may be determined from a cholesterol standard curve. EXAMPLE 1 : HOMOGENEOUS ADJUVANT FORMULATION PREPARATION: A PROCESS USING MICROFLUIDIC-MIXING WITH SAPONIN ENABLING STERILE FILTRATION

Liposomal adjuvant drug product of sizes 30-200 nm amenable to sterile filtration were generated using a microfluidic-mixing process. The liposomes were formed by mixing lipids (DMPG, DMPC, cholesterol and MPLA) at 0.8X to 2X concentration in organic solvent (ethanol, isopropyl alcohol or other solvent) by sonication, heat or combination of both (organic phase), and the organic phase mixed with aqueous phase containing saponin QS-21 . The mixing happens in a microfluidic mixer. The design of the mixer could be Y-shaped, T-shaped or coaxial (Y-junction mixer from Precision Nanosystem, T-junction mixer from IDEX and coaxial mixer) or any other configuration where turbulent flow is generated. A schematic representation of the process is shown in FIG. 1.

The lipids in organic solvent (organic phase) were heated up to 65 °C. The organic phase was pumped into a buffer (aqueous phase) kept at RT (22 °C-25 °C). The buffer (aqueous phase) is 10 mM phosphate at pH 6.2 containing 150 mM NaCI and containing 0.07-0.35 mg/mL QS-21 . The lipids (organic phase) were introduced to the aqueous phase within a microfluidic chip with a Y-shaped mixing junction. Syringe pumps were used to control the total flow rate at approximately 12 mL/min at a 3:1 Aqueous:Organic volumetric ratio. The size of the liposomal adjuvant drug product formed was about 107 nm and 0.16 PDI by controlling the concentration of QS-21 in the Aqueous phase at 0.07 mg/mL while holding the Aqueous:Organic volumetric ratio constant at 3:1 and holding the total flowrate constant at 12 mL/min. Table 1 below provides data from the liposomal adjuvant drug product formed. The organic solvent in the liposomes may be removed via tangential flow filtration (TFF) using hollow fiber membranes, cassette membranes, or spin centrifugation membranes. The concentration of the final product may be adjusted by controlling the ultrafiltration and diafiltration steps. The molecular weight cut-off (MWCO) of the membranes used for TFF range from 100-500 kDa. Following TFF, the liposomal adjuvant drug product are resuspended in buffer and passed through a bioburden reduction filter (0.45 micron) and sterile filtered (0.22 micron) before use. This process allows sterile filtration of final liposomal adjuvant drug product. The above described process can be continuous or non-continuous and allows sterile filtration of the formed liposomal adjuvant drug product. Wherein “continuous process” means all the unit operations of liposome formation by microfluidic mixing of organic and aqueous phase, followed by removal of organic phase and concentration in TFF, followed by sterile filtration can be done as one continuous series of steps prior to fill finish. While in “non-continuous process”, the unit operations of microfluidic mixing, TFF and sterile filtration can be independent process steps prior to fill finish.

Table 1

EXAMPLE 2: HOMOGENEOUS ADJUVANT FORMULATION PREPARATION: A PROCESS USING MICROFLUIDIC-MIXING WITH SAPONIN ENABLING STERILE FILTRATION - ALTERNATE METHOD

Liposomes of sizes 30-200 nm were generated using a microfluidic-mixing process. The liposomes were formed by dissolving lipids (DMPG, DMPC, cholesterol and MPLA) at 0.8X to 2X concentration in organic solvent (ethanol, isopropyl alcohol or other solvent) (see Table 2) by sonication, heat or combination of both (organic phase), and mixing it with aqueous buffer. The mixing results in formation of intermediate liposomes. Once these intermediate liposomes formed, the final liposomal adjuvant drug product formed by inline addition with saponin QS-21 which is dissolved in aqueous phase (i.e buffer system). The mixing happens in a microfluidic mixer. The design of the mixer could be Y- shaped, T-shaped or co-axial (Y-junction mixer from Precision Nanosystem, T-junction mixer from IDEX and coaxial mixer) or any other configuration where turbulent flow is generated .The lipids in organic solvent (organic phase) were heated up to 65 °C. The organic phase was pumped into a buffer (aqueous phase) kept at RT (22 °C-25 °C). The buffer (aqueous phase) is 10 mM phosphate at pH 6.2 containing 150 mM NaCI . The lipids (organic phase) were introduced to the aqueous phase within a microfluidic chip with a Y-shaped, T-shaped or coaxial mixing junction. Syringe pumps, peristaltic pumps or HPLC pumps were used to control the total flow rate at approximately 12 mL/min to 240 mL/min at a 3:1 Aqueous:Organic volumetric ratio to form intermediate liposomes. QS-21 dissolved in the Aqueous phase or buffer is added to this intermediate liposome inline to form the final liposomal adjuvant drug product as depicted in Fig-2. The organic solvent in the liposomes may be removed via tangential flow filtration (TFF) using hollow fiber membranes, cassette membranes, or spin centrifugation membranes. The concentration of the final product may be adjusted by controlling the ultrafiltration and diafiltration steps. The molecular weight cut-off (MWCO) of the membranes used for TFF range from 100-500 kDa. Following TFF, the liposomal adjuvant drug product are resuspended in buffer and passed through a bioburden reduction filter (0.45 micron) and sterile filtered (0.22 micron) before use. This process allows sterile filtration of final liposomal adjuvant drug product. A schematic representation of the process is shown in FIG. 2.

The above described process can be continuous or non-continuous and allows sterile filtration of the formed liposomal adjuvant drug product. Wherein “continuous process” means all the unit operations of liposome formation by microfluidic mixing of organic and aqueous phase, followed by removal of organic phase and concentration in TFF, followed by sterile filtration can be done as one continuous series of steps prior to fill finish. While in “non- continuous process”, the unit operations of microfluidic mixing, TFF and sterile filtration can be independent process steps prior to fill finish.

EXAMPLE 3: HOMOGENEOUS ADJUVANT FORMULATION PREPARATION USING MICROFLUIDIC MIXING

Preparation of the intermediate liposome (30-200 nm)

FIG. 3 provides a brief overview of the process of manufacturing these homogeneous LiNA-2 adjuvants. The process steps involve lipid preparation in organic phase, preparation of aqueous phase, forming liposome with microfluidic mixing both solutions, removing the organic phase using tangential flow filtration followed by filtering the liposomes and compounding (l.e. a form of mixing) it with sterile filtered QS-21 solution to form the final homogeneous adjuvant drug product. The final homogeneous adjuvant drug product formed is then filled into glass vials.

Liposomes having a size range of 50-200 nm were generated using a microfluidicmixing process. The liposomes were formed by dissolving lipids (DMPG, DMPC, cholesterol and MPLA) at either 0.8X or 2X concentration in organic solvent (ethanol) by sonication, heat or combination of both (organic phase) according to Table 2. The lipids in organic solvent (organic phase) were heated up to 65 °C. The organic phase was pumped into a buffer (aqueous phase) kept at room temperature (22°C - 25°C). The buffer (aqueous phase) is 10 mM phosphate at pH 6.2 containing 150 mM NaCI or other suitable buffer system as described herein. The lipids (organic phase) were introduced to the aqueous phase using Y-shaped mixing junction or a T-shaped mixing junction with characteristic lengths ranging from 300-500 pm. The total flowrate ranged from 12 mL/min to 240 mL/min or up to 3L/min and Aqueous:Organic volumetric ratio was varied from 1 :1 to 3:1 .

The formation of intermediate liposomes was screened throughout a design space having dimensions of mixer geometry (Y-shaped, T-shaped, Co-axial or any other microfluidic mixer), Aqueous:Organic ratio, lipid concentration in the organic phase, and flowrate. hTe results are summarized in Table 3.

TABLE 2: LIPID STOCK (IN 200 PROOF ETHANOL) [ORGANIC PHASE]

TABLE 3: Conditions Tested

The organic solvent in the liposome intermediate may be removed via tangential flow filtration using hollow fiber membranes, cassette membranes, or spin centrifugation membranes. The concentration of the final product may be adjusted by controlling the ultrafiltration and diafiltration steps. The molecular weight cut-off (MWCO) of the membranes used for TFF range from 100-500 kDa. Following TFF, the intermediate liposomes are resuspended in buffer and passed through a bioburden reduction filter (0.45 micron) and a sterile filter (0.22 micron).

EXAMPLE 4: ADJUVANT FORMULATIONS

The present invention provides methods for making scalable amounts of MPLA-liposomal adjuvant formulations comprising a saponin (e.g. QS-21). Exemplary MPLA-liposomal adjuvant formulations comprising a saponin (e.g. QS-21) include, but are not limited to, the homogeneous adjuvant formulations of this invention which include a homogeneous adjuvant formulation known as LiNA-2 (Liposomal Novel Adjuvant-2) which may be at a 0.5X LiNA-2 concentration or 0.8X LiNA-2 or 1XLiNA-2 concentration or a 2XLiNA-2 concentration or any other concentration variation of 1XLiNA-2. The composition of these adjuvant formulations is set forth in Table 4 below:

TABLE 4: Adjuvant Formulations in final Drug Product

EXAMPLE 5: IMMUNOGENICITY OF C. DIFFICILE VACCINE ANTIGENS FORMULATED WITH DIFFERENT LINA-2 ADJUVANTS

The relative immunogenicity of C. difficile toxoid antigens formulated with aluminum hydroxide (AI(OH)₃) and different LiNA-2 adjuvant formulations (homogeneous and heterogeneous) was compared in rats. Homogeneous and heterogeneous LiNA-2 adjuvants are described herein and in Table 5. Final rat LiNA-2 adjuvant doses were prepared by diluting 1X concentration of LiNA-2 at a 1 :5 dilution using PBS buffer at pH 6.2. Table 5. LiNA-2 adjuvant formulation

The toxin neutralization assay (TNA) described below was used to measure the functional cytotoxic activity of sera at multiple time points following immunization.

Wistar Han rats (10 per Group, 8-10 weeks old, Charles River Laboratories) were immunized intramuscularly (IM) according to the study design in Table 6. Group 1 received C. difficile vaccine antigens formulated with AI(OH)₃. Groups 2 and 3 received the C. difficile vaccine antigens formulated with the homogeneous and heterogeneous LiNA-2 adjuvant, respectively.

Sera were collected at multiple time points and the ability to neutralize toxin cytotoxic activity was measured in TNA’s. Neutralization titers in sera from individual animals for Toxin B are shown in FIG. 4 and illustrate that toxoids formulated with homogeneous and heterogeneous LiNA-2 elicited similar immune responses able to neutralize Toxin B cytotoxicity.

Table 6. Rat study design with LiNA-2 adjuvanted formulations

Toxin Neutralization Assay (TNA)

Immune response induced by administering the composition of the present invention may be determined using a toxin neutralization assay (TNA), ELISA, or more preferably, a cytotoxicity assay, such as that described in WIPO Patent Application WO/2012/143902, U.S. Patent No. 9187536, and WIPO Patent Application WO/2014/060898, which are each incorporated by reference herein in their respective entireties.

A toxin neutralization assay (TNA) may be used to quantitate neutralizing antibodies to C. difficile toxin. In this assay, serial diluted serum may be incubated with a fixed amount of C. difficile toxin A or B. Test cells (e.g., Vero cells) may then then added and serum- toxin-cell mixture incubated under appropriate conditions (e.g., 37 °C for 6 days). The ability of the sera to neutralize the cytotoxic effect of the C. difficile toxin may be determined by and correlated to the viability of the cells. The assay utilizes the accumulation of acid metabolites in closed culture wells as an indication of normal cell respiration. In cells exposed to toxin, metabolism and CO2 production is reduced; consequently, the pH rises (e.g., to 7.4 or higher) as indicated by the phenol red pH indicator in the cell culture medium. At this pH, the medium appears red. Cell controls, or cells exposed to toxin which have been neutralized by antibody, however, metabolize and produce CO₂ in normal amounts; as a result, the pH is maintained (e.g., at 7.0 or below) and at this pH, the medium appears yellow. Therefore, C. difficile toxin neutralizing antibodies correlate with the ability of the serum to neutralize the metabolic effects of C. difficile toxin on cells as evidenced by their ability to maintain a certain pH (e.g., of 7.0 or lower). The color change of the media may be measured (e.g., at 562 nm to 630 nm) using a plate reader to further calculate the antitoxin neutralizing antibody titer at 50% inhibition of the C. difficile toxin-mediated cytotoxicity. In one aspect, the composition induces a toxin neutralizing antibody titer that is at least greater than 1-fold, such as, for example, at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 32- fold, or higher in the subject after receiving a dose of the composition than a toxin neutralizing antibody titer in the subject prior to receiving said dose, when measured under identical conditions in a toxin neutralization assay.

Briefly, in this Example, a 384-well microtiter plate was seeded with IMR-90 cells serving as the target of toxin-mediated cytotoxicity. Each test serum sample was analyzed separately for the ability to neutralize Toxin A or Toxin B. Four serial dilutions of test sera were mixed with fixed concentrations of Toxin A (TcdA) or Toxin B (TcdB) for 60 minutes in a humidified incubator (37°C/5% CO2) to allow for neutralization of the toxins to occur. All plates included a reference standard and quality controls which consisted of antitoxin antibodies of known titer to monitor assay performance. After the 60-minute incubation, the toxin-antiserum mixture was applied to the IMR-90 cell monolayers and the plates were incubated for an additional 72 hours. Viability of the IMR-90 cell monolayers was then tested using the luciferase-based CellTiter-Glo® reagent which provides a measure of ATP levels in metabolically active cells and was reported as relative luminescence units (RLU). A high ATP level indicates high cell viability and antibody mediated neutralization of TcdA or TcdB. The neutralizing antibody concentration was determined by comparing the RLU value of a test sample to the calibration curve from the antitoxin A or B reference standard using a custom Statistical Analysis System (SAS®) program. The functional antibody concentrations were expressed as arbitrary units per mL (or neutralizing units/mL) of serum. The lower limit of quantitation (LLOQ) for the TcdA and TcdB TNA assays are 75.9 and 249.7 neutralizing units/mL of serum, respectively.

EXAMPLE 6: INTERMEDIATE LIPOSOME PREPARATION USING A COAXIAL MICROFLUIDIC MIXER Intermediate liposomes were fabricated using a coaxial mixing device (depicted in FIG. 5) over a range of total flowrates and aqueous phase:organic phase volumetric ratios. The goals of the experiment were first, to test the effects of mixing parameters on the size of the intermediate liposomes and second, to demonstrate that the intermediate liposomes could be fabricated using this coaxial mixing technology in contrast to the traditional impingement jet mixers (/.e. T-mixers).

In this experiment, the intermediate liposomes were fabricated using 14:0 cardiolipin (Avanti® Polar Lipids, CAS Number 63988-21-6) as a 1 :1 molar substitute for 3D PHAD (Avanti® Polar Lipids, CAS Number 1699735-79-9). In orderto support the use of the 14:0 cardiolipin material, control experiments were run using a low-volume NanoAssembler™ Ignite™ device to ensure that the size of the intermediate liposomes generated using the 1 :1 molar substitute, that is 14:0 cardiolipin, was comparable to intermediate liposomes fabricated using 3D-PHAD.

In brief, the intermediate liposomes were formed by dissolving lipids (DMPG, DMPC, cholesterol, and 3D-PHAD or cardiolipin) in organic solvent (USP-grade 200 proof ethanol) by sonication, heat or a combination of both to form the organic phase. The lipids in the organic phase were heated up to 65 °C. The organic phase was pumped into a buffer (aqueous phase) and kept at room temperature (22°C - 25°C). The buffer (aqueous phase) was 10 mM phosphate at pH 6.2 containing 150 mM NaCI. The lipids (organic phase) were introduced to the aqueous phase using a NanoAssembler™ Ignite™ mixer device with syringe pumps. The aqueous phase:organic phase ratio was held constant at 3. Total flowrates of 6, 12, and 18 (mL/minute) were tested for both 3D-PHAD and cardiolipin.

As shown in Table 7 below, the particle size (Z-average, mean, and D90) and the PDI of the intermediate liposomes containing either 3D-PHAD or cardiolipin was measured.

TABLE 7: CONTROL EXPERIMENT TO COMPARE 3D-PHAD AND 14:0 CARDIOLIPIN

This control experiment demonstrated that intermediate liposomes fabricated with a 1 :1 molar substitute for 3D-PHAD, that is 14:0 cardiolipin, were reasonably comparable in terms of particle size, although minor differences were observed which may be attributed to assay variation.

Based on this result, a follow up experiment to fabricate the intermediate liposomes using the coaxial mixing device and 14:0 cardiolipin was completed.

In brief, the intermediate liposomes were formed by dissolving lipids (DMPG, DMPC, cholesterol and cardiolipin in organic solvent (USP-grade 200 proof ethanol) by sonication, heat or a combination of both to form the organic phase according to Table 8. The lipids in the organic phase were heated up to 65 °C. The organic phase was pumped into a buffer (aqueous phase) and kept at room temperature (22°C - 25°C). The buffer (aqueous phase) was 10 mM phosphate at pH 6.2 containing 150 mM NaCI. TABLE 8: LIPID STOCK (IN 200 PROOF ETHANOL) [ORGANIC PHASE]

The lipids (organic phase) were introduced to the aqueous phase using a coaxial mixer with peristaltic pumps. The coaxial mixer used is depicted in FIG. 5 and has the following characteristics: the mixer is constructed of stainless steel and consists of an outer chamber wherein the aqueous phase flows and a parallel, coaxial inner chamber wherein the 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 microns and a length of 30.7 mm. The difference in length between the outer and inner chambers is the volume in which the nanoprecipitation reaction initially occurs.

The aqueous phase:organic phase ratios tested were 3, 4, and 5. Different aqueous phase: organic phase ratios were achieved by varying the relative flowrate of the aqueous and organic phases to the mixer via the peristaltic pump. Total flowrates of 300, 500, 700, and 900 (mL/minute) were tested. As shown in Table 9 below, the parameters measured were z- average particle size, PDI, mean particle size, and D90.

TABLE 9: PARAMETERS OF INTERMEDIATE LIPOSOMES PRODUCED IN A COAXIAL MIXER

The DLS response surface plot is provided in FIG. 6. The z-average particle size vs. total flowrate vs. aqueous phase:organic phase ratio (v/v) relationship is depicted at a constant lipid concentration of 2X, using a 1 :1 molar replacement of cardiolipin for 3D-PHAD. The results demonstrated that the liposome size (z-average) could be tuned during fabrication by adjusting the aqueous phase:organic phase volumetric ratio and total flowrate.

The following clauses describe additional aspects of the disclosure:

C1 . A method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(v) concentrating the saponin-containing liposome of step (iv);

C2.The method of C1 , wherein the saponin is selected from the group consisting of QS-7, QS- 18, QS-21 , or a mixture thereof.

C3.The method of C2, wherein the saponin is QS-21.

C4.The method of any one of C1-C3, wherein the saponin is in an amount from about 0.07 mg/ml to about 0.35 mg/ml.

C5.The method of C4, where in the saponin is in an amount of about 0.07 mg/ml.

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

C7.The method of any one of C1 -C6, wherein in step (i) the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat or a combination thereof.

C8.The method of C7, wherein the organic solvent is ethanol or isopropyl alcohol.

C9.The method of C7 or C8, wherein the organic phase is heated to a temperature of between 45°C to 65°C.

C10. The method of any one of C1 -C9, wherein the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2.

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

C12. The method of any one of C1 -C11 , wherein the flowrate of step (ii) is 12 mL/min to 240 mL/min or up to 3 L/min.

C13. The method of any one of C1 -C12, wherein the mass ratio of aqueous phase of step (ii) to organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1 . C14. The method of any one of C1-C13, wherein the microfluidic mixer of step (iii) uses a pump or syringe injection.

C15. The method of C14, wherein the microfluidic mixer of step (iii) is a Y-junction, T-junction or coaxial microfluidic mixer.

C16. The method of C15, wherein the microfluidic mixer of step (iii) has an internal diameter size ranging from 300 pm to 1 ,000 pm.

C17. The method of any one of C1 -C16, wherein removing the organic phase of the saponin- containing liposome of step (iv) is by Tangential Flow Filtration (TFF).

C18. The method of C17, wherein the TFF is TFF d iafi Itratio n .

C19. The method of C17 or C18, wherein the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa.

C20. The method of any one of C1 -C19, wherein the concentrating of step (v) is by TFF, wherein TFF comprises diafiltration, ultrafiltration or both.

C21 . The method of any one of C1 -C20, wherein the filtering of step (vi) comprises a bioburden reduction filter and a sterile filter.

C22. The method of C21 , wherein the bioburden reduction filter is up to 0.45 microns.

C23. The method of C21 or C22, wherein the sterile filter is up to 0.22 microns.

C24. The method of any one of C1 -C23, wherein the concentrating of step (v) and filtering of step (vi) occur at room temperature.

C25. A method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(v) concentrating the saponin-containing liposome of step (iv);

C26. The method of C25, wherein the saponin is selected from the group consisting of QS-7, QS- 18, QS-21 , or a mixture thereof.

C27. The method of C26, wherein the saponin is QS-21 .

C28. The method of any one of C25-C27, wherein the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

C29. The method of any one of C25-C28, wherein in step (i) the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, or a combination thereof.

C30. The method of C29, wherein the organic solvent is ethanol or isopropyl alcohol.

C31 . The method of C29 or C30, wherein the organic phase is heated to a temperature of between 45°C to 65°C. C32. The method of any one of C25-C31 , wherein the buffer comprises 10 mM phosphate and 150 mM NaCl at pH 6.2.

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

C34. The method of any one of C25-C33, wherein the flowrate of step (ii) is 12 mL/min to 240 ml/min or up to 3L/min.

C35. The method of any one of C25-C34, wherein the mass ratio of the aqueous phase of step (ii) to the organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1 .

C36. The method of any one of C25-C35, wherein the microfluidic mixer of step (ii) uses a pump or syringe injection.

C37. The method of C36, wherein the microfluidic mixer of step (ii) is a Y-junction, T-junction or coaxial microfluidic mixer.

C38. The method of any one of C25-C37, wherein the microfluidic mixer of step (ii) has a length ranging from 300 pm to 1 ,000 pm.

C39. The method of any one of C25-C38, wherein removing the organic phase of the saponin- containing liposome of step (iv) is by Tangential Flow Filtration (TFF).

C40. The method of C39, wherein the TFF is TFF diafiltration.

C41 . The method of C39 or C40, wherein the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa.

C42. The method of any one of C25-C41 , wherein the concentrating of step (v) is by TFF, wherein the TFF comprises diafiltration, ultrafiltration or both.

C43. The method of any one of C25-C42, wherein the filtering of step (vi) comprises a bioburden reduction filter and a sterile filter.

C44. The method of C43, wherein the bioburden reduction filter is up to 0.45 microns. C45. The method of C43 or C44, wherein the sterile filter is up to 0.22 microns.

C46. The method of any one of C25-C45, wherein the concentrating of step (v) and the filtering of step (vi) occur at room temperature.

C47. A method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(iii) removing the organic phase of the intermediate liposome of step (ii);

(iv) concentrating the intermediate liposome of step (iii);

(v) sterile filtering the intermediate liposome of step (iv);

(vi) sterile filtering the saponin; and

C48. The method of C47, wherein the saponin is selected from the group consisting of QS-7, QS- 18, QS-21 , or a mixture thereof.

C49. The method of C48, wherein the saponin is QS-21 . C50. The method of any one of C47-C49, wherein the liposome composition comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).

C51 . The method of any one of C47-C50, wherein in step (i) the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, or a combination thereof.

C52. The method of C51 , wherein the organic solvent is ethanol or isopropyl alcohol.

C53. The method of C51 or C52, wherein the organic phase is heated to a temperature between 45°C to 65°C.

C54. The method of any one of C47-C53, wherein the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2.

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

C56. The method of any one of C47-C55, wherein the microfluidic mixer of step (ii) uses a pump or syringe injection.

C57. The method of C56, wherein the microfluidic mixer of step (ii) is a Y-junction, T-junction or coaxial microfluidic mixer.

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

C59. The method of any one of C47-C58, wherein the flowrate of step (ii) is 12 mL/min to 240 ml/min or up to 3L/min.

C60. The method of any one of C47-C59, wherein the mass ratio of the aqueous phase of step (i) to the organic phase of step (ii) ranges from 8:1 to 3:1 , 5:1 to 3:1 .

C61 . The method of any one of C47-C60, wherein removing the organic phase of the intermediate liposome of step (iii) and concentrating of step (iv) are by Tangential Flow Filtration (TFF).

C62. The method of C61 , wherein the TFF is TFF diafiltration, ultrafiltration or both. C63. The method of C61 or C62, wherein the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa.

C64. The method of any one of C47-C63, wherein the sterile filtering of step (v) and step (vi) comprise a bioburden reduction filter and a sterile filter.

C65. The method of C64, wherein the bioburden reduction filter is up to 0.45 microns.

C66. The method of C64 or C65, wherein the sterile filter is up to 0.22 microns.

C67. A homogeneous adjuvant formulation produced by any one of the methods according to C1-66.

C68. An adjuvant formulation obtained by the method of any one of C1-C24.

C69. An adjuvant formulation obtainable by the method of any one of C1-C24.

C70. An adjuvant formulation obtained by the method of any one of C25-C46.

C71 . An adjuvant formulation obtainable by the method of any one of C25-C46.

C72. An adjuvant formulation obtained by the method of any one of C47-C67.

C73. An adjuvant formulation obtainable by the method of any one of C47-C67.

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

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

C76. An adjuvant formulation consisting essentially of 3D-PHAD, QS-21 , DMPC, DMPG, and cholesterol. C77. An adjuvant formulation comprising 3D-PHAD, QS-21 , DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

C78. An adjuvant formulation consisting essentially of 3D-PHAD, QS-21 , DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

C79. An adjuvant formulation consisting of 3D-PHAD, QS-21 , DMPC, DMPG, cholesterol, phosphate buffer, and sodium chloride.

C80. 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.

C81 . An adjuvant formulation consisting essentially of 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.

C82. An adjuvant formulation consisting of 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.

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

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

C85. An adjuvant formulation consisting of 3D-PHAD, QS-21 , DMPC, DMPG, cholesterol, 10 mM phosphate buffer, and 150 mM sodium chloride.

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

C87. The adjuvant formulation of any one of C68 to C85, comprising 3D-PHAD at a concentration of about 0.4 mg/ml. C88. The adjuvant formulation of any one of C68 to C85, comprising 3D-PHAD at a concentration between about 0.4 mg/ml and about 1 .2 mg/ml.

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

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

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

C92. The adjuvant formulation of any one of C68 to C89, comprising DMPC at a concentration between about 14 mg/ml and about 42 mg/ml.

C93. The adjuvant formulation of any one of C68 to C89, comprising DMPC at a concentration of about 28 mg/ml.

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

C95. The adjuvant formulation of any one of C68 to C93, comprising DMPG at a concentration of about 1.6 mg/ml.

C96. The adjuvant formulation of any one of C68 to C93, comprising DMPG at a concentration between about 1 .6 mg/ml and about 4.8 mg/ml.

C97. The adjuvant formulation of any one of C68 to C93, comprising DMPG at a concentration of about 3.2 mg/ml.

C98. The adjuvant formulation of any one of C68 to C97, comprising cholesterol at a concentration between about 5 mg/ml and about 17 mg/ml.

C99. The adjuvant formulation of any one of C68 to C97, comprising cholesterol at a concentration of about 11 mg/ml. C100. The adjuvant formulation of any one of C68 to C97, comprising cholesterol at a concentration between about 10 mg/ml and about 34 mg/ml.

C101 . The adjuvant formulation of any one of C68 to C97, comprising cholesterol at a concentration of about 22 mg/ml.

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

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

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

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

C106. The adjuvant formulation of any one of C68 to C105, comprising DMPC and DMPG at a mole ratio of DMPC to DMPG (mol/mol) of between about 10:1 and about 8:1 .

C107. The adjuvant formulation of any one of C68 to C105, comprising DMPC and DMPG at a mole ratio of DMPC to DMPG (mol/mol) of about 9:1 .

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

C109. The adjuvant formulation of any one of C68 to C107, comprising DMPC, DMPG, and 3D-PHAD, wherein the 3D-PHAD:phospholipid mole ratio is about 1 :88.

C110. The adjuvant formulation of any one of C68 to C109, wherein the adjuvant formulation has a mole ratio of cholesterol: phospholipids of greater than 1. C111 . The adjuvant formulation of any one of C68 to C109, wherein the adjuvant formulation has a mole ratio of cholesterol: phospholipids between about 55:50 and about 55:40.

C112. The adjuvant formulation of any one of C68 to C109, wherein the adjuvant formulation has a mole ratio of cholesterol: phospholipids of about 55:45.

C113. The adjuvant formulation of any one of C68-C112, wherein the liposomes in the adjuvant formulation range in size from between about 30 nm to about 400 nm.

C114. The adjuvant formulation of any one of C68-C112, wherein the liposomes in the adjuvant formulation range in size from between about 30 nm and about 200 nm.

C115. The adjuvant formulation of any one of C68-C112, wherein the liposomes in the adjuvant formulation have a size of less than about 200 nm.

C116. The adjuvant formulation of any one of C68-C115, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) between about 0.05 and about 0.5.

C117. The adjuvant formulation of any one of C68-C115, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) between about 0.05 and about 0.3.

C118. The adjuvant formulation of any one of C68-C115, wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) of less than about 0.3.

C119. The adjuvant formulation of any one of C68-C112, wherein the liposomes in the adjuvant formulation range in size from between about 30 nm and about 1400 nm.

C120. The adjuvant formulation of any one of C68-C112, wherein the liposomes in the adjuvant formulation range in size from between about 300 nm and about 1000 nm.

C121. The adjuvant formulation of any one of C68-C112, wherein the liposomes in the adjuvant formulation have a size of more than about 300 nm.

C122. The adjuvant formulation of any one of C68-C112 or C119-C121 , wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) between about 0.4 and about 1 . C123. The adjuvant formulation of any one of C68-C112 or C119-C121 , wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) of more than about 0.5.

C124. The adjuvant formulation of any one of C68-C112 or C119-C121 , wherein the liposomes in the adjuvant formulation have a polydispersity index (PDI) of more than about 0.4.

C125. An immunogenic composition comprising a combination of the adjuvant formulation of any one of C68-C124 and an immunogen.

C126. A method of inducing the immune response of a subject, comprising administering the immunogenic composition of C125 to a subject.

C127. The method of C126, wherein neutralizing antibody titers specific for the immunogen are increased in the subject after the administration.

C128. The method of C127, wherein the neutralizing antibody titers are increased by at least about 10-fold, or more in the subject.

C129. The method of C127, wherein the neutralizing antibody titers are increased by at least about 100-fold, or more in the subject.

C130. The method of any one of C125-C129, wherein the immunogen is specific for C. difficile.

Claims

1 . A method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(ii) mixing a buffer or water with the saponin to form an aqueous phase;

(v) concentrating the saponin-containing liposome of step (iv);

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

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

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

5. The method of claim 4, where in the saponin is in an amount of about 0.07 mg/ml.

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

7. The method of any one of claims 1-6, wherein in step (i) the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat or a combination thereof.

8. The method of claim 7, wherein the organic solvent is ethanol or isopropyl alcohol.

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

10. The method of any one of claims 1 -9, wherein the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2.

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

12. The method of any one of claims 1 -11 , wherein the flowrate of step (ii) is 12 mL/min to 240 mL/min or up to 3 L/min.

13. The method of any one of claims 1 -12, wherein the mass ratio of aqueous phase of step (ii) to organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1 .

14. The method of any one of claims 1 -13, wherein the microfluidic mixer of step (iii) uses a pump or syringe injection.

15. The method of claim 14, wherein the microfluidic mixer of step (iii) is a Y-junction, T- junction or coaxial microfluidic mixer.

16. The method of claim 14 or 15, wherein the microfluidic mixer of step (iii) has an internal diameter size ranging from 300 pm to 1 ,000 pm.

17. The method of any one of claims 1-16, wherein removing the organic phase ofthe saponin- containing liposome of step (iv) is by Tangential Flow Filtration (TFF).

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

19. The method of claim 17 or 18, wherein the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa.

20. The method of any one of claims 1 -19, wherein the concentrating of step (v) is by TFF, wherein TFF comprises diafiltration, ultrafiltration or both.

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

22. The method of claim 21 , wherein the bioburden reduction filter is up to 0.45 microns.

23. The method of claim 21 or 22, wherein the sterile filter is up to 0.22 microns.

24. The method of any one of claims 1 -23, wherein the concentrating of step (v) and filtering of step (vi) occur at room temperature.

25. A method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(ii) mixing the organic phase of step (i) into an aqueous phase, wherein the aqueous phase comprises a buffer or water, in a microfluidic mixer at a specific flowrate and at a specific ratio of the organic phase to the aqueous phase to form an intermediate liposome; (iii) mixing the intermediate liposome of step (ii) with a saponin, wherein the saponin is first dissolved in a buffer or water, to form a saponin-containing liposome;

(v) concentrating the saponin-containing liposome of step (iv);

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

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

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

29. The method of any one of claims 25-28, wherein in step (i) the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, or a combination thereof.

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

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

32. The method of any one of claims 25-31 , wherein the buffer comprises 10 mM phosphate and 150 mM NaCI at pH 6.2.

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

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

35. The method of any one of claims 25-34, wherein the mass ratio of the aqueous phase of step (ii) to the organic phase of step (i) ranges from 8:1 to 3:1 , 5:1 to 3:1.

36. The method of any one of claims 25-35, wherein the microfluidic mixer of step (ii) uses a pump or syringe injection.

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

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

39. The method of any one of claims 25-38, wherein removing the organic phase of the saponin-containing liposome of step (iv) is by Tangential Flow Filtration (TFF).

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

41 . The method of claim 39 or 40, wherein the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa.

42. The method of any one of claims 25-41 , wherein the concentrating of step (v) is by TFF, wherein the TFF comprises diafiltration, ultrafiltration or both.

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

44. The method of claim 43, wherein the bioburden reduction filter is up to 0.45 microns.

45. The method of claim 43 or 44, wherein the sterile filter is up to 0.22 microns.

46. The method of any one of claims 25-45, wherein the concentrating of step (v) and the filtering of step (vi) occur at room temperature.

47. A method for producing a homogeneous adjuvant formulation comprising a liposome bilayer comprising (a) monophosphoryl lipid A (MPLA), (b) a saponin, and (c) a liposome 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), distearyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), distearyl phosphatidylglycerol (DSPG), and a combination thereof, and (ii) cholesterol wherein the mole percent concentration of the cholesterol in the liposome composition is greater than 50% (mol/mol), said method comprising the steps of:

(iii) removing the organic phase of the intermediate liposome of step (ii);

(iv) concentrating the intermediate liposome of step (iii);

(v) sterile filtering the intermediate liposome of step (iv);

(vi) sterile filtering the saponin; and

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

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

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

51 . The method of any one of claims 47-50, wherein in step (i) the phospholipids, cholesterol and MPLA are dissolved in the organic solvent by sonication, heat, or a combination thereof.

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

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

54. The method of any one of claims 47-53, wherein the buffer of step (ii) comprises 10 mM phosphate and 150 mM NaCI at pH 6.2.

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

56. The method of any one of claims 47-55, wherein the microfluidic mixer of step (ii) uses a pump or syringe injection.

57. The method of claim 56, wherein the microfluidic mixer of step (ii) is a Y-junction, T-junction or coaxial microfluidic mixer.

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

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

60. The method of any one of claims 47-59, wherein the mass ratio of the aqueous phase of step (i) to the organic phase of step (ii) ranges from 8:1 to 3:1 , 5:1 to 3:1 .

61 . The method of any one of claims 47-60, wherein removing the organic phase of the intermediate liposome of step (iii) and concentrating of step (iv) are by Tangential Flow Filtration (TFF).

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

63. The method of claim 61 or 62, wherein the TFF comprises membranes having a molecular weight cut-off (MWCO) ranging from 100-500 kDa.

64. The method of any one of claims 47-63, wherein the sterile filtering of step (v) and step (vi) comprise a bioburden reduction filter and a sterile filter.

65. The method of claim 64, wherein the bioburden reduction filter is up to 0.45 microns.

66. The method of claim 64 or 65, wherein the sterile filter is up to 0.22 microns.

67. A homogeneous adjuvant formulation produced by any one of the methods according to claims 1-66, 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 of claim 67 or 68, wherein the adjuvant formulation has a polydispersity index between about 0.05 and about 0.2.

70. The adjuvant formulation of any one of claims 67-69, wherein the adjuvant formulation has a polydispersity index of about 0.1 .

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

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

73. The adjuvant formulation of any one of claims 67-72, 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 of any one of claims 67-73, wherein the adjuvant formulation comprises DMPC at a concentration of about 14 mg/ml or about 28 mg/ml.

75. The adjuvant formulation of any one of claims 67-74, 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 of any one of claims 67-75, wherein the adjuvant formulation comprises cholesterol at a concentration of about 11 mg/ml or about 22 mg/ml.

77. The adjuvant formulation of any one of claims 67-76, 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 of any one of claims 67-77, wherein the adjuvant formulation comprises DMPC and DMPG at a mole ratio of DMPC to DMPG (mol/mol) of between about 10:1 and about 8:1 .

79. The adjuvant formulation of any one of claims 71-78, wherein the adjuvant formulation has a mole ratio of cholesterol: phospholipids between about 55:50 and about 55:40.

80. The adjuvant formulation of any one of claims 71 -79, wherein the adjuvant formulation has a 3D-PHAD:phospholipid mole ratio between about 1 :80 and about 1 :95.

81 . An immunogenic composition comprising a combination of the adjuvant formulation of any one of claims 67-80 and an immunogen.

82. A method of inducing an immune response in a subject, comprising administering the immunogenic composition of claim 81 to a subject.