Processes for making stable nanoemulsion adjuvants

Abstract

The present disclosure provides a process to make a stable nanoemulsion (SNE) comprising: (a) preparing or obtaining a formulated component material (organic phase) and a formulated aqueous solvent (aqueous phase), wherein the component material comprises squalene and one or more emulsifiers; (b) mixing the organic phase and the aqueous phase in a nanoprecipitation mixing device, wherein the organic phase and the aqueous phase are liquid streams each with controllable flow rates, wherein the organic phase and the aqueous phase i) separately enter the nanoprecipitation mixing device, ii) mix in the nanoprecipitation device, and iii) exit the nanoprecipitation device as a single product stream containing an intermediate SNE; and (c) filtrating the intermediate SNE to generate the final SNE.

Description

25948 PROCESSES FOR MAKING STABLE NANOEMULSION ADJUVANTS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 63 / 650,662, filed May 22, 2024, and U.S. Provisional Application No.63 / 783,476 filed April 04, 2025, each of which is hereby incorporated by reference in its entirety. FIELD

[0002] This disclosure relates to processes for making stable nanoemulsions (SNEs) useful as vaccine adjuvants. BACKGROUND

[0003] Nanoemulsion preparation traditionally involves the combination of oils, emulsifiers, and an aqueous solution followed by high energy homogenization techniques to form nanoemulsion particles in the nanometer size scale. This is a highly developed, studied, and documented practice, and while robust in nature, does present drawbacks (Montes de Oca- Avalus, J.M. et al., Opinion in Food Science (2017) 16, 1-6). Homogenized nanoemulsions contain particles with particle size dependent nearly entirely upon the oil and emulsifier formulation with limited ability to control the size of the product nanoemulsion which can alter filtration and downstream processing parameters.

[0004] It is desirable to have control over particle size in stable nanoemulsion (SNE) formulations which allows for better control over product characteristics and potentially aid in downstream manufacturing operations. SUMMARY

[0005] The present disclosure provides a process to make a stable nanoemulsion (SNE) comprising: (a) preparing or obtaining a formulated component material (organic phase) and a formulated aqueous solvent (aqueous phase), wherein the component material comprises squalene and one or more emulsifiers; (b) mixing the organic phase and the aqueous phase in a nanoprecipitation mixing device, wherein the organic phase and the aqueous phase are liquid streams each with controllable flow rates, wherein the organic phase and the aqueous phase i) separately enter the nanoprecipitation mixing device, ii) mix in the nanoprecipitation device, and iii) exit the nanoprecipitation device as a single product stream containing an intermediate SNE; and (c) filtrating the intermediate SNE to generate the final SNE.

[0006] In one aspect of the process, the nanoprecipitation mixing device is selected from a T- Tube mixer (T-mixer), a staggered herringbone mixer (SHM) and a multi-inlet vortex mixer (MIVM).

[0007] In another aspect of the process, the flow rate of the organic stream and the aqueous stream are controlled to a specific flow rate ratio.

[0008] In another aspect of the process, the component material is squalene, one or more emulsifiers and one or more lipids.

[0009] In another aspect of the process, the emulsifier is selected from sorbitan trioleate (SPAN-85), polysorbate-20 (PS-20) and polysorbate-80 (PS-80).

[0010] In another aspect of the process, the component materials are formulated at specific concentration ratios.

[0011] In another aspect of the process, the aqueous solvent is Tris, Bis-Tris, Citrate, HEPES, Phosphate, and L-Histidine.

[0012] In another aspect of the process, the aqueous solvent is L-Histidine.

[0013] In another aspect of the process, the single stream is stabilized with an in-line dilution of a stabilizing buffer.

[0014] In another aspect of the process, the step (c) filtration is tangential flow filtration followed by bioburden reduction filtration.

[0015] In another aspect, the SNEs are adjuvant formulations.

[0016] In another aspect, the SNEs generated using the instant process are useful as vaccine adjuvants.

[0017] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG.1 shows a traditional homogenization process diagram for making an SNE.

[0019] FIG.2 shows a T-mixing process diagram for making an SNE.

[0020] FIG.3 shows Cryo-EM images of an SNE produced via homogenization (left panel) and T-mixing (right panel).

[0021] FIG.4 shows T-mixed SNE particle size as a function of tee mix ratio (TMR).

[0022] FIG.5 shows that increasing pH has an impact on the particle size of SNE produced at varying tee mix ratios.

[0023] FIG.6 shows particle size as a function of tee mix ratio for a complex SNE.

[0024] FIG.7 shows average particle size can be controlled by preparation concentration factors and staggered herringbone mixing flow ratio, the MTR.

[0025] FIG.8 shows the impact of pH and buffer species (sodium citrate vs. histidine) on TMR and particle size relationship for the exemplified nanoemulsion.

[0026] FIG.9 shows the impact of pH for the exemplified nanoemulsion composed of squalene, PS-20, and SPAN-85 in a 20mM histidine buffer on the relationship between TMR and particle size.

[0027] FIG.10 shows the impact of histidine buffer concentration (all buffers at pH 5.5) on TMR and particle size relationship for the exemplified nanoemulsion at pH 5.5.

[0028] FIG.11 shows the impact that changing both the squalene: PS-20 and the squalene: SPAN-85 ratios has on the TMR and particle size relationship.

[0029] FIG.12 shows the impact that changing the squalene: PS-20 ratio has on the relationship between TMR and particle size while keeping the squalene: SPAN-85 ratio constant at 10:1.

[0030] FIG.13 shows no observed impact when changing the squalene: SPAN-85 ratio to 5:1 and 3:1 on the TMR and particle size relationship (while keeping the squalene: PS-20 ratio constant at 10:1).

[0031] FIG.14 shows a demonstration of scalability from pilot scale mixing and small scale mixing for the exemplified nanoemulsion.

[0032] FIG.15 shows the bioburden reduction filtration pressure profile for a nanoemulsion composed of approximately 10:1:1 squalene: PS-20:SPAN-85 nanoemulsion in 20 mM histidine, pH 5.8 buffer.

[0033] FIG.16 shows the impact that a squalene: functional lipid ratio and a squalene: each emulsifier ratio has on the relationship between TMR and particle size. DETAILED DESCRIPTION

[0034] The present disclosure provides a process to make a stable nanoemulsion (SNE) comprising: (a) preparing or obtaining a formulated component material (organic phase) and a formulated aqueous solvent (aqueous phase), wherein the component material comprises a terpene and one or more emulsifiers; (b) mixing the organic phase and the aqueous phase in a nanoprecipitation mixing device, wherein the organic phase and the aqueous phase are liquid streams each with controllable flow rates, wherein the organic phase and the aqueous phase i) separately enter the nanoprecipitation mixing device, ii) mix in the nanoprecipitation device, andiii) exit the nanoprecipitation device as a single stream containing an intermediate SNE; and (c) filtrating the intermediate SNE to generate the final SNE.

[0035] In one aspect of the process, the nanoprecipitation mixing device is selected from a T- Tube mixer (T-mixer), a staggered herringbone mixer (SHM) and a multi-inlet vortex mixer (MIVM).

[0036] In another aspect of the process, the flow rate of the organic stream and the aqueous stream are controlled to a specific flow rate ratio. The flow rate ratio is selected based on the particular and / or specific component materials.

[0037] In another aspect of the process, the component material is squalene, one or more emulsifiers and one or more lipids.

[0038] In another aspect of the process, the emulsifier is selected from sorbitan trioleate (SPAN-85), polysorbate-20 (PS-20) and polysorbate-80 (PS-80).

[0039] In another aspect of the process, the component materials are formulated at specific concentration ratios. The specific concentration ratios are selected based on the particular and / or specific component materials.

[0040] In another aspect of the process, the aqueous solvent is Tris, Bis-Tris, Citrate, HEPES, Phosphate, and L-Histidine.

[0041] In another aspect of the process, the aqueous solvent is L-Histidine.

[0042] In another aspect of the process, the single stream is stabilized with an in-line dilution of a stabilizing buffer.

[0043] In another aspect of the process, the step (c) filtration is tangential flow filtration followed by bioburden reduction filtration.

[0044] In another aspect, the SNEs are adjuvant formulations.

[0045] In another aspect, the SNEs generated using the instant process are useful as vaccine adjuvants.

[0046] In another aspect, the present disclosure provides a method of making a nanoemulsion formulation that comprises squalene, polysorbate 20 (PS-20) and sorbitan trioleate (SPAN-85), comprising: a) preparing or obtaining (i) an organic solution that comprises squalene, PS-20, and SPAN-85, and (ii) an aqueous solution; and b) mixing the organic solution and the aqueous solution in a tee mixing device, wherein the organic solution is a liquid stream and has an particular flow rate and the aqueous solution is a liquid stream and has a particular flow rate, wherein the organic solution and the aqueous solution (i’) separately enter the tee mixing device at a tee mix ratio of about 0.5 to 5.0, (ii’) mix in the tee mixing device, and (iii’) exit the tee-mixing device as a single liquid stream; wherein the single liquid stream contains particles that have an average size from about 80 nm to about 200 nm.

[0047] In one aspect of the method, the mass ratio of squalene to SPAN-85 is between 12:1 or 11:1 or 10:1 or 9:1 or 8:1 or 3:1.

[0048] In another aspect of the method, the mass ratio of squalene to PS-20 to SPAN-85 is about 10:1:1.

[0049] In one aspect of the method, the tee mix ratio is about 1.0 to about 3.0.

[0050] In another aspect of the method, the tee mix ratio is about 1.0 to about 1.8.

[0051] In another aspect of the method, the tee mix ratio is about 1.4.

[0052] In another aspect of the method, the particles have an average size from about 150 nm to about 170 nm.

[0053] In another aspect of the method, the particles have an average size of about 160 nm.

[0054] In another aspect of the method, the aqueous solution contains Tris, Bis-Tris, Citrate, HEPES, Phosphate, or L-Histidine.

[0055] In another aspect of the method, the aqueous solution contains L-Histidine.

[0056] In another aspect of the method, the nanoemulsion formulation is an adjuvant formulation.

[0057] In another aspect of the method, the nanoemulsion formulation is useful in vaccine formulations. DEFINITIONS

[0058] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0059] 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. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0060] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0061] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable).

[0062] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0063] An "adjuvant," as defined herein, refers to a lipid or lipid formulation or SNE or SNE formulation or a nanoemulsion formulation that serves to enhance the immunogenicity of a vaccine. An adjuvant may i) enhance an immune response to an antigen (for example, a pneumococcal polysaccharide) that is weakly immunogenic when administered alone, e.g., inducing no or weak antibody titers or cell-mediated immune response, ii) increase antibody titers to the antigen, and / or iii) lower the dose of the antigen effective to achieve an immune response in the individual.

[0064] As used herein, the term “antigen” refers to any antigen that can generate one or more immune responses. An antigen may be a protein, peptide or polypeptide. An antigen may a lipid or a carbohydrate. An antigen may be a polysaccharide. An antigen may be a pneumococcal polysaccharide. An antigen may be an S. pneumoniae polysaccharide. An antigen may be one that generates a humoral and / or CTL immune response.

[0065] As used herein, the term “immunogenic” or “immunogenicity” refers to the ability of an antigen (for example, an S. pneumoniae polysaccharide) to provoke an immune response in a subject.

[0066] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0067] As used herein, the term “SNE” means a nanoemulsion formulation comprising emulsifiers and / or solubilizers and / or surfactants and / or lipids that have adjuvant properties in a vaccine. For example, an SNE refers to an SNE adjuvant formulation comprising 1) sorbitan trioleate (SPAN-85); 2) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); and 3) squalene. For example, an SNE comprises 0.001 mg / mL to 60 mg / mL SPAN-85, 0.001 mg / mL to 60 mg / mL25948 PS-20 and 0.01 mg / mL to 200 mg / mL squalene. In further embodiments, an SNE comprises 0.01 mg / mL to 10 mg / mL SPAN-85, 0.01 mg / mL to 10 mg / mL PS-20 and 0.03 mg / mL to 30 mg / mL squalene. In still further embodiments, an SNE comprises 0.2 mg / mL to 10 mg / mL SPAN-85, 0.2 mg / mL to 10 mg / mL PS-20 and 0.5 mg / mL to 20 mg / mL squalene. For example, an SNE refers to an SNE adjuvant formulation comprising 1) sorbitan trioleate (SPAN-85); 2) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); 3) squalene; and a 4) lipid. For example, an SNE comprises 0.01 µg / mL to 1000 µg / mL lipid, 0.001 mg / mL to 60 mg / mL SPAN-85, 0.001 mg / mL to 60 mg / mL PS-20 and 0.01 mg / mL to 200 mg / mL squalene. In further embodiments, an SNE comprises 100 µg / mL or 80 µg / mL lipid, 0.01 mg / mL to 10 mg / mL SPAN-85, 0.01 mg / mL to 10 mg / mL PS-20 and 0.03 mg / mL to 30 mg / mL squalene. In still further embodiments, an SNE comprises 80 µg / mL or 16 µg / mL or 4 µg / mL or 0.5 µg / mL lipid, 0.2 mg / mL to 10 mg / mL SPAN-85, 0.2 mg / mL to 10 mg / mL PS-20 and 0.5 mg / mL to 20 mg / mL squalene.

[0068] As used herein, the term “SNE intermediate” means any product stream formed utilizing the instant process before the filtration step. For example, any product stream formed utilizing the instant process before final bioburden filtration is performed.

[0069] As used herein, the term “final SNE” means the final state of the SNE which is formed after the filtration step. For example, the final state of the SNE which is formed after the product stream has been processed through the final bioburden reducing filtration step.

[0070] As used herein, the term “vaccine” or “vaccine composition” refers to a biological preparation used to stimulate the production of antibodies and provide immunity against an infectious disease. Component Materials and Formulated Component Materials

[0071] An SNE primarily contains squalene combined with an emulsifier (including one or more emulsifiers) and may also include lipids in a formulation. Squalene is a branched, unsaturated terpenoid which is readily available commercially and is derived from either an animal source (shark-based) or a plant-based source or can be synthetically derived. Emulsifiers can be ionic or non-ionic, with non-ionic being preferred.

[0072] Examples of emulsifiers include the polyoxyethylene sorbitan esters emulsifiers (commonly referred to as the Tweens, especially PS-20 and PS-80), copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol(IGEPAL CA-630 / NP-40); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span-85, Tween-85 or [2-[(2R,3S,4R)-4- hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec-9-enoyl]oxyethyl] (Z)-octadec- 9-enoate) and sorbitan monolaurate.

[0073] Preferred examples of emulsifiers include Tweens and polysorbates such as polysorbate- 20 (PS-20) and polysorbate-80 (PS-80); sorbitan esters (commonly known as Spans), such as sorbitan trioleate (SPAN-85), sorbitan monoleate (SPAN 80) and sorbitan monolaurate (SPAN 20).

[0074] Emulsifiers of particular interest include polysorbates, specifically polysorbate-20 (PS- 20, polysorbate-80 (PS-80) and sorbitan trioleate (SPAN-85).

[0075] A preferred example of component materials include squalene, PS-20 and SPAN-85.

[0076] A preferred example of component materials include a lipid (specifically disclosed herein), squalene, PS-20 and SPAN-85.

[0077] Lipids can also be included in the SNEs. Example of a lipids are disclosed in U.S. Patent Application Publication Nos. US 2008 / 0085870, US 2008 / 0057080, US 2009 / 0263407, US 2009 / 0285881, US 2010 / 0055168, US 2010 / 0055169, US 2010 / 0063135, US 2010 / 0076055, US 2010 / 0099738, US 2010 / 0104629, US 2013 / 0017239, and US 2016 / 0361411, International Patent Application Publication No. WO2011 / 022460 A1; WO2012 / 040184, WO2011 / 076807, WO2010 / 021865, WO 2009 / 132131, WO2010 / 042877, WO2010 / 146740, WO2010 / 105209, and in U.S. Pat. Nos.5,208,036, 5,264,618, 5,279,833, 5,283,185, 6,890,557, and 9,669,097.

[0078] Further examples of lipids are: (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1- amine, or (6Z,9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,29-tetraen-18-amine, or N,N- dimethyl-1-((1S,2R)-2-octylcyclopropyl)heptadecan-8-amine.

[0079] Further examples of lipids are: DLinDMA; DLinKC2DMA; DLin-MC3-DMA; CLinDMA; S-Octyl CLinDMA; (2S)-l-{7-[(3P)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4- en-l- yloxy]-N,N-dimethylpropan-2-amine; (2R)-l-{4-[(3P)-cholest-5-en-3-yloxy]butoxy}-3- [(4Z)-dec-4-en-l-yloxy]-N,N-dimethylpropan-2-amine; l-[(2R)-l-{4-[(3β)-cholest-5-en-3- yloxy]butoxy}-3-(octyloxy)propan-2- yl]guanidine; l-[(2R)-l-{7-[(3β)-cholest-5-en-3- yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine; l- [(2R)-l-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]propan-2-amine; (2S)-l-({6-[(3P))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3- [(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (3β)-3-[6-{[(2S)-3-[(9Z)-octadec-9-en-l-yloxyl]-2-(pyrrolidin-l-yl)propyl]oxy}hexyl)oxy] cholest-5-ene; (2R)-l-{4-[(3P)-cholest-5-en-3- yloxy]butoxy}-3-(octyloxy)propan-2-amine; (2R)-l-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-(pentyloxy)propan-2-amine; (2R)-l-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- 3-(heptyloxy)-N,N- dimethylpropan-2-amine; (2R)-l-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(2Z)-pent- 2-en-1-yloxy]propan-2-amine; (2S)-l-butoxy-3-({8-[(3P)-cholest-5- en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine; (2S-1-({8-[(3P)-cholest-5-en-3- yloxy]octyl}oxy)-3-[2,2, 3,3,4,4,5, 5,6, 6,7, 7, 8, 8,9, 9-hexadecafluorononyl)oxy]-N,N- dimethylpropan-2-amine; 2-amino-2-{[(9Z, 12Z)-octadeca-9, 12-dien-1-yloxy]methyl} propane- 1,3-diol; 2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]nonyl}oxy)-2-{[(9Z,12Z)- octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol; 2-amino-3-({6-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest- 5-en-3-yloxy]nonyl}oxy)-2-{[(9Z)-octadec-9-en- 1-yloxy]methyl}propan-1-ol; (20Z,23Z)-N,N- dimethylnonacosa-20,23-dien-10-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-8-amine; (13Z,16Z)-N,N-dimethyldocosa-13,16- dien-5-amine; (12Z,15Z)-N,N-dimethylhenicosa-12,15 -dien-4-amine; (14Z,17Z)-N,N- dimethyltricosa-14,17-dien-6-amine; (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine; (18Z,21 Z)-N,N-dimethylheptacosa-18,21-dien-10-amine; (15Z,18Z)-N,N-dimethyltetracosa- 15,18-dien-5-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine; (19Z,22Z)-N,N- dimethyloctacosa-19,22-dien-9-amine; (18Z,2lZ)-N,N-dimethylheptacosa-18,21-dien-8-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine; (16Z,19Z)-N,N-dimethylpentacosa- 16,19-dien-6-amine; (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine; (21 Z,24Z)- N,N-dimethyltriaconta-21,24-dien-9-amine; (18Z)-N,N-dimethylheptacos-18-en-10-amine; (17Z)-N,N-dimethylhexacos-17-en-9-amine; (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7- amine; Ν,Ν-dimethylheptacosan-10-amine; (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien- 10-amine; 1-[(1 lZ,14Z)-l-nonylicosa-l l,14-dien-l-yl]pyrrolidine; (20Z)-N,N-dimethylheptacos- 20-en-10-amine; (15Z)-N,N-dimethylheptacos-15-en-10-amine; (14Z)-N,N-dimethylnonacos-14- en-10-amine; (17Z)-N,N-dimethylnonacos-17-en-10-amine; (24Z)-N,N-dimethyltritriacont-24- en-10-amine; (20Z)-N,N-dimethylnonacos-20-en-10-amine; (22Z)-N,N-dimethylhentriacont-22- en-10-amine; (16Z)-N,N-dimethylpentacos-16-en-8-amine; (12Z,15Z)-N,N-dimethyl-2- nonylhenicosa-12,15-dien-1-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1- amine; N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]heptadecan-8-amine; l-[(l S,2R)-2- hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine; Ν,Ν-dimethyl-1-[(1S,2R)-2- octylcyclopropyl]nonadecan-10-amine; N,N-dimethyl-21 -[(1S,2R)-2- octylcyclopropyl]henicosan-10-amine; N,N-dimethyl-l-[(lS,2S)-2-{[(lR,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine; N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]hexadecan-8-amine; N,N-dimethyl-l-[(lR,2S)-2- undecylcyclopropyl]tetradecan-5-amine; N,N-dimethyl-3-{7-[(l S,2R)-2- octylcyclopropyl]heptyl}dodecan-l-amine; 1-[(1R,2S)-2-heptylcyclopropyl]-N,N- dimethyloctadecan-9-amine; 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine; N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]pentadecan-8-amine; and (1lE,20Z,23Z)-N,N- dimethylnonacosa-l 1,20,23-trien-10-amine.

[0080] Further examples of lipids are: (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3- d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; (S)-N- (5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)- 3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; (S)-1-(4-(4-((5-amino-7-((1- hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxyphenyl)piperazin-1-yl)octadecan-1-one; N-(5-(4-(4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5- oxopentyl)stearamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide; N-(5-(4-(4-((5- amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1- yl)-5-oxopentyl)oleamide; (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3- d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12- dienamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide; N-(5-(4-(4-((5-amino-7- (butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5- oxopentyl)stearamide; N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide; 1-(4-((5-amino-7- (butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3- stearamidopropyl)piperidine-4-carboxamide; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N- octadecylcyclobutane-1-carboxamide; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N- hexadecylcyclobutane-1-carboxamide; N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3- d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide; N-(7-(4-(4- ((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide; N-(3-(2-(4-(4-((5-amino-7- (butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2- oxoethyl)cyclobutyl)stearamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-25948 d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5- oxopentyl)stearamide; N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide; 1-(4-(4-((5- amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1- yl)-5-(octadecyloxy)pentan-1-one; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3- d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one; N- (5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; (9Z,12Z)-N-(4-((4-((5-amino-7- (butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide; N-(4-((4-((5-amino-7- (butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide; N-(4-((4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide; N- (4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)stearamide; N-(4-((4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4- oxobutyl)stearamide; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (4-((4-((5-amino-7- (butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)carbamate; 4-(4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1- carboxamide; 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin- 2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate; N-(5-(4-(4-((5-amino-7-(butylamino)- 2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6- trifluorohexyl)stearamide; N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide; and (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl (5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3- d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate; or pharmaceutically acceptable salts thereof.

[0081] In a particular aspect, the process disclosed herein is further useful to make SNEs containing lipids that are insoluble or only slightly soluble in aqueous solutions and / or squalene “insoluble lipids” and are not suitable to be combined into an SNE using typical homogenization / microfludization processes. These insoluble lipids can be dissolved in an organic solvent (i.e., ethanol) and can be included in the component material formulation.25948

[0082] Mass ratios of squalene to emulsifiers can vary depending on the particular component material formulation. A particular mass ratio is between 3:1 or 4:1 or 5:1 or 6:1 or 7:1 or 8:1 or 9:1 or 10:1. The mass ratio of squalene to cationic lipid can vary from 1:1 or 10:1 or 100:1 or 500:1 or 1000:1 or 2000:1 or 3000:1 or 4000:1.

[0083] Mass ratios of squalene to PS-20 can vary depending on the particular component material formulation. A particular mass ratio is between about 25:1 or about 10:1 or about 3:1. Another particular mass ratio is between 25:1 or 10:1 or 3:1.

[0084] Mass ratios of squalene to SPAN-85 can vary depending on the particular component material. A particular mass ratio is between about 12:1 or about 11:1 or about 10:1 or about 9:1 or about 8:1 or about 3:1. Another particular mass ratio is between 12:1 or 11:1 or 10:1 or 9:1 or 8:1 or 3:1.

[0085] Mass ratios of squalene to PS-20 to SPAN-85 can vary depending on the particular component material. A particular mass ratio is between about 25:1:1 or about 12:1:1 or about 11:1:1 or about 10:1:1 or about 9:1:1 or about 8:1:1 or about 3:1:1. Another particular mass ratio is between 25:1:1 or 12:1:1 or 11:1:1 or 10:1:1 or 9:1:1 or 8:1:1 or 3:1:1.

[0086] The particular mass ratios highlighted above are utilized in the instant process to generate particles that have an average size of about 80 nm to about 200 nm.

[0087] The particular mass ratios highlighted above are utilized in the instant process to generate particles that have an average size of about 100 nm to about 200 nm.

[0088] The particular mass ratios highlighted above are utilized in the instant process to generate particles that have an average size of about 150 nm to about 200 nm.

[0089] The particular mass ratios highlighted above are utilized in the instant process to generate particles that have an average size of about 150 nm to about 170 nm.

[0090] The particular mass ratios highlighted above are utilized in the instant process to generate particles that have an average size of about 160 nm. Aqueous and Component Solvents

[0091] To enable nanoprecipitation and SNE particle formation all components of the SNE must be first dissolved in an appropriate agent. Selection of an organic solvent for SNE component dissolution must first be based on the solubility of the SNE components in various solvents. Once a group of organic solvents are identified which provide appropriate solubility, further selection criteria is based on solvent safety handling, availability, and environmental impact. Ethanol, methanol, and isopropanol or combinations thereof would be first line25948 candidates for the component solvent. This is aligned with the practices used in liposome and lipid nanoparticle solvent selection as well.

[0092] An aqueous solvent is required in the nanoprecipitation process primarily to act as an organic solvent diluent. The dilution of organic solvent content in the mixed solution will reduce the solubility of the SNE components as mixing occurs and this lowered solubility drives the preferential component-component interactions. As component-component interactions become more preferential this induces nanoparticle nucleation, expansion, and formation of the final SNE all within microsecond timescales during the mixing process with formed SNE exiting the nanoprecipitation mixing device. The composition of the aqueous solvent is selected for compatibility and process robustness based on the SNE components. Criteria to consider would be desired pH range, buffer species, and ionic strength of the aqueous solvent. Potential aqueous solvent buffer species include Tris, Bis-Tris, Citrate, HEPES, Phosphate, and L-Histidine. The presence or absence of charged SNE components would be the primary driver in the pH and ionic strength selection of the aqueous solvent. Nanoprecipitation Mixing Devices (T-Tube Mixer, Staggered Herringbone Mixer (SHM), Multi- Inlet Vortex Mixer (MIVM)) and Use Thereof

[0093] Nanoprecipitation by means of rapid mixing of organic and aqueous liquids with various components dissolved in one or both solutions is a well understood and described method in the spontaneous formation of liposomes and lipid nanoparticles (Liu, Y. et al., Ind. Eng. Chem. Res. (2020) 59, 9, 4134-4149). Liposomes and lipid nanoparticles are two closely related types of membrane based nanoparticles with related morphologies and overlapping component materials. Literature describes production of liposomes and lipid nanoparticles with a wide variety of mixing geometries to produce countless different formations and iterations of these types of nanoparticles (Shepherd, S.J. et al., Biomaterials (2021) 274, 1-36). The application of these practices have been used in academic settings as well as in the manufacture of commercial pharmaceutical products. While also a type of nanoparticle, stable nanoemulsions use a completely distinct set of component materials and are morphologically distinct entities in comparison to liposomes and lipid nanoparticles. The techniques established for liposome and LNP preparation are less suitable for the preparation of SNEs.

[0094] Here we describe the preparation of a variety of SNE formulations using nanoprecipitation-based preparation processes with multiple nanoprecipitation mixing devices. The three different types of mixing technologies detailed in this invention include a T-tube style (T-mixer), multi-inlet-vortex-mixer (MIVM), and staggered herringbone mixing systems (SHM).25948 All three systems require an organic side and aqueous solution side which are combined within the designated mixing system at a specific ratio of aqueous to organic flowrate. The pumping systems used can be syringe style pumps, low pulsation HPLC pumps, and centrifugal pumps which can deliver a non-pulsatile flowrate to the mixing device. The specific ratio of aqueous to organic phase is termed the tee-mix-ratio (TMR) and is a critical parameter to determine SNE size as detailed in the subsequent examples. A T-tube style mixer contains the aqueous and organic inlets 180 degrees, but does not need to be exactly 180, rotated such that the flows are impinging upon each other at the point of impact. The combined flow then turns 90 degrees, but does not need to be exact, and heads out of the mixing device. The MIVM mixing system is circular in shape with at least 2 ports on top but more typically 4 ports. Typically, 1-3 inlet ports are used for feeding the aqueous buffer and 1-2 ports are used for feeding the organic phase to the mixing system. Inside the flows combine as they move in a circular direction towards the center of the device and then exit through a single central outlet on the bottom of the unit. MIVM devices have been applied for lipid nanoparticle (LNP) processing and are commercially available. The staggered herringbone mixing system (SHM) is composed of herringbone grooves in one or more of the walls of the flowpath channel. The aqueous and organic streams are directed towards the inlet of the channel and combine at the entrance where mixing occurs as fluid passes over the grooves, creating passive mixing. SHM mixing devices are widely used for (LNP) processing and are commercially available. T-Mixer Process

[0095] For the T-mixing process, important parameters include the T-Mix Ratio (TMR), total flow rate through the tee, and dilution factor or dilution flow rate. The TMR is defined as theratio of the aqueous to organic flow rates such that ^^^^^^ ൌ ^^௨^^௨^ ^^^௪ ^^௧^^^^^^^^ ^^^௪ ^^௧^. TMRs may be between 0.5-5.0 or between 0.5-2.0 for nanoemulsion processes, andoften require adjustment when different nanoemulsion formulations, pH, and buffer species are used. Preferred TMRs for mixing depend on these factors and on the target SNE particle size. The total flow rate through the T-mixer is another important factor and varies based on the inner diameter or size of the T-mixer being used. The dilution pump flow rate or dilution factor is set from 2-4x or from 1- 3x the total flow rate from the T-mixer and the purpose of this step is to reduce the ethanol content from the SNE intermediate. In other aspects of the invention, TMRs are between about 1.0 to about 3.0, or are between about 1.0 to about 1.8, or are about 1.4. In another aspect of the invention, TMRs are between 1.0-3.0, or are between 1.0-1.8, or are 1.4.25948 MIVM Process

[0096] For the MIVM process one important parameter is the Mixing Ratio (MR), which is similar to TMR but since the MIVM has multiple inlets ports this mixing ratio is termed total aqueous flowrate divided by the total organic flow rate feeding to the mixer. Additional parameters that are important include total flow rate through the mixer, dilution factor or dilution flow rate, and configuration of inlet ports (more than 2 ports allows for various combinations of organic and aqueous stream configurations). Ideally MRs are between 0.5-3.0 for nanoemulsion processes using a MIVM, and MRs often require adjustment when nanoemulsion formulation, pH, and buffer species are used. Preferred MRs for mixing depend on these factors and on the target SNE particle size. The total flow rate through the MIVM is another important factor and varies based on the internal dimensional characteristics of the mixer being used. The dilution pump flow rate or dilution factor is set from 0-4x or from 0-3x the total flow rate from the MIVM and the purpose of this step is to reduce the ethanol content from the SNE intermediate. Preferred configuration of inlet ports would be a 4 port inlet with organic streams next to each other and aqueous inlet ports next to each other. SHM Process

[0097] For the SHM process Mixing Ratio (MR) is also important and is termed the same as the MIVM. Additional parameters that are important include total flow rate through the mixer, and dilution factor or dilution flow rate. Ideally MRs are between 0.5-3.0 for nanoemulsion processes using a SHM, and MRs often require adjustment when nanoemulsion formulation, pH, and buffer species are used. Preferred MRs for mixing depend on these factors and on the target SNE particle size. The total flow rate through the SHM is another important factor and varies based on the internal dimensional characteristics of the mixer being used. The dilution pump flow rate or dilution factor is set from 0-4x or from 0-3x the total flow rate from the SHM and the purpose of this step is to reduce the ethanol content from the SNE intermediate. Stabilizing Buffers and Use Thereof

[0098] The liquid stream (the single product stream) exiting the nanoprecipitation mixing device typically has organic solvent concentrations that could range from 5% to 55% depending on the selected nanoprecipitation mixing device and mixing parameters selected. At high concentrations the organic solvent content can be destabilizing to the formed SNE intermediate. To counteract this, the organic solvent content must be lowered by dilution with a stabilizing buffer. This dilution results in the lowering of the organic solvent content to 20% or below and25948 inhibits the destabilizing effects of the organic solvent on the formed nanoparticles. The dilution with stabilizing buffer is performed through in-line dilution as the formed SNE intermediate exits the nanoprecipitation mixing device for the most immediate reduction in organic solvent content. As the primary goal of this stabilizing buffer addition is to reduce organic solvent content, a variety of buffering agents and compositions can be utilized based on the particular SNE composition. Examples of potential stabilizing buffer agents are Tris, Bis-Tris, Citrate, HEPES, Phosphate, L-Histidine, among others, with the selection based on the optimal pH range and compatibility with a given SNE. Later processing steps can replace this buffer, so it is possible to use a buffer not intended to be in the final formulation of the SNE. Filtration

[0099] Following nanoprecipitation in the nanoprecipitation mixing device, the product SNE intermediate is concentrated, exchanged into a new buffer, and filtered. To concentrate and exchange the background buffer of the SNE intermediate, tangential flow filtration is used. Following the tangential flow filtration step, a normal filtration is performed with a sterilizing grade filter to produce the SNE intermediate.

[0100] Tangential flow filtration (TFF) is performed using a hollow fiber modified polyethersulfone (mPES) membrane with a 500kDa molecular weight cut off. To start TFF, an initial volume is fed from the nanoprecipitation device mixed product container to a working vessel; this volume is the diavolume. The mixed product is recirculated across the membrane using a centrifugal, peristaltic, or other positive-displacement pump. The crossflow rate set to achieve a target shear rate. In the first step of TFF, the mixed product is concentrated in fed-batch mode using constant flux permeation. Peristaltic addition and permeation pumps are used to control the flow rates of the mixed product fed over into the working vessel and waste permeated respectively. The addition pump flowrate is modified throughout concentration and diafiltration to maintain a constant working vessel volume. The permeate flow rate may be decreased below the target during the run to maintain positive pressure as needed. Volumetric concentration factors can range from 10-20x depending on the desired final concentration of the SNE intermediate. The second step of TFF is fed batch diafiltration in which buffer is continuously fed into the working vessel to exchange the buffer of the SNE intermediate. The primary purpose of this step is to remove ethanol from the mixed product and exchange the buffer species and / or concentration if desired. Typically, 20 mM histidine, pH 5.8 buffer is used. Different pH buffers, concentrations, and buffer components can also be used to achieve better process conditions and SNE stability. The diafiltration endpoint is determined based on the number of diavolumes25948 permeated and typical targets are 8-12x the SNE diavolume. After diafiltration is completed an additional batch concentration step may be performed to further concentrate the SNE intermediate further 2-4x.

[0101] Bioburden reduction filtration (BRF) is performed following TFF to remove large particles that may have formed throughout the process and prepare the final SNE for storage at 2- 8°C. The TFF product is filtered using a normal filtration process to produce the final SNE. Using a constant flux of 20-150 LMH, the TFF product is filtered through a Pall Supor dual layer filter with a 0.8µm layer followed by 0.2µm layer. Inlet pressure during the filtration is monitored and the flow rate may be decreased to avoid over pressurization. Alternate filters may be used depending on the final SNE to optimize throughput and yield. SNE Adjuvant Formulations

[0102] In some embodiments of the invention, a formulation is provided wherein the SNE comprises components which are selected from a surfactant, a mixture of surfactants, a phospholipid, a terpene, a terpenoid, a triterpene or a combination thereof.

[0103] In some embodiments of the invention, the surfactant includes, but is not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially PS- 20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span-85, Tween-85 or [2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec- 9-enoyl]oxyethyl] (Z)-octadec-9-enoate) and sorbitan monolaurate.

[0104] In some embodiments of the invention, mixtures of surfactants are used, e.g., PS- 20 / Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t- octylphenoxypolyethoxyethanol (Triton X-100) are also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

[0105] In some embodiments of the invention, the amounts of surfactants or emulsifiers are: polyoxyethylene sorbitan esters (such as PS-20) 0.01 to 10 mole %, in particular about 1 to 425948 mol%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 10 mol%, in particular about 1 to 4 mol%; w / v, in particular 0.01 to 0.1% w / v; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 mol%, preferably 0.5 to 10 mol% and in particular 1 to 4 mol% or about 10 % by mass.

[0106] In some embodiments of the invention, the phospholipid is selected from, a natural phospholipid including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacyl-phosphatidylcholine (lyso PC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), N-Acyl-PE, a phosphoinositide, and a phosphosphingolipid. Phospholipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), phosphatidylserine (DOPS). Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18: l), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0 and lethicin. In certain embodiments of the invention, the phospholipid is phosphatidylserine, l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2- dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0107] In some embodiments of the invention, the terpene is selected from a monoterpene, such as geraniol, terpeneol, limonene, myrcene, linalool or pinene. In some embodiments, the formulation comprises a sesquiterpene consisting of humulene, a farnesene, or farnesol; a diterpene such as cafestol, kahweol, cembrene or taxadiene; A triterpene such as squalene or squalante; a tetraterpene such as acyclic lycopene, monocyclic gamma-carotene, or bicyclic alpha- and beta-carotenes; a polyterpene or a norisoprenoids.

[0108] The disclosure provides an adjuvant formulation that comprises 1) one or more lipids; 2) one or more sorbitan-based surfactants; and 3) one or more terpenes.

[0109] The disclosure provides an adjuvant formulation that comprises 1) squalene; 2) polysorbate-20 (PS-20); and sorbitan trioleate (SPAN-85).

[0110] The disclosure provides an adjuvant formulation that comprises 1) one or more lipids; 2) sorbitan trioleate (SPAN-85); 3) polysorbate-20 (PS-20) or polysorbate-80 (PS-80), and 4) squalene.

[0111] The disclosure provides an adjuvant formulation that comprises 1) one or more lipids; 2) SPAN-85; 3) PS-20, and 4) squalene.25948

[0112] The disclosure provides an adjuvant formulation that comprises 1) a lipid; 2) SPAN-85; 3) PS-20 or PS-80, and 4) squalene.

[0113] The disclosure provides an adjuvant formulation that comprises 1) a lipid; 2) SPAN-85; 3) PS-20, and 4) squalene.

[0114] In some embodiments, an SNE formulation is provided comprising: 1) about 10-14 mol% of a lipid; 2) about 1-4 mol% of SPAN-85; 3) about 1-4 mol% of PS-20 or PS-80; and 4) about 50-80 mol% of squalene.

[0115] In some embodiments, an SNE formulation is provided comprising: 1) about 30-65 mol% of a lipid; 2) about 5-30 mol% of SPAN-85; 3) about 0.5-4 mol% of PS-20 or PS-80; and 4) about 10-40 mol% of squalene.

[0116] In some embodiments, an SNE formulation is provided comprising: 1) about 55-65 mol% of a lipid; 2) about 5-15 mol% SPAN-85; 3) about 1-2.5 mol% of PS-20 or PS-80; and 4) about 25-35 mol% of squalene.

[0117] In some embodiments, an SNE formulation is provided comprising: 1) about 13-45 mol% of a lipid; 2) about 2-4 mol% SPAN-85; 3) about 1.5-3 mol% of PS-20 or PS-80; and 4) about 50-82 mol% of squalene.

[0118] In some embodiments, an SNE formulation is provided comprising: 1) about 13-14 mol% of a lipid; 2) about 1-2 mol% SPAN-85; 3) about 1-2 mol% PS-20 or PS-80; and 4) about 79-81 mol% of squalene.

[0119] In some embodiments, an SNE formulation is provided comprising: about 1-60 mol% of a lipid; 3) about 1-4 mol% of PS-20 or PS-80; and 4) about 32-97 mol% of squalene.

[0120] In some embodiments, an SNE formulation is provided comprising s: 1) about 0-45 mol% of a lipid; 2) about 1-10 mol% of a non-ionic surfactant; and 3) about 50-85 mol% of squalene. In one aspect of this embodiment, the non-ionic surfactant comprises a mixture of SPAN-85 and PS-20 or PS-80.

[0121] In some embodiments, an SNE formulation is provided comprising: 1) about 10-14 mol% of a lipid; 2) about 1-4 mol% of SPAN-85; 3) about 1-4 mol% of PS-20; and 4) about 50- 80 mol% of squalene.

[0122] In some embodiments, an SNE formulation is provided comprising: 1) about 30-65 mol% of a lipid; 2) about 5-30 mol% of SPAN-85; 3) about 0.5-4 mol% of PS-20; and 4) about 10-40 mol% of squalene.

[0123] In some embodiments, an SNE formulation is provided comprising: 1) about 55-65 mol% of a lipid; 2) about 5-15 mol% SPAN-85; 3) about 1-2.5 mol% of PS-20; and 4) about 25- 35 mol% of squalene.25948

[0124] In some embodiments, an SNE formulation is provided comprising: 1) about 13-45 mol% of a lipid; 2) about 2-4 mol% SPAN-85; 3) about 1.5-3 mol% of PS-20; and 4) about 50- 82 mol% of squalene.

[0125] In some embodiments, an SNE formulation is provided comprising: 1) about 13-14 mol% of a lipid; 2) about 1-2 mol% SPAN-85; 3) about 1-2 mol% PS-20; and 4) about 79-81 mol% of squalene.

[0126] In some embodiments, an SNE formulation is provided comprising: about 1-60 mol% of a lipid; 2) about 1-4 mol% SPAN-85; 3) about 1-4 mol% of PS-20; and 4) about 32-97 mol% of squalene.

[0127] In some embodiments, an SNE formulation is provided comprising: 1) about 0-45 mol% of a lipid; 2) about 1-10 mol% of a non-ionic surfactant; and 3) about 50-85 mol% of squalene. In one aspect of this embodiment, the non-ionic surfactant comprises a mixture of SPAN-85 and PS-20.

[0128] In some embodiments, an SNE formulation is provided comprising: 0.01-0.1 mol% of a lipid; 2) 1.5-15.0 mol% SPAN-85; 3) 1-10.0 mol% of PS-20; and 4) 80.0-98.0 mol% of squalene.

[0129] In some embodiments, an SNE formulation is provided comprising: 0.1-1.0 mol% of a lipid; 2) 8.0-11.9 mol% SPAN-85; 3) 6-10 mol% of PS-20; and 4) 78-85 mol% of squalene.

[0130] In some embodiments, an SNE formulation is provided comprising: 0.01-0.1 mol% of a lipid; 2) 8.0-11.9 mol% SPAN-85; 3) 6-10 mol% of PS-20; and 4) 78-85 mol% of squalene.

[0131] In some embodiments, an SNE formulation is provided comprising: 1) 8-16 mg of SPAN-85; 2) 8-16 mg of PS-20; and 3) 40-80 mg of squalene.

[0132] In some embodiments, an SNE formulation is provided comprising: 1) 0.6-3.6 mg of SPAN-85; 2) 0.6-3.6 mg of PS-20; and 3) 2.0-12.0 mg of squalene.

[0133] In some embodiments, an SNE formulation is provided comprising: 1) 0.15-0.18 mg of SPAN-85; 2) 0.15-0.18 mg of PS-20; and 3) 0.5-0.6 mg of squalene.

[0134] In some embodiments, an SNE formulation is provided comprising: 1) 0.15-0.95 mg of SPAN-85; 2) 0.15-0.95 mg of PS-20; and 3) 0.5-3.0 mg of squalene.

[0135] In some embodiments, an SNE formulation is provided comprising: 1) 0.24-1.3 mg of SPAN-85; 2) 0.24-1.3 mg of PS-20; and 3) 0.8-4.0 mg of squalene.

[0136] In some embodiments, an SNE formulation is provided comprising: 1) 1.2-6.5 mg of SPAN-85; 2) 1.2-6.5 mg of PS-20; and 3) 4.0-20.0 mg of squalene.

[0137] In some embodiments, an SNE formulation is provided comprising: 1) 5-8.0 mg of SPAN-85; 2) 5-8.0 mg of PS-20; and 3) 20.0-40.0 mg of squalene.25948

[0138] In some embodiments, an SNE formulation is provided comprising: 200-400 µg of a lipid; 2) 8-16 mg of SPAN-85; 3) 8-16 mg of PS-20; and 4) 40-80 mg of squalene.

[0139] In some embodiments, an SNE formulation is provided comprising: 10-20 µg of a lipid; 2) 8-16 mg of SPAN-85; 3) 8-16 mg of PS-20; and 4) 40-80 mg of squalene.

[0140] In some embodiments, an SNE formulation is provided comprising: 0.5-3.0 µg of a lipid; 2) 0.6-3.6 mg of SPAN-85; 3) 0.6-3.6 mg of PS-20; and 4) 2.0-12.0 mg of squalene.

[0141] In some embodiments, an SNE formulation is provided comprising: 0.5-3.0 µg of a lipid 2) 0.15-0.18 mg of SPAN-85; 3) 0.15-0.18 mg of PS-20; and 4) 0.5-0.6 mg of squalene.

[0142] In some embodiments, an SNE formulation is provided comprising: 0.5-3.0 µg of a lipid; 2) 0.15-0.95 mg of SPAN-85; 3) 0.15-0.95 mg of PS-20; and 4) 0.5-3.0 mg of squalene.

[0143] In some embodiments, an SNE formulation is provided comprising: 4.0-20 µg of a lipid; 2) 0.24-1.3 mg of SPAN-85; 3) 0.24-1.3 mg of PS-20; and 4) 0.8-4.0 mg of squalene.

[0144] In some embodiments, an SNE formulation is provided comprising: 4.0-20 µg of a lipid; 2) 1.2-6.5 mg of SPAN-85; 3) 1.2-6.5 mg of PS-20; and 4) 4.0-20.0 mg of squalene.

[0145] In some embodiments, an SNE formulation is provided comprising: 20-100 µg of a lipid; 2) 1.2-6.5 mg of SPAN-85; 3) 1.2-6.5 mg of PS-20; and 4) 4.0-20.0 mg of squalene.

[0146] In some embodiments, an SNE formulation is provided comprising: 20-40 µg of a lipid; 2) 5-8.0 mg of SPAN-85; 3) 5-8.0 mg of PS-20; and 4) 20.0-40.0 mg of squalene.

[0147] In some embodiments, any one of the formulations provided above comprise a particular lipid selected from: (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; (S)-N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; (S)-1-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide; (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide;25948 N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide; N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide; 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N- (3-stearamidopropyl)piperidine-4-carboxamide; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide; N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide; N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide; N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide; N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-(octadecyloxy)pentan-1-one; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one; N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)oleamide; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)stearamide; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (4-((4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate;25948 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N- (3-stearamidopropyl)piperazine-1-carboxamide; 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazine-1-carboxylate; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3- methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide; N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5- dimethoxybenzyl)(methyl)amino)butyl)stearamide; and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (5-(4-(4-((5-amino-7-(butylamino)-2H- pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5- oxopentyl)carbamate. Vaccines

[0148] Vaccines are well known in the art and include adenovirus vaccines, anthrax vaccines, cholera vaccines, dengue vaccines, diphtheria vaccines, hepatitis vaccines, Haemophilus influenzae type b (Hib) vaccines, human papillomavirus (HPV) vaccines, seasonal influenza (Flu) vaccines, Japanese encephalitis vaccines, measles vaccines, meningococcal vaccines, mumps vaccines, pertussis vaccines, pneumococcal vaccines, polio vaccines, rabies vaccines, rotavirus vaccines, rubella vaccines, shingles vaccines, smallpox vaccines, tetanus vaccines, tuberculosis vaccines, typhoid fever vaccines, varicella vaccines and yellow fever vaccines.

[0149] Pneumococcal vaccines or compositions are well known (for example PNEUMOVAX®, Merck & Co., Inc., Rahway, NJ, USA). Pneumococcal conjugate vaccines or compositions have been previously disclosed. See WO2011 / 100151, WO2019 / 139692 and WO2020 / 131763.

[0150] In some embodiments of the invention, a formulation is provided wherein the SNE further comprises one or more additional components which are selected from a surfactant, a mixture of surfactants, a phospholipid, a terpene, a terpenoid, a triterpene or a combination thereof.

[0151] In some embodiments of the invention, the surfactant includes, but is not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially PS- 20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); nonylphenol ethoxylates, such as25948 the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span-85, Tween-85 or [2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec- 9-enoyl]oxyethyl] (Z)-octadec-9-enoate) and sorbitan monolaurate.

[0152] In some embodiments of the invention, mixtures of surfactants are used, e.g., PS- 20 / Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t- octylphenoxypolyethoxyethanol (Triton X-100) are also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

[0153] In some embodiments of the invention, the amounts of surfactants or emulsifiers are: polyoxyethylene sorbitan esters (such as PS-20) 0.01 to 10 mole %, in particular about 1 to 4 mol%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 10 mol%, in particular about 1 to 4 mol%; w / v, in particular 0.01 to 0.1% w / v; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 mol%, preferably 0.5 to 10 mol% and in particular 1 to 4 mol% or about 10 % by mass.

[0154] In some embodiments of the invention, the phospholipid is selected from, a natural phospholipid including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacyl-phosphatidylcholine (lyso PC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), N-Acyl-PE, a phosphoinositide, and a phosphosphingolipid. Phospholipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), phosphatidylserine (DOPS). Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18: l), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0 and lethicin. In certain embodiments of the invention, the phospholipid is phosphatidylserine, l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2- dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0155] In some embodiments of the invention, the terpene is selected from a monoterpene, such as geraniol, terpeneol, limonene, myrcene, linalool or pinene. In some embodiments, the formulation comprises a sesquiterpene consisting of humulene, a farnesene, or farnesol; aditerpene such as cafestol, kahweol, cembrene or taxadiene; A triterpene such as squalene or squalante; a tetraterpene such as acyclic lycopene, monocyclic gamma-carotene, or bicyclic alpha- and beta-carotenes; a polyterpene or a norisoprenoids. EXAMPLES

[0156] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Example 1: A TRADITIONAL SNE PROCESS (FIGURE 1) PREPARATION OF A STABLE NANOEMULSION (SNE) ADJUVANT SYSTEM WITH AND WITHOUT THE CATIONIC LIPID: (13Z,16Z)-N, N-DIMETHYL-3-NONYLDOCOSA-13,16-DIEN-1-AMINE)

[0157] The SNE adjuvant can be prepared with and without cationic lipids, for example, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine), or (6Z,9Z,26Z,29Z)-N,N- dimethylpentatriaconta-6,9,26,29-tetraen-18-amine, or N,N-dimethyl-1-((1S,2R)-2- octylcyclopropyl)heptadecan-8-amine. The SNE is a multi-component emulsion formulation consisting of 3 stabilizing ingredients; squalene, sorbitan trioleate (SPAN-85), and polysorbate- 20 (PS-20) with a cationic lipid, see Table 1, and without a cationic lipid (see Table 2). This formulation was prepared by combining and mixing the cationic lipid (if used), squalene, SPAN- 85 and PS-20 or similar (e.g., surfactants, oils, and solubilizers) components together (Table 1 and Table 2 and Figure 1). Once mixed and blended, a histidine buffer was added and mixed with the initial emulsion components. Blended emulsion components were first subjected to course homogenization followed by fine homogenization, as described below. The resulting formulation was subjected to a final 0.2 μm filtration step. Several process parameters within each step, such as order of addition, mixing time, pH, temperature, concentration of components, homogenization, microfluidization were controlled to yield an emulsion system with desired attributes. Table 1: Composition of a Representative SNE Coarse Homogenization Content of Molecular Content of EachPS-20 polysorbate-20 1.72-2.68 1228 4.55-7.14 Buffer 20 mM Histidine, pH 5.8 N / AContent of Molecular Component Description Each Lipid Weight Content of Each Lipid (Mass %)

[0158] The squalene and solubilizer formulation (referred to as the oil phase) of the stable emulsion was prepared by addition of squalene, SPAN-85, PS-20 and the cationic lipid to a vessel. The oil phase was then mixed using magnetic stirring at 100-1000 RPM for 10 to 120 minutes. After mixing of these components, an aqueous phase comprised of 20 mM Histidine pH 5.8, was slowly added to the oil phase while being mixed using a magnetic stir bar. This formulation was then mixed again for 1 hour. Coarse Homogenization

[0159] The oil and aqueous phase mixture (referred to as the pre-homogenized emulsion or PHE) was then homogenized and size reduced to form a rough emulsion using a rotor stator homogenizer at ambient temperature. The homogenizer arm tip was submerged into the PHE and held in place near the bottom of the formulation vessel and was operated at 6 to 10 kRPM for 5- 15 minutes. This process resulted in a homogenous micro-emulsion (ME) suspension of squalene emulsion particles in the 4 to 20 µm diameter range which were suitable for additional size reduction by microfluidization in a high-pressure homogenizer to create a stable nanoemulsion (SNE). Fine Homogenization to Produce the Stable Nanoemulsion (SNE)

[0160] After coarse homogenization, the emulsion was further processed using a high-pressure homogenizer / microfluidizer to produce stable nanometer-sized emulsion particles. The ME was introduced to a high-pressure homogenizer such as the Microfluidics low volume Microfluidizer25948 ®, the GEA Group PandaPlus 2000 or Bee International, NanoDeBEE and a recirculation loop is established. A counter-flow heat exchanger, fed by a Controlled Temperature Unit with a set point of 5℃, is included in the recirculation loop to neutralize the heat generated through high pressure homogenization. For the production of emulsion particles of desired size and processability, 20 kPSI was selected as the operating set point for this process step. The high- pressure homogenizer operates at a constant and unalterable flow rate through the established recirculating loop. Using this measured flow rate and the volume of ME to be processed, the theoretical time required for the entirety of the formulation to make a single pass through the recirculation loop was calculated. Given this calculated single pass time, the high-pressure homogenizer was usually operated until the desired pass count of at least 10 was reached, yielding either the SNE or CLA-SNE. Bioburden Reduction Filtration

[0161] After formulation, the SNE (without the cationic lipid) or the SNE (with the cationic lipid) was passed through a 0.8 / 0.2 µm PES filter. A flux of 42 LMH through the filter was selected given its optimal mass yield and particle stability through filtration.

[0162] A laser diffraction or static light scattering (SLS) technique using a Malvern Panalytical Ltd. MS3000 instrument was utilized to measure the volume-weighed size distribution of a nanoemulsion during preparation. This data was then analyzed to calculate the size of the particles that created the scattering pattern. Sample fractions of pre-homogenized emulsion (PHE), micro-emulsion (ME), and stable nanoemulsion (SNE) were obtained. These emulsion formulations were diluted to target an obscuration of 3% into 5 mM Histidine pH 5.8 and 2.5 mM NaCl buffer and SLS was performed and collected under recirculation of 1200 RPM. Sample data sets were collected with a scan of 30 seconds per data set. Although 20 mM Histidine pH 5.8 buffer was a perfectly suitable formulation for the stability of the bulk during process and when stored in polymeric containers (e.g., plastic), upon storage in glass, non- specific absorption of the SNEs to the surface of the glass was observed. A screen evaluating surfactants / solubilizers, buffers and salts was evaluated, and multiple formulations show success in eliminating this stability issue with the selected formulation of 20 mM Histidine 0.05% PS-20 and 75 mM NaCl being selected as the stabilizing formulation (data not shown). Example 2: Preparation of an SNE in a Nanoprecipitation Mixing Device (T-mixer) (Figure 2): Preparation of a Stable Nanoemulsion (SNE) Adjuvant System With and Without the Cationic lipid: (13Z,16Z)-N, N-dimethyl-3-nonyldocosa-13,16-dien-1-amine)25948 Formulation Preparation

[0163] Squalene, SPAN-85, and PS-20 were added to a vessel followed by ethanol. This mixture was mixed using magnetic stirring at 100-1000 RPM for 10 to 120 minutes and once complete referred to as the oil packet or the formulated component mixture (or organic phase). A buffer of 20 mM L-Histidine pH 5.8 was prepared and was used as the aqueous packet (or aqueous phase), stabilizing buffer, and diafiltration buffer in downstream operations. T-mixing

[0164] Once the oil and aqueous packets (the organic and aqueous phases) were formulated, T- mixing was initiated. Individual pumps control the volumetric flow rate of the oil (organic) and aqueous phases and these liquid streams meet in a T-mixer at 180° to each other, and once they meet, exit the T-mixer at 90° to their original flow. The ratio of the organic and aqueous flowrates were tightly controlled and referred to as the T-Mix Ratio (TMR). The TMR is defined as the aqueous phase flow rate divided by the organic phase flow rate. In this example, the TMR was set to 1.6, and the combined flow rate of both aqueous phase and organic phase streams through a 0.25 mm inner diameter T-mixer was 30 mL / min. Upon exiting the T-mixer the liquid product stream was stabilized using a 4-fold in-line dilution with a stabilizing buffer. The particle size of the SNE intermediate was measured by DLS using a Malvern Panalytical Nano ZS. Tangential Flow Filtration

[0165] After T-mixing the SNE intermediate is diluted in a non-optimal buffer matrix containing 5-20% ethanol from the oil (organic) phase. To concentrate and buffer exchange the SNE intermediate to a formulation suitable for pharmaceutical applications (the final SNE) tangential flow filtration was performed. A hollow fiber membrane with 500kDa molecular weight cutoff (MWCO) was flushed and prepared for SNE intermediate filtration. Fed-batch concentration was performed to concentrate the SNE intermediate by a factor of 10-20. Following SNE intermediate concentration, a volume of diafiltration buffer equal to 8-12x the SNE intermediate volume at the end of concentration (diavolume) was added to exchange the SNE intermediate buffer and remove ethanol. At the end of diafiltration, additional batch concentration can be performed to achieve the final target SNE concentration. The particle size of the SNE intermediate was measured by DLS using a Malvern Panalytical Nano ZS.25948 Bioburden Reduction Filtration

[0166] After formulation, the SNE (without the cationic lipid) or SNE (with the cationic lipid) was passed through a 0.8 / 0.2 µm PES filter. A flux of 42 LMH through the filter was selected given its optimal mass yield and particle stability through filtration.

[0167] Upon completion of the final SNE preparation process, particle size of the final SNE was measured by DLS. Comparison between SNE particle sizes produced by methods described in Example 1 and this example (Example 2) are shown in Table 3; similar particle sizes and polydispersity can be achieved using these methods. Cryo-EM images of final SNE produced via homogenization and T-mixing are shown in Figure 3 and no disenable differences can be distinguished in the final product SNE between these two preparation processes. Table 3. Comparison of particle size of SNE produced via homogenization and T-mixing Preparation Method Particle Size by DLS (nm) Polydispersityp y g g T-mixing Process

[0168] Using the T-mixing process, the SNE particle size can be controlled by altering a single process parameter, namely the tee mix ratio (TMR). The TMR is the ratio of aqueous flow (the aqueous stream) divided by the lipid flow (the organic stream). The T-mixing process as described in Example 2 was performed with total flow through a T-mixer maintained and the TMR altered from 1.0 to 3.0. DTMP was collected and analyzed by DLS using a Malvern Panalytical Nano ZS. As shown in Figure 4, TMR has an inverse correlation with particle size; as the TMR increases, the particle size decreases in a controlled and reproducible manner.

[0169] In addition to TMR, particle size can also be controlled by changing the pH of the aqueous buffer used in T-mixing. In the example shown in Figure 5, increasing the pH shifts the TMR curve to the left meaning that lower TMRs are required to achieve a target particle size. Example 4: Preparation of an SNE Adjuvant with Inclusion of an Insoluble Lipid Formulation Using a T-mixing Device

[0170] An SNE comprising an insoluble lipid is considered a multi-component complex emulsion formulation consisting of emulsifier ingredients, squalene and an insoluble lipid, for example the insoluble lipid: N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-25948 2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide. This formulation was prepared, in a similar manner to Example 2, by combining and mixing emulsifiers (which include, SPAN-85 and PS-20) with squalene and the insoluble lipid after dissolving in 100% EtOH together. The aqueous buffer was 20mM Histidine pH 5.8.

[0171] The process of making the SNE consists of 5 steps: 1) solution preparation of a component mixture that includes 3 stabilizing ingredients and the insoluble lipid: SPAN-85, PS- 20, squalene, and N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2- yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide; 2) SNE intermediate formation by means of controlled precipitation process utilizing a T-mixing device; 3) ultra- filtration; 4) bioburden reduction filtration; and 5) sterile filtration and vial filling. The following example will highlight the precipitation mixing device (the T-mixer) and ability to control particle size via variation of flowrates entering the T-mixing device. Solution preparation of stabilizer / compound mixture and mixing system

[0172] The stabilizer / compound components were weighed and combined before being dissolved in ethanol and then heated at 40ºC for 30 min and sterile filtered to form the organic phase. The concentration of N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin- 2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide was 0.00625- 1.25 mg / mL. The concentration of PS20 was 5-9 mg / mL. The concentration of SPAN-85 was 5-9 mg / mL. The concentration of squalene was 25 mg / mL. The organic phase was then combined with the aqueous phase, consisting of 20 mM Histidine buffer pH 5.8, using the T-mixer. The flowrate of the aqueous phase divided by the organic phase is termed the tee mix ratio. This ratio was varied from a low value of 1.35 to 2. The stream exiting the apparatus was immediately diluted 3:1 with 20 Histidine pH 5.8, and then collected as the formed SNE intermediate.

[0173] Tangential flow filtration, bioburden reduced filtration, sterile filtration and vial filling processes were similar to Example 2. As shown in Figure 6, an increase in aqueous to ethanol (organic) flow ratio from 1.35 to 2 results in a decrease in particle size of the SNE. Example 5: Preparation of an SNE Using a Staggered Herringbone Mixing Device

[0174] The SNE is a multi-component emulsion formulation consisting of emulsifier ingredients and squalene. This formulation is prepared by combining and mixing emulsifiers (which include, SPAN-85 and PS-20) with squalene after dissolving in 100% EtOH together.

[0175] The process of making the SNE consists of 5 steps: 1) solution preparation of a component mixture that includes the 3 stabilizing ingredients: SPAN-85, PS-20 and squalene; 2)25948 SNE formation by means of a SHM device; 3) ultra-filtration; 4) bioburden reduced filtration; and 5) sterile filtration and vial filling. Solution preparation of stabilizer / compound mixture

[0176] The stabilizer / compound components were weighed and combined before being dissolved in ethanol and then heated at 40ºC for 30 min and sterile filtered to form the component mixture. The component mixture and Histidine buffer were then combined at adjacent ends of the SHM device using a flow ratio of 1.5; 2 and 5 of aqueous to ethanol (organic) streams. The product stream exiting the apparatus was immediately diluted 1:1 with 20 Histidine pH 5.8, and then collected as the formed SNE intermediate. Buffer exchange by dialysis

[0177] The SNE intermediate was then subjected to Dialysis in order to both concentrate the material approximately 10-fold as well as buffer exchange the material against 20 mM Histidine, 0.05% (w / v) PS-20 and 75 mM NaCl, pH 5.8 or 20 mM Histidine pH 5.8 or 20 mM Histidine 0.05% PS-20 pH 5.8. After the dialysis, there was a final concentration step performed to achieve final target concentration. Histidine pH 5.8, PS-20, sodium chloride, L-met, and EDTA solutions were prepared and added to a formulation vessel. The adjuvant bulk SNE was added to the formulation vessel. During the addition of the final SNE to the formulation buffer, the vessel was mixed to ensure homogeneity using a magnetic sir bar or magnetic impeller. After all additions were made and the solution was stirred the formulations were filled into plastic syringes, glass syringes, or vials. As shown in Figure 7, an increase in aqueous to ethanol (organic) flow ratio from 1 to 5 results in a decrease in particle size of the nano-emulsion. Example 6: Preparation of a Nanoemulsion Formulation

[0178] To prepare the nanoemulsion, an organic phase (organic solvent) and aqueous phase (aqueous solvent) were mixed at specific controlled flowrates to produce a desired particle size. The organic phase was prepared by combining squalene, PS-20, and SPAN-85 in a 100% ethanol solution. The organic phase was composed of 25 mg / mL squalene, 5.0 mg / mL PS-20, and 2.5 mg / mL SPAN-85, or at a ratio of 25: 5: 2.5 squalene: PS-20: SPAN-85. After mixing, the organic phase was filtered through a sterilizing grade filter. The aqueous phase was prepared as 20 mM L-histidine, pH 5.5.

[0179] Precipitation mixing was performed using a stainless steel tee with 0.75 mm inner diameter. The aqueous and organic phases met at 180°C, and the tee outlet was positioned 90°25948 from both the aqueous and organic phase inlets. Downstream of the stainless steel tee (Tee #1), a second plastic tee (Tee #2) was used for inline dilution of the output of Tee #1. To determine the flow rates of the organic and aqueous phases, the tee mix ratio (TMR) is defined as the aqueous phase flow rate divided by the organic phase flow rate. The TMR used for this process was approximately 1.4. The Tee #2 dilution buffer was the same formulation as the aqueous phase buffer.

[0180] The tee mix product was concentrated and exchanged into fresh 20 mM L-histidine, pH 5.8 buffer via an ultrafiltration process. To start ultrafiltration, an initial diavolume of the tee mix product was dispensed into the working vessel and then recirculated across a hollow fiber filter. The tee mix product was concentrated and then diafiltration was performed using 20 mM histidine, 0.05% PS-20, 10 mM L-methionine, 0.1 mM EDTA, pH 5.8 buffer. After diafiltration, a final batch concentration step was performed to further concentrate the material, making the overall ultrafiltration concentration factor approximately 40x from the tee mix to ultrafiltration product.

[0181] Following ultrafiltration, a normal filtration was performed to reduce bioburden using a sterilizing grade filter. Impact of pH and buffer species (sodium citrate vs. histidine) on TMR and particle size

[0182] As shown in Figure 8, to determine the impact of pH and buffer species on nanoemulsion tee mixing, the pH of the aqueous buffer and dilution buffer was varied from 3 to 7. The organic phase composition was 25: 2.5: 2.5 mg / mL squalene: PS-20: SPAN-85 (10:1 squalene: each emulsifier). The concentration of all buffers was held constant at 20 mM L- histidine. At pH 6, the relationship between TMR and particle size is comparable for sodium citrate and histidine buffer. When the pH of the sodium citrate buffer is decreased to 3 and 4, the TMR curve does not shift significantly. When the pH was increased above 6 for the L-histidine buffer, the TMR curves shifted to the left; this was further investigated (see figure 9). This data shows that both L-histidine and sodium citrate are useful buffers for forming stable nanoemulsions using a tee mixing process. Impact of pH on TMR and particle size

[0183] As shown in Figure 9, to further investigate the impact of pH, 20 mM histidine buffers from pH 5 to 7 were investigated for a different nanoemulsion composition. The organic phase composition was 25: 5: 2.5 mg / mL squalene: PS-20: SPAN85 (5:1 squalene: PS-20 and 10:1 squalene: SPAN-85). The pH was adjusted for the buffer fed into Tee #1 and Tee #2. When the25948 pH was varied between 5-6, no significant impact was observed. However, when the pH was increased to 6.5 and 7, the TMR curve shifts to the left, meaning a lower TMR was required to produce a given particle size. In a separate test, when the Tee #1 aqueous phase was pH 7 and the Tee #2 dilution buffer was pH 6, the relationship matches the pH 7 overall condition (data not shown). This indicates that the aqueous buffer used as the Tee #1 feed has greater control over the TMR and particle size relationship. This knowledge can be leveraged to optimize Tee #1 conditions for robustness, but the final pH of the tee mixed product can be changed via the input of Tee #2. Impact of histidine buffer concentration on TMR and particle size

[0184] As shown in Figure 10, histidine buffer concentration in the aqueous phase impacts the relationship between TMR and particle size. The organic phase composition was 25: 5: 2.5 mg / mL squalene: PS-20: SPAN-85 (5:1 squalene: PS-20 and 10:1 squalene: SPAN85) and all aqueous buffers were made with L-histidine at pH 5.5. As the histidine concentration was varied from 5-50 mM, the TMR and particle size relationship for TMR > 1.25 was comparable. However, when the histidine concentration was decreased to 2 mM and 1 mM, the left side of the TMR curve is flattened for TMRs between 1-1.4. This provides a potential process advantage if the target particle size and formulation combination is between 130-200 nm because slight variations in flowrates (and therefore in the TMR) will not cause as dramatic of an impact on the particle size when the aqueous phase has lower histidine concentration. When the Tee #2 dilution buffer histidine concentration is modified, the TMR and particle size relationship does not change; the Tee #1 aqueous buffer determines this relationship. If a lower histidine concentration is used as the input to Tee #1, a higher histidine concentration can be used to improve the overall buffering capacity of the tee mix product without impacting the TMR and particle size relationship. Impact of changing the squalene: PS-20 to squalene: SPAN-85 ratio has on TMR and particle size

[0185] As shown in Figure 11, increasing the concentration of emulsifiers relative to squalene in the organic phase enables the formation of smaller particle sizes at a given TMR. To determine the impact of changing the squalene: PS-20 and squalene: SPAN-85 ratio has on TMR and particle size the emulsifier concentrations were increased to 5:1 and 3.33:1 (squalene: each emulsifier). As the overall emulsifier concentration is increased, the TMR curve shifts down meaning that smaller particles can be formed at a specific TMR and the overall minimum particle25948 size is smaller. This aligns with previous observations in literature that increasing the emulsifier content in homogenized emulsions results in smaller particles. Impact of changing the squalene: PS-20 ratio (keeping the squalene: SPAN-85 ratio constant at 10:1) has on TMR and particle size

[0186] To determine the impact of changing the squalene: PS-20 ratio has on the relationship between TMR and particle size, the squalene: SPAN85 ratio was held constant and the PS-20 in the organic phase was modified. As shown in Figure 12, when the squalene: SPAN85 ratio was held constant at 10:1 and the amount of PS-20 was increased, the minimum particle size that can be produced decreases to a limit. When the amount of PS-20 was reduced, the minimum particle size increases and the particle size produced at a given TMR increases relative to formulations with higher squalene: PS-20 ratios. Modifying the ratio of squalene: PS-20 can be advantageous if targeting a specific particle size. For example, if targeting a particle size of 100 nm, using a 5:1 or 3:1 ratio is required. Alternately, if a particle size of 155-175 nm is desired, using the 20:1 ratio is advantageous because a wide range of TMRs can be used to produce these particles (2.5- 4.0). Having a wide range of TMR that will produce the target particle size is advantageous because slight deviations in the aqueous and organic flow rates will not produce particle sizes out of specification. If high or low TMRs are difficult to achieve on a particular mixing system, changing pH or squalene: PS-20 ratio may be advantageous to optimize the TMR needed to produce a given particle size. This shows that in addition to the TMR, the PS-20 concentration is another parameter that can be used to control particle size in tee mixing. Impact that changing the squalene: SPAN-85 ratio (keeping the squalene: PS-20 ratio constant at 10:1) has on TMR and particle size

[0187] To determine the impact that changing the squalene: SPAN-85 ratio has on the relationship between TMR and particle size, the squalene and PS-20 concentrations were held constant and the SPAN85 concentration in the organic phase was increased. As shown in Figure 13, when the squalene: PS-20 ratio is held constant at 10:1 and the SPAN-85 in the formulation is increased, the relationship between the TMR and particle size did not change indicating that PS- 20 has a greater control over this relationship. However, SPAN-85 does play a role in the TMR and particle size relationship because increasing the concentrations of both emulsifiers together produces a slightly different curve than produced when changing PS-20 concentration alone (compare to curves in Figure 11).25948 Effect of scalability on TMR and particle size

[0188] As shown in Figure 14, a scale comparison was performed by preparing nanoemulsions at different scales. An organic phase with a squalene: PS-20: SPAN-85 composition of 25: 2.5: 2.5 mg / mL was used with an aqueous phase of 20 mM histidine, pH 5.5. The pilot scale plot was generated using a 0.75 mm tee with a centrifugal pump system with a total flow rate of 270 mL / min out of Tee #1. The small scale plot was generated using a 0.15 mm tee and a dual syringe pump with a total flow rate of 10.8 mL out of Tee #1. Error bars show the standard deviation (n=2) for TMRs less than 3. Although there is a small offset between the pilot scale and small scale mixes (approximately 10 nm), the standard deviation between mixes with the same conditions is low (often less than 1 nm). This suggests that although small differences in particle sizes may be observed between the two scales, individual mixes produce nanoemulsions with highly consistent particle size distributions. Bioburden reduction filtration pressure profile

[0189] As shown in Figure 15, bioburden reduction filtration was performed for nanoemulsion made using a traditional homogenization process as described in Example 1 and nanoemulsion made using a tee mixing process as described in Example 6. Both nanoemulsions were composed of approximately 10:1:1 squalene: PS-20: SPAN-85 nanoemulsion in 20mM histidine, pH 5.8 buffer. Emulsion loading was calculated using the sum of the estimated squalene, PS-20, and SPAN-85 concentrations in the filtration feed. The particle sizes of the product of both batches were within 10 nm of each other. Compared to the homogenization process, the tee mix process has significantly lower filtration pressure and higher throughput. This suggests that a tee mixing process may significantly improve the sterile filtration of nanoemulsions compared to traditional manufacturing methods for the same emulsion composition. Comparison of a tee mixing process v. a traditional process

[0190] A comparison of a tee mixing process and a traditional process was conducted. As shown in Table 4, tee mixing can be used to form nanoemulsions with the same average particle size and polydispersity as nanoemulsions made using traditional methods. Additionally, tee mixing can be used to form nanoemulsions within a range of particle sizes from approximately 100 to 200 nm without changes the nanoemulsion composition, while microfluidization and homogenization processes can produce one average particle size. Typical squalene mass yields are higher for the tee mixing process, mostly attributed to a significant improvement in filterability as demonstrated in Figure 15.25948

[0191] Table 4. Demonstration showing that a tee mixing process can be used for form stable nanoemulsions with analytical comparability to traditional homogenization and microfluidization processes. Method Average Particle Size (nm) Polydispersity Typical Process Squalene Yield (%)R and particle size

[0192] To determine the impact that a squalene: functional lipid ratio and a squalene: emulsifier ratio has on the relationship between TMR and particle size, three different nanoemulsion compositions were produced at a range of TMRs. As shown in Figure 16, the first ratio listed shows the squalene: functional lipid ratio, and the second ratio gives the squalene: each emulsifier ratio. Similar to the emulsion formulation without the functional lipid, increasing the relative amount of emulsifiers causes the TMR curve to shift down and enables the formation of smaller particles. When the functional lipid was increased (from the 1000:1 to 40:1 formulation), the TMR curve shifted significantly to the left showing that the concentration of the functional lipid has an impact on the TMR and particle size relationship over this range. This suggests that above a certain relative concentration threshold the functional lipid has an impact on the TMR and particle size relationship.

[0193] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims. Embodiments Disclosed

[0194] A process to make a stable nanoemulsion (SNE) comprising: a. preparing or obtaining a formulated component material (organic phase) and a formulated aqueous solvent (aqueous phase), wherein the component material comprises squalene and one or more emulsifiers; b. mixing the organic phase and the aqueous phase in a nanoprecipitation mixing device, wherein the organic phase and the aqueous phase are liquid streams each with25948 controllable flow rates, wherein the organic phase and the aqueous phase i) separately enter the nanoprecipitation mixing device, ii) mix in the nanoprecipitation device, and iii) exit the nanoprecipitation device as a single stream containing an intermediate SNE; and c. filtrating the intermediate SNE to generate the final SNE.

[0195] The process above, wherein the nanoprecipitation mixing device is selected from a T- Tube mixer (T-mixer), a staggered herringbone mixer (SHM) and a multi-inlet vortex mixer (MIVM).

[0196] The process above, wherein the flow rate of the organic stream and the flow rate of the aqueous stream are controlled to a specific flow rate ratio.

[0197] The process above, wherein the component material is squalene, one or more emulsifiers and one or more lipids.

[0198] The process above, wherein the emulsifier is selected from sorbitan trioleate (SPAN- 85), polysorbate-20 (PS-20) and polysorbate-80 (PS-80).

[0199] The process above, wherein the component materials are formulated at specific concentration ratios.

[0200] The process above, wherein the aqueous solvent is Tris, Bis-Tris, Citrate, HEPES, Phosphate, and L-Histidine.

[0201] The process above, wherein the aqueous solvent is a L-Histidine.

[0202] The process above, wherein the single stream is stabilized with an in-line dilution of a stabilizing buffer.

[0203] The process above, wherein the step (c) filtration is tangential flow filtration followed by bioburden reduction filtration.

Claims

25948 WHAT IS CLAIMED IS:

1. A method of making a nanoemulsion formulation that comprises squalene, polysorbate-20 (PS-20) and sorbitan trioleate (SPAN-85), comprising: a. preparing or obtaining (i) an organic solution that comprises squalene, PS-20, and SPAN-85, and (ii) an aqueous solution; b. mixing the organic solution and the aqueous solution in a tee mixing device, wherein the organic solution is a liquid stream and has an particular flow rate and the aqueous solution is a liquid stream and has a particular flow rate, wherein the organic solution and the aqueous solution (i’) separately enter the tee mixing device at a tee mix ratio of about 0.5 to 5.0, (ii’) mix in the tee mixing device, and (iii’) exit the tee- mixing device as a single liquid stream; wherein the single liquid stream contains particles that have an average size from about 80 nm to about 200 nm.

2. The process of claim 1, wherein the mass ratio of squalene to SPAN-85 is between 12:1 or 11:1 or 10:1 or 9:1 or 8:1 or 3:

1.

3. The process of claim 1, wherein the mass ratio of squalene to PS-20 to SPAN-85 is about 10:1:

1.

4. The process of claim 1, wherein the tee mix ratio is about 1.0 to about 3.

0.

5. The process of claim 1, wherein the tee mix ratio is about 1.0 to about 1.

8.

6. The process of claim 1, wherein the tee mix ratio is about 1.

4.

7. The process of claim 1, wherein the particles have an average size from about 150 nm to about 170 nm.

8. The process of claim 1, wherein the particles have an average size of about 160 nm.

9. The process of claim 1, wherein the aqueous solution contains Tris, Bis-Tris, Citrate, HEPES, Phosphate, or L-Histidine.25948 10. The process of claim 9, wherein the aqueous solution contains L-Histidine.

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