Supercritical fluid chromatography (SFC) purification of saponins

Abstract

The present application is directed to methods, systems, and compositions related to and obtained from the supercritical fluid chromatography -based purification of saponins. In some embodiments, the present application is directed to methods of producing a purified saponin composition, the methods comprising applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system. In some embodiments, the present application is directed to such methods and applying an elution condition to the SFC stationary phase to produce the purified saponin composition. Also provided herein are SFC systems for practicing the methods described herein, as well as compositions and medicaments produced by the methods described herein.

Description

SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) PURIFICATION OF SAPONINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 564,805, filed on March 13, 2024, the contents of which are hereby incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] Aspects of the present disclosure relate to methods and compositions for purifying saponins using supercritical fluid chromatography (SFC). In some embodiments, the present disclosure is directed to methods of producing a purified saponin composition from a Quillaja saponin source composition using SFC. Certain aspects of the present disclosure further relate to purified saponin compositions, including purified saponin compositions comprising QS-21, and their use in products such as vaccines.BACKGROUND

[0003] Saponins have a wide range of commercial and medicinal uses. For example, the saponin QS-21 has gained a reputation as a premier adjuvant enabling protein subunit vaccines. QS-21 is found in the approved vaccines Shingrix® (herpes zoster), Mosquirix® (malaria), and Arexvy® (RSV) all from Glaxo Smith Kline (GSK). While saponin mixtures can be used for a similar purpose, specific types of saponins, such as QS-21, have been found to be particularly effective at enhancing or inducing immune response.

[0004] Saponins, which are steroid or triterpenoid glycosides that are amphipathic, may be derived from natural sources and synthetic approaches. For example, the tree Quillaja saponaria is an evergreen tree that is native to Chile. Q. saponaria contains saponins, such as QS-21.Current tree -based production methods for saponins, e.g., QS-21, primarily use the bark of mature Q. saponaria trees from natural forests. This means that saponin production is reliant on a slow-growing and limited natural resource. The bark of mature Q. saponaria trees is used to produce an extract known as Quil-A, which is a purified mixture of over 25 different saponins and can be used to isolate individual saponins such as QS-21.

[0005] At the time of filing this application, conventional sources of QS-21 purified to over 90% purity (such as those useful as vaccine adjuvants) are from purification processes involving reversed-phase (RP) high pressure liquid chromatography (HPLC) techniques (RP-HPLC). RP- HPLC is useful for purifying QS-21 as this saponin contains numerous sugar moieties and an acyl chain that increase its polar characteristic relative to other types of saponins. While effective at purifying QS-21, such kilogram- sc ale RP-HPLC purification schemes are extremely expensive, use very large amounts of the hazardous organic chemical acetonitrile, and require special explosion proof cGMP facilities. Specifically, the acetonitrile using in RP-HPLC is volatile and flammable, easily ignited by heat, sparks, or flames and gives off highly toxic hydrogen cyanide fumes when heated. Moreover, acetonitrile can react with water, steam, or acids to produce flammable vapors that can form explosive mixtures when exposed to air. The only FDA licensed facility to produce cGMP QS-21 was built in 2019 at a cost of $100M and was licensed by the EPA to discard a maximum of 300,000 gallons of acetonitrile per year meaning that production is capped.BRIEF SUMMARY

[0006] In some aspects, provided herein is a method of producing a purified saponin composition, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system; and applying an elution condition to the SFC stationary phase to produce the purified saponin composition.

[0007] In some embodiments, the Quillaja saponin source composition is from Quillaja saponaria. In some embodiments, the Quillaja saponin source composition is from Quillaja brasiliensis . In some embodiments, the method comprises processing a plant material to produce the Quillaja saponin source composition. In some embodiments, the plant material is selected from the group consisting of bark, cells, callus, leaf, root, plantlet, and laboratory-derived or bioreactor-derived plant material. In some embodiments, the method further comprises processing spent plant growth media derived from a Quillaja saponin source plant material. In some embodiments, the Quillaja saponin source composition is a synthetic Quillaja saponin source composition.

[0008] In some embodiments, the method further comprises collecting at least one elution fraction from the SFC stationary phase.

[0009] In some embodiments, the purified saponin composition comprises one or more of QS-21, QS-7, QS-17, or QS-18. In some embodiments, the purified saponin composition comprises QS-21. In some embodiments, QS-21 is QS-21 -apiose and / or QS-21 -xylose. In some embodiments, QS-21 is a mixture of QS-21-apiose and QS-21-xylose at a ratio of about 1:20 to about 20:1.

[0010] In some embodiments, the purified saponin composition comprises at least about 90% QS-21 relative to components in the purified saponin composition with a molecular weight of about 1985 Da to about 1995 Da.

[0011] In some embodiments, the SFC stationary phase is a polar stationary phase. In some embodiments, the SFC stationary phase comprises silica, polysaccharide, zirconia, polystyrene, divinylbenzene, or porous graphitic carbon. In some embodiments, the SFC stationary phase comprises particles. In some embodiments, the particles have a particle size of about 0.5 pm to about 5 pm. In some embodiments, the particles have an average pore size of about 50 A to about 250 A. In some embodiments, the SFC stationary phase comprises Ethylene Bridged Hybrid particles, Charged Surface Hybrid particles, High Strength Silica particles, or Solid-Core particles. In some embodiments, the SFC stationary phase is a Viridis BEH column, Viridis BEH 2-ethylpyridine column, Torus DEA column, Torus 2-PIC column, Torus Diol compound, Torus 1-AA column, or Viridis HSS C18 SB column.

[0012] In some embodiments, the applying the Quillaja saponin source composition to the SFC stationary phase comprises use of a mobile phase comprising CO2. In some embodiments, the mobile phase comprises an organic modifier and / or a pH adjuster. In some embodiments, the organic modifier is methanol, acetonitrile, ethanol, isopropanol (IPA), butanol, tetrahydrofuran (THF), or dimethylsulfoxide (DMSO), or a combination thereof. In some embodiments, the pH adjuster is formic acid (FA), trifluoroacetic acid (TFA), diethylamine (DEA), dimethylethanolamine (DMEA), triethylamine (TEA), ammonia (NH3), or acetate buffers, or a combination thereof. In some embodiments, the applying the elution condition to the SFC stationary phase comprises subjecting the SFC station phase to a gradient-based elution. In some embodiments, the gradient-based elution comprises increasing a percentage of anorganic modifier in a mobile phase. In some embodiments, the organic modifier is methanol. In some embodiments, the applying the elution condition to the SFC stationary phase comprises subjecting the SFC station phase to an isocratic-based elution. In some embodiments, the mobile phase comprises CO2, methanol, and formic acid. In some embodiments, the formic acid is present at a concentration of about 0.01% to about 1% v / v. In some embodiments, the mobile phase used in the applying a Quillaja saponin source composition to the SFC stationary phase comprises less than about 2% v / v water. In some embodiments, the mobile phase comprises less than about 0.1% v / v water.

[0013] In some embodiments, the SFC system comprises an automatic back pressure regulator capable of maintaining a constant back pressure. In some embodiments, the SFC system exhibits a column flow rate between 1-2 mL / min. In some embodiments, the column flow rate is about 1.25 mL / min. In some embodiments, the method further comprises degassing the elute from the SFC stationary phase.

[0014] In some embodiments, the method further comprises analyzing the purified saponin composition. In some embodiments, the analysis is performed using a mass spectrometry technique.

[0015] In other aspects, provided herein is a method of producing a purified saponin composition comprising QS-21, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system, wherein the SFC stationary phase comprises inorganic-organic ethylene -bridged hybrid particles, wherein the Quillaja saponin source composition is applied using a mobile phase comprising CO2and an organic modifier and / or a pH adjuster; and applying an elution condition to the SFC stationary phase to produce the purified saponin composition comprising QS-21.

[0016] In other aspects, provided herein is a system for producing a purified saponin composition, the system comprising a SFC system comprising a SFC stationary phase, a SFC mobile phase, and a Quillaja saponin source composition.

[0017] In other aspects, provided herein is purified Quillaja saponin composition produced by a method described herein. In some embodiments, the purified Quillaja saponin composition comprises QS-21.

[0018] In other aspects, provided herein is a vaccine comprising a purified Quillaja saponin composition described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows the juxtaposition of chromatograms from the electrospray ionization (ESI) negative total ion current (TIC) and single ion recording (SIR) of a commercial QS-21 standard. The SIR chromatogram was obtained with the QS-21 1988 m / z channel selected.

[0020] FIG. 2 shows the mass spectrum of the two isomer peaks found in the QS-21 standard.

[0021] FIG. 3 shows the ESI negative TIC and SIR (m / z 1988) chromatograms from Quil- A.

[0022] FIG. 4 shows the ESI negative TIC and SIR (m / z 1988) chromatograms from a Quil- A preparation spiked with a commercial QS-21 standard.

[0023] FIG. 5 shows the TIC chromatograms of a commercial QS-21 standard, Quil-A, and Quil-A spiked with a QS-21 standard.

[0024] FIG. 6 shows the TIC chromatograms of a commercial QS-21 standard, Quil-A, and Quil-A spiked with a QS-21 standard.

[0025] FIG. 7 shows a chromatogram from the analytical HPLC method separating Flash21.

[0026] FIG. 8 shows a chromatogram from the analytical UPLC method separating Flash21.

[0027] FIG. 9 shows a chromatogram from SFC of Flash21 analyzed on an analytical diol column.

[0028] FIG. 10 shows a chromatogram from SFC of Flash21 analyzed on an analytical amino-phenyl column.

[0029] FIG. 11 shows elution of Flash21 on a 2.5 cm diameter diol column.

[0030] FIG. 12 shows the UV trace of a fraction of preparative SFC on an amino phenyl column.

[0031] FIG. 13 shows 400mg of the lyophilized product QS-21.

[0032] FIG. 14 shows a comparison of chromatograms of different QS-21 preparations.

[0033] FIG. 15 shows high resolution mass spectrometry of a common QS-21 preparation contaminant.

[0034] FIG. 16 shows high resolution mass spectrometry of a QS-21 preparation contaminant unique to our SFC method.

[0035] FIG. 17 shows high resolution mass spectrometry of a QS-21 preparation contaminant unique to our QS-21 source materials.

[0036] FIG. 18 shows HPLC analysis of QS-21 purified from Flash21 using SFC.

[0037] FIG. 19 shows HPLC analysis of QS-21 purified from Quil-A using SFC.

[0038] FIG. 20 shows HPLC analysis of QS-18 purified from bark-derived Quil-A (upper) and our sustainable Quillaja saponaria plant material processed into Flash21 (lower).DETAILED DESCRIPTION

[0039] Provided herein, in some aspects, are methods of producing a purified saponin composition, such as a highly purified (e.g., at least about 90%) saponin composition comprising QS-21, using supercritical fluid chromatography (SFC). For example, in some embodiments, described herein is a method of producing a purified saponin composition, the method comprising: applying a saponin source composition, such as a Quillaja saponin source composition, to a supercritical fluid chromatography (SFC) stationary phase of a SFC system; and applying an elution condition to the SFC stationary phase to produce the purified saponin composition. In other aspects, further provided herein are products produced by practicing the taught methods of producing a purified saponin composition, such as compounds or medicines, as wells as kits and systems for practicing the methods thereof.

[0040] The disclosure provided herein is based, at least in part, on the inventor’s unique perspectives and findings unexpectedly demonstrating that supercritical fluid chromatography (SFC) efficiently purifies saponins from Quillaja saponin sources, such as QS-21. Prior to these findings, SFC was traditionally used for the separation of chiral compounds and relatively nonpolar compounds. Specifically, there are well-known compatibility issues limiting the application of SFC to the separation of polar compounds, such as saponins from Quillajasaponin sources. For example, relative to other saponins, QS-21 contains numerous sugar moieties and an acyl chain thereby increasing its polar characteristic. As demonstrated in the Examples, the inventor surprisingly found that SFC can be used to efficiently purify highly polar saponins, such as QS-21 (e.g., gradients around 6 minutes provided excellent separation of QS- 21 resulting in highly pure saponin compositions exceeding purities of 90%). As also demonstrated herein, it was found that the taught SFC techniques provide a high resolving power capable of separating the isomers of QS-21, which has not been observed with reversed-phased liquid chromatography. The SFC techniques taught herein can be readily performed economically and on a commercial scale. Moreover, the taught techniques do not require the hazardous chemicals used in traditional RP-HPLC purification of saponins. Furthermore, the SFC techniques taught herein allow for the purification of polar saponins without the addition of water to the mobile phase, further simplifying post-purification procedures as water does not need to be removed from the eluate thereby avoiding another energy intensive process needed in traditional RP-HPLC saponin purification. The findings presented herein represent a significant advancement to the field.

[0041] Thus, in some aspects, provided herein is a method of producing a purified saponin composition, the method comprising performing supercritical fluid chromatography (SFC) on a saponin source composition, such as a Quillaja saponin source composition, to produce the purified saponin composition.

[0042] In some aspects, provided herein is a method of producing a purified saponin composition, the method comprising applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system and applying an elution mobile phase to the SFC stationary phase to produce the purified saponin composition, such as a purified saponin composition comprising QS-21.

[0043] In some aspects, provided herein is a method of producing a purified saponin composition comprising QS-21, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system, wherein the SFC stationary phase comprises inorganic-organic ethylene -bridged hybrid particles; wherein the Quillaja saponin source composition is applied using a mobile phase comprising CO2and an organic modifier and / or a pH adjuster; and applying an elutioncondition to the SFC stationary phase to produce the purified saponin composition comprising QS-21.

[0044] In other aspects, provided herein is a system for producing a purified saponin composition, the system comprising a SFC system comprising a SFC stationary phase, and a Quillaja saponin source composition.

[0045] In other aspects, provided herein is a purified Quillaja saponin composition produced by the methods described herein. In some embodiments, the purified Quillaja saponin composition comprises QS-21.

[0046] In other aspects, provided herein is a medicament comprising a purified Quillaja saponin composition produced by the methods described herein. In some embodiments, the medicament is a vaccine.

[0047] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.I. Definitions

[0048] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0049] The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (z.e., contain only) components A, B, and C, or can contain not only components A, B, and C but alsoone or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0050] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0051] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0052] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.II. Supercritical Fluid Chromatography (SFC) purification of saponins

[0053] In certain aspects, provided herein are methods for producing a purified saponin composition using supercritical fluid chromatography (SFC). In some embodiments, the purified saponin composition comprises one or more saponins, and use of the term “purified” in purified saponin composition is merely intended to describe that, as compared to the saponin source solution applied to a SFC stationary phase, one or more target saponins, such as QS-21, has an increased relative presence to another component of the saponin source composition or the purified saponin composition.

[0054] In some embodiments, the method provided herein comprises performing supercritical fluid chromatography (SFC) on a saponin source composition, such as a Quillaja saponin source composition, to produce the purified saponin composition. In some embodiments, the method provided herein comprises applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system. In some embodiments, the method provided herein comprises applying an elution condition, such as an elution mobile phase, to a SFC stationary phase to produce a purified saponin composition.In some embodiments, the method provided herein comprises collecting at least one elution fraction from the SFC stationary phase.

[0055] Certain aspects of the methods taught herein are discussed in more detail in a modular fashion below. One of ordinary skill in the art will readily understand how the aspects of the present description can be combined to practice any SFC saponin purification method encompassed by the teachings provided herein. The discussion of SFC saponin purification methods, including components and configurations thereof, in a modular fashion does not limit the scope of the description encompassed herein.A. Saponin source compositions

[0056] Provided herein, in certain aspects, are methods of producing a purified saponin composition from a saponin source composition. The saponin source compositions described herein comprise one or more desired saponins that the SFC methods taught herein are used to purify. In some embodiments, the saponin source composition is a Quillaja saponin source composition. In some embodiments, the Quillaja saponin source composition refers to a composition comprising one or more saponins, or analogs thereof, found in the tree family Quillajacae. The use of the term Quillaja saponin source composition does not imply that the source composition is necessary obtained from a tree in the Quillajacae family. For example, saponins known to originate from Quillajacae, such as QS-21, can be synthetically produced and such sources are included within the scope of the term “Quillaja saponin source composition.” Moreover, such saponins that would define a Quillaja saponin source composition do not have to be solely found in a tree in the Quillajacae family.

[0057] Saponin source composition may be obtained from a variety of natural and / or synthetic sources. Saponins are naturally derived glycosides with medicinal and industrial applications. Of particular importance is the role of QS-21, a purified saponin composition, as an adjuvant in commercially available vaccines. Saponins can be obtained from plants in numerous botanical families, including, but not limited to, Quillajaceae. In certain embodiments, the Quillaja saponin source composition is from Quillaja saponaria. In some embodiments, the Quillaja saponin source composition is Quillaja brasiliensis . In some embodiments, the Quillaja saponin source composition is not from Ilex latifolia., Panaxquinquefolius L., Panax ginseng, orDioscorea zingiberensis. In certain embodiments, the Quillaja saponin source composition does not contain a substantial amount of a furostanol saponin (such as 5% or less, including any of 4% or less, 3% or less, 2% or less, 1% or less, or an amount below the limit of detection as a target saponin to be purified). In some embodiments, the Quillaja saponin source composition does not contain a substantial amount of a saikosaponin (such as 5% or less, including any of 4% or less, 3% or less, 2% or less, 1% or less, or an amount below the limit of detection as a target saponin to be purified). In some embodiments, the Quillaja saponin source composition does not contain a substantial amount of a matrine saponin (such as 5% or less, including any of 4% or less, 3% or less, 2% or less, 1% or less, or an amount below the limit of detection as a target saponin to be purified). In some embodiments, the Quillaja saponin source composition does not contain a substantial amount of a ginsenoside (such as 5% or less, including any of 4% or less, 3% or less, 2% or less, 1% or less, or an amount below the limit of detection as a target saponin to be purified). In some embodiments, the Quillaja saponin source composition does not contain a substantial amount of a spirosterol saponin (such as 5% or less, including any of 4% or less, 3% or less, 2% or less, 1% or less, or an amount below the limit of detection as a target saponin to be purified). Additional saponin source compositions are contemplated herein, including other natural sources of saponins, such as Caryophyllaceae, Sapindaceae, Aceraceae, and Hippocastanaceae.

[0058] In some embodiments, the methods provided herein comprise obtaining a saponin source solution, which may involve growing plants and / or culturing techniques and one or more subsequent processing steps to obtain a composition containing one or more desired saponins to be purified. For example, conventional saponin extraction processes use mature Q. saponaria trees at least 5 years of age, and up to 25 years of age or older. Currently, natural forests are the principal source (about 98%) of raw material for Q. saponaria extraction. These trees are cut down, and then the bark from the trees is removed and processed. Processing may include drying, grinding, treatment with liquids (e.g., solvents), filtering, and chromatography (e.g., U.S. Pat. No. 8,252,346; U.S. Pat No. 5,057,540; U.S. Pat. No. 9,540,416, each of which is hereby incorporated herein by reference in its entirety). In addition to bark, saponins may also be produced from branches and leaves, which contain up to 2.58% w / w of saponins (Schlotterbeck, T.; Castillo-Ruiz, M.; Canon- Jones, H.; San Martin, R. 2015; which is hereby incorporated herein by reference in its entirety). The use of leaves from young trees of Quillaja saponaria(Molina) plantations can also be a source of saponins (Economic Botany 69: 262-272, which is hereby incorporated herein by reference in its entirety). Further, saponin extracts may be produced from Q. saponaria cells or tissues (e.g., differentiated plant material) maintained in laboratory conditions. For example, Q. saponaria callus may be cultivated on semi-solid media and Q. saponaria cell cultures or cell suspensions may be cultivated in liquid media. Exemplary methods of cultivating Q. saponaria cells or tissues are provided in U.S. Pat. No. 5,716,848, which is hereby incorporated herein by reference in its entirety. As another example, saponins may be obtained (such as to form a saponin source composition) from Q. saponaria bark using one or more extraction techniques, such as Soxhlet extraction. In some embodiments, the method comprises one or more enrichment steps, e.g., flash chromatography. In some embodiments, the method comprises obtaining a saponin source composition, such as a Quillaja saponin source composition, the obtaining comprising performing an extraction technique (such as Soxhlet extraction) on a plant material from a tree in the Quillajaceae family, and then performing a flash chromatography step.

[0059] Mature Quillaja saponaria trees may be grown in an uncultivated state, such as a forest, where they may be managed using standard forestry practices. Q. saponaria trees may also be grown from seedlings in a nursery and then established in an orchard. In natural conditions the regeneration of plants from seed is rare. In these conditions the Q. saponaria tree regenerates from stump suckers. Under greenhouse conditions, Q. saponaria regenerates primarily from seeds. For example, the seeds are collected in autumn and then dried and later germinated; for example, the seeds are dried at 40 °C for 8 h. The dry seeds are then pretreated in cold water for 72 h and germinated at 25 °C for 11 h and 10 °C for 13 h. Additionally, Q. saponaria can be propagated vegetatively through the use of cuttings. Indole butyric acid may be used to achieve rooting of the cuttings in clay soil. Tissue of Q. saponaria plants may be used to produce a callus that is grown on semi- solid media or a cell suspension that is grown on liquid media. Q. saponaria plant material produced using these different growth methods may be processed to obtain saponin extracts. Traditionally, saponin extracts are produced from wild growing trees or trees obtained by methods described above. Recently, in vitro produced Q. saponaria plants have been used to reforest overexploited areas of this species for commercial purposes.

[0060] The Quillaja genus belongs to the Quillajaceae family in the Fabales order of the Rosid clade. In this genus, there are two species: Quillaja saponaria Molina and Quillaja brasiliensis (St. A.-Hil. & Tul.) Mart. Both of these are evergreen tree species native to South America, and both contain saponins in the bark, wood, and leaves of mature trees. Q. saponaria is also known as quillay (meaning “to wash”) or the soap bark tree, as powder from Q. saponaria bark foams in water and can be used as a soap. Of the two Quillaja species, Q. saponaria is primarily used as a source for commercial saponin production, because it has a relatively high content of saponins: about 1.2% in branches, and about 16-20% of dry weight in bark (Guerra, Fernando, & Sepulveda, Sebastian. (2021). Saponin production from Quillaja genus species. An insight into its applications and biology. Scientia Agricola, 78(5), e20190305. Epub August 24, 2020.).

[0061] In some embodiments, the methods provided herein comprise producing a saponin source composition, such as a Quillaja saponin source composition, e.g., growing Quillaja saponaria plantlets in a bioreactor (or other laboratory setting) and producing saponin- containing extracts from the plantlets (or parts thereof) and / or the spent liquid media from the plant growth. For example, methods for producing a saponin source composition may comprise, e.g., growing Quillaja saponaria plantlets in a bioreactor or other growth vessel comprising a liquid media for a growth period in the bioreactor comprising about 2-14 weeks, immersing the plantlets in the liquid media at least once, such as once, twice, three times, four times, or five times, per day (such as with an immersion time of about 1-10 minutes) during the growth period, collecting the liquid media at the end of the growth period or at the end of a portion of the growth period, and processing the liquid media to produce a first saponin-containing extract and / or processing the plant material (i.e. plantlets) at the end of the growth period or at the end of a portion of the growth period. In some embodiments, the methods provided herein comprise producing a saponin source composition using transgenic organisms expressing genes for the production of Quillaja saponins. See Martin, L.B.B., Kikuchi, S., Rejzek, M. et al. Nat Chem Biol (2024).

[0062] In some embodiments, the methods provided herein comprise producing a saponin source composition, such as a Quillaja saponin source composition, e.g., synthesizing a Quillaja saponin source composition. Synthetic production of QS-21 is described in US Patent10,906,926 and US Patent 11,274,116, the entire contents of which are hereby incorporated herein in their entirety by reference.

[0063] Encompassed in the methods provided herein are also methods of purifying intermediate compounds in the synthesis of a saponin source composition. Although such intermediates may not be found in a plant source, one of ordinary skill will readily understand the scope of such additional methods as the intermediate will be used to produce (directly or indirection) a saponin source composition described herein. For example, in some embodiments, the method comprises: applying a synthetic Quillaja saponin intermediate to a supercritical fluid chromatography (SFC) stationary phase of a SFC system and applying an elution condition to the SFC stationary phase to produce the purified saponin composition. SFC can also be applied in such situations, especially when the intermediates have similar properties (such as via structural similarity) with a saponin of a saponin source composition described herein. In some embodiments, the saponin source composition comprises prosapogenin, a triterpene and branched trisaccharide. From this molecule, the remainder of QS-21 can be synthesized, resulting in a semi-synthetic source material.

[0064] In some embodiments, the method comprises processing a starting material to produce a saponin source composition, such as a Quillaja saponin source composition. In some embodiments, the starting material may be Quillaja-derived materials, such as Quillaja tree bark, lab grown Quillaja-derived materials, such as callus, cell and plantlets, transgenic organisms expressing genes for the production of Quillaja saponins, chemically Quillaja synthesized saponins, and the like. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, is Quil-A. In some embodiments, the processing the starting material does not comprise a step of reversed-phase liquid chromatography.

[0065] The saponin source compositions, such as a Quillaja saponin source composition, encompassed by the present application include a diverse array of compositions. In some embodiments, the one or more saponins (including all saponins being targeted for purification) in the saponin source composition each comprise at least 3 sugar moieties, including at least any of 4, 5, 6, or 7 sugar moieties. For example, the one or more saponins in the saponin source composition can each comprise 3-7 sugar moieties, including 3-6 sugar moieties, 3-5 sugar moieties, 3-4 sugar moieties, 4-7 sugar moieties, 4-6 sugar moieties, 4-5 sugar moieties, 5-7 sugar moieties 5 6 sugar moieties or 6 7 sugar moieties In some embodiments the saponinsource composition, such as a Quillaja saponin source composition, comprises one or more saponins with a structure containing aglycone and R groups: RO, Rl, R2, R3, and R4. In some embodiments, the aglycone structure is comprised of quillaic acid, quillaic acid with 22β-OH, phytolaccinic acid, or echynocystic acid. In some embodiments, a quillaic acid moiety is in one or more (including all) saponins in a saponin source composition. In some embodiments, the RO group is comprised of xylose, hydroxyl, or rhamnose. In some embodiments, the Rl group is comprised of apiose-xylose, xylose-apiose, xylose-xylose, xylose, or hydroxyl. In some embodiments, the R2 group is comprised of glucopyranose, hydroxyl, or 2-methylbutanoyl. In some embodiments, the R3 group is comprised of rhamnose, glucopyranose, glucopyranoseacetyl, acetyl, hydroxyl, or Fa-Ara-rhamnose. In some embodiments, the R4 group is comprised of Fa- Ara, hydroxyl, 2-methylbutanoyl, 3-hydroxy-4-methylhexanoyl, or acetyl. Fleck el al. (2019) Molecules 24(1): 171, the entire contents of which are hereby incorporated herein by reference. In some embodiments, the saponin source composition comprising one or more saponins comprising an acyl chain. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins from Table 1.Table 1. Example saponins in saponin source compositions.

[0066] As used in Table 1 above, Q: quillaic acid, Q-OH: quillaic acid, 22-OH, P: phytolaccinic acid, P-Ac: phytolaccinic acid, 23-O-Ac, E: echynocystic acid. Fa-Ara: see structure below, MeBu: 2-methylbutanoyl, and OHMeHex: 3-hydroxy-4-methylhexanoyl.

[0067] Per Table 1, R0, Rl, R2, R3, and R4 are as depicted in the structure below. In some embodiments, Ra is CHO, CH2OH, CH2OCOCH3, or CH3. In some embodiments, Rb is OH or H. In some embodiments, Rc is H or OH. In some embodiments, Rc is CH3, COOCH3. In some embodiments, wherein the saponin comprises quillaic acid, Ra is CHO, Rb is OH, Rc is H, and Rd is CH3.

[0068] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is apiose-xylose, R2 is hydroxyl, and R3is hydroxyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is apiose-xylose, R2 is hydroxyl, and R3 is glucopyranose. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid and RO is hydroxyl.

[0069] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid and RO is rhamnose. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid and RO is xylose. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is hydroxyl, R1 is xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl.

[0070] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, R0 is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, R0 is rhamnose, R1 is xylose-xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, R0 is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, R0 is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranose, and R4 is acetyl.

[0071] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, R0 is xylose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, R0 is rhamnose, R1 is hydroxyl, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaicacid, RO is xylose, R1 is hydroxyl, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranoseacetyl, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranose-acetyl, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose-acetyl, and R4 is acetyl.

[0072] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose-acetyl, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, thesaponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose- xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa-Ara.

[0073] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is hydroxyl, R1 Is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is hydroxyl, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is Fa-Ara- Rhamnose, and R4 is hydroxyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is 3-hydroxy-4- methylhexanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is 3-hydroxy-4-methylhexanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone t t i h t l i i id RO i h d l R1 i h d l R2 i h d l R3 iglucopyranose, and R4 is methylbutanoyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is echynocystic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is phytolaccinic acid- acetyl, RO is hydroxyl, R1 is hydroxyl, R2 is methylbutanoyl, R3 is glucopyranose, and R4 is methylbutanoyl.

[0074] In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the saponin source composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. See, e.g., Fleck et al. (2019) Molecules 24(1 ): 171, the entire contents of which are herein incorporated by reference.

[0075] In some embodiments, the saponin source composition comprises triterpene saponins. In some embodiments, the saponin(s) targeted for purification are triterpene saponin(s).

[0076] In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins with a molecular weight of about any of 1512 Da, 1674 Da, 824 Da, 970 Da, 956 Da, 1436.6 Da, 1582.7 Da, 1568.7 Da, 1714.7 Da,1700.7 Da, 1714.7 Da, 1700.7 Da, 1598.7 Da, 1584.7 Da, 1628.7 Da, 1614.7 Da, 1730.7 Da,1716.7 Da, 1760.8 Da, 1746.7 Da, 1640.7 Da, 1626.7 Da, 1802.8 Da, 1788.7 Da, 1744.8 Da,1730.7 Da, 1876.8 Da, 1862.8 Da, 1870.9 Da, 1856.9 Da, 2002.9 Da, 1988.9 Da, 2002.9 Da,1988.9 Da, 1912.9 Da, 1898.9 Da, 2045 Da, 2030.9 Da, 2045 Da, 2030.9 Da, 2033 Da, 2018.9Da, 2165 Da, 2151 Da, 2165 Da, 2151 Da, 1886.9 Da, 2018.9 Da, 2297 Da, 1656.7 Da, 1642.7 Da, 1788.8 Da, 1774.8 Da, 1978.9 Da, 1964.9 Da, 1392.7 Da, 1732.8 Da, or 1560.7 Da. In some embodiments, the one or more saponins targeted for purification (such as triterpene saponins) have a molecular weight of at least about 1600 Da to about 3000 Da, such as at least about 1700 to about 2500 Da, or at least about 1800 Da to about 2300 Da.

[0077] In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins with a molecular weight of at least about 1000 Da, at least about 1100 Da, at least about 1200 Da, at least about 1300 Da, at least about 1400 Da, at least about 1500 Da, at least about 1600 Da, at least about 1700 Da, or at least about1800 Da. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins with a molecular weight of about 1000 Da to about 2500 Da, about 1100 Da to about 2500 Da, about 1200 Da to about 2500 Da, about 1300 Da to about 2500 Da, about 1400 Da to about 2500 Da, about 1500 Da to about 2500 Da, about 1600 Da to about 2500 Da, about 1700 Da to about 2500 Da, about 1800 Da to about 2500 Da, about 1850 Da to about 2500 Da. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins with a molecular weight of about 1988 Da.

[0078] Hydrophilicity of compounds can be quantified as a LogP value, wherein P = the concentration of a compound that dissolves in the organic phase divided by the concentration of a compound that dissolves in the aqueous phase of an immiscible biphasic system of lipid and water. A negative value for LogP means the compound has a higher affinity for the aqueous phase (it is more hydrophilic); when LogP = 0 the compound is equally partitioned between the lipid and aqueous phases; a positive value for LogP denotes a higher concentration in the lipid phase (i.e., the compound is more lipophilic). LogP = 1 means there is a 10:1 partitioning in Organic:Aqueous phases. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins with a LogP value less than about -4.00, less than about -5.00, less than about -6.00, or less than about -6.50. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises one or more saponins with a LogP value of about -6.92.

[0079] In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises a synthetic saponin such as one or more of synthetic QS-21, synthetic QS1-0101, synthetic QS1-0102, or synthetic QS1-0103. In certain embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises a synthetic QS in which the labile ester groups within the acyl chain are replaced by more hydrolytically robust amide linkages. In certain embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises a synthetic QS in which the lipophilic acyl chains are glycosylated or nonglycosylated. See, e.g., Ragupathi et al. (2013) Expert Rev Vaccines 10(4): 463-470, the entire contents of which are incorporated herein by reference.

[0080] In some embodiments, the saponin source composition, such as a Quillaja saponin source composition comprises a semi synthetic QS saponin variant In some embodiments, thesemi- synthetic QS saponin variant is an amide-based acyl chain variant. In some embodiments, the semi-synthetic QS saponin variant does not contain the branched trisaccharide domain. See, e.g., Fernandez-Tejada, et al. Nat Chern, (2015) 6(7): 635-643, Fernandez-Tejada, et al. Bioorg Med Chem. (2014) 22(21): 5917-5923, and Pifferi, et al. Chem Sci. 2023 Mar 1 ; 14(13):3501- 3513, the entire contents of which are incorporated herein by reference,

[0081] In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises triterpene glycoside saponin analogues and intermediates, such as those described in the art (see, e.g., Zeng, et al, (2020) Org Lett. 22(21 ):8613-8617 and U.S. Pat. No. 10,906,926, the entire contents of which are herein incorporated by reference). In certain embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises truncated triterpene saponin analogues, which are described in further detail in, e.g., US 11,274,116, the entire contents of which are hereby incorporated herein by reference.

[0082] In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, comprises QB-80 or QB-90 derived from 0. brasiliensis . See, e.g., Cibulski, et al. (2018) Vaccine 36(1 ):55-65, and Wallace, et al. (2019) Phytochem Anal. 30(6):644-652, the entire contents of which are incorporated herein by reference.

[0083] Techniques are known in the art for assessing compositions to determine whether it is a saponin source composition as described herein, e.g., by identifying a saponin known or discovered to be from a tree in the Quillajacae family. For example, mass spectrometry methods, such as those demonstrated in the examples of this application, may be used to assess a source composition to determine a saponin therein.B. SFC stationary phases and mobile phases

[0084] The methods of SFC described herein may utilize numerous types of stationary phases, such as in the form of a SFC column, which vary in composition, size or volume, particle attributes, and mobile phases. In some embodiments, two or more SFC columns may be utilized, such as two or more SFC columns arranged in series. For example, in some embodiments, the method comprises applying a Quillaja saponin source composition, or an aspect thereof such as a fraction obtained from a purification technique of a Quillaja saponinsource, to a first SFC stationary phase of a SFC system, and applying one or more fractions from the first SFC stationary phase to a second SFC stationary phase of the SFC system. In some embodiments, the first SFC stationary phase and the second SFC stationary phase are the same type of SFC stationary phase. In some embodiments, the first SFC stationary phase and the second SFC stationary phase are different types of SFC stationary phase.

[0085] In some embodiments, the SFC stationary phase is a polar stationary phase. In some embodiments, the SFC stationary phase comprises silica, polysaccharide, zirconia, polystyrene, divinylbenzene, or porous graphitic carbon. In some embodiments, the SFC stationary phase comprises particles. These particles may vary in size, pore size, or chemistry to modify the way in which analytes interact with the column.

[0086] In some embodiments, the SFC stationary phase comprises Ethylene Bridged Hybrid particles, Charged Surface Hybrid particles, High Strength Silica particles, or Solid-Core particles. In some embodiments, the SFC stationary phase is a Viridis BEH column, Viridis BEH 2-ethylpyridine column, Torus DEA column, Torus 2-PIC column, Torus Diol compound, Torus 1-AA column, Viridis HSS C18 SB column, YMC- Actus Triart Diol column, or GreenSep Amino Phenyl column.

[0087] In some embodiments, the particles have a particle size of about 0.5 pm to about 5 pm, such as about any of 0.5 pm, 0.75 pm, 1 pm, 1.25 pm, 1.5 pm, 1.75 pm, 2 pm, 2.25 pm, 2.5 pm, 2.75 pm, 3 pm, 3.25 pm, 3.5 pm, 3.75 pm, 4 pm, 4.25 pm, 4.5 pm, 4.75 pm, or 5 pm. In certain embodiments, the particles have a particle size of about 1.7 pm. In some embodiments, the SFC stationary phase is a particle having an average pore size of about 50 A to about 250 A, such as about any of 50 A, 75 A, 100 A, 125 A, 150 A, 175 A, 200 A, 225 A, or 250 A.

[0088] In some embodiments, the SFC stationary phase comprises dihydroxypropyl particles, a 5 pM particle size, a pore size of 120 A, and a carbon content of 12%.

[0089] In some embodiments, the SFC stationary phase comprises amino-phenyl particlesm a 5 pM particle size, and a pore size of 120 A.

[0090] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) particles, a 1.7 pM particle size, a surface area of 185 m2 / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0091] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) particles, a 3.5 pM particle size, a surface area of 185 m2 / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0092] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid bonded with 2-pyridylethyl ligand (BEH 2-EP) particles, a 1.7 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0093] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid bonded with 2-pyridylethyl ligand (BEH 2-EP) particles, a 3.5 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0094] In some embodiments, the SFC stationary phase comprises spherical high-strength silica particles, a 1.8 pM particle size, a surface area of 230 m / g, a pore size of 100 A, and a 0.70 cc / g pore volume.

[0095] In some embodiments, the SFC stationary phase comprises spherical high-strength silica particles, a 3.5 pM particle size, a surface area of 230 m / g, a pore size of 100 A, and a 0.70 cc / g pore volume.

[0096] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and diethylamine particles, a 1.7 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0097] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and diethylamine particles, a 5 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0098] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and 2-picolylamine particles, a 1.7 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0099] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and 2-picolylamine particles, a 5 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0100] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and high-density diol particles, a 1.7 pM particle size, a surface area of 185 m / g, a pore size of 130 A, and a 0.70 cc / g pore volume.

[0101] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and high-density diol particles, a 5 pM particle size, a surface area of 185 m / g, a pore size of 130 Å, and a 0.70 cc / g pore volume.

[0102] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and 1 -aminoanthracene particles, a 1.7 pM particle size, a surface area of185 m / g, a pore size of 130 Å, and a 0.70 cc / g pore volume.

[0103] In some embodiments, the SFC stationary phase comprises spherical ethylene- bridged hybrid (BEH) and 1 -aminoanthracene particles, a 5 pM particle size, a surface area of185 m / g, a pore size of 130 Å, and a 0.70 cc / g pore volume.

[0104] In some embodiments, the SFC stationary phase comprises ethylene bridge hybrid (BEH) particles, such as described in US Patent 6,686,035, US Patent 7,223,473, and US Patent 7,250,214, each of which are hereby incorporated herein by reference in their entirety. BEH particles are inorganic -organic ethylene-bridged hybrid particles. In some embodiments, the BEH comprises one or more amino containing compounds as a support material.

[0105] In some embodiments, the SFC stationary phase comprises an additional chemical group bound to the particles described herein. In some embodiments, the additional chemical group is a 2-picolylamine. In some embodiments, the additional chemical group is a diethylamine. In some embodiments, the additional chemical group is a diol. In some embodiments, the additional chemical group is a 1 -aminoanthracene. In some embodiments, the additional chemical group is an amylose tris-(3, 5-dimethylphenylcarbamate). In some embodiments, the additional chemical group is a cellulose tris-(3, 5-dimethylphenylcarbamate). In some embodiments, the additional chemical group is a cellulose tris-(3-chloro-4- methylphenylcarbamate). In some embodiments, the additional chemical group is a 2- ethylpyridine. In some embodiments, the additional chemical group is a CSH fluoro-phenyl. In some embodiments, the additional chemical group is a Cis.

[0106] In some embodiments, the SFC stationary phase comprises an open-tubular capillary column. In some embodiments, the open-tubular capillary column chemistry comprisesmethylpoly siloxane. In some embodiments, the open-tubular capillary column chemistry comprises phenylmethylpoly siloxane. In some embodiments, the phenylmethylpoly siloxane chemistry comprises phenyl(5)methyl(95)polysiloxane or phenyl(50)methyl(50)polysiloxane. In some embodiments, the biphenylmethylpoly siloxane chemistry comprises biphenyl(30)methyl(70)polysiloxane. In some embodiments, the open-tubular capillary column chemistry comprises cyanopropylmethylpoly siloxane. In some embodiments, the open-tubular capillary column chemistry comprises octylmethylpolysiloxane. In some embodiments, the octylmethylpoly siloxane chemistry comprises octyl(50)methyl(50)polysiloxane. In some embodiments, the open-tubular capillary column chemistry comprises polyethylene glycol. In some embodiments, the open-tubular capillary column chemistry comprises silica. In some embodiments, the open-tubular capillary column chemistry comprises an alkyl chain. In some embodiments, the alkyl-chain chemistry comprises Cl 8. In some embodiments, the open-tubular capillary column chemistry comprises polystyrene.

[0107] In some embodiments, the SFC stationary phase comprises a packed column. In some embodiments, the packed column chemistry comprises an alkyl-chain. In some embodiments, the alkyl chain comprises C4, C8, C16, C18, or C30. In some embodiments, the packed column chemistry comprises silica. In some embodiments, the silica chemistry comprises silica or hybrid silica. In some embodiments, the packed column chemistry comprises diol. In some embodiments, the diol chemistry comprises diol or propanediol. In some embodiments, the packed column chemistry comprises pentahydroxyl. In some embodiments, the packed column chemistry comprises ethyl pyridine. In some embodiments, the ethyl pyridine chemistry comprises 2-ethyl pyridine or 4-ethyl pyridine. In some embodiments, the packed column chemistry comprises 2-picolylamine. In some embodiments, the packed column chemistry comprises 1 -amino anthracene. In some embodiments, the packed column chemistry comprises an amine moiety. In some embodiments, the amine moiety chemistry comprises amine, aminopropyl, or diethylamine. In some embodiments, the packed column chemistry comprises an amide moiety. In some embodiments, the amide moiety chemistry comprises amide or diamide. In some embodiments, the packed column chemistry comprises a cyano moiety. In some embodiments, the cyano moiety chemistry comprises cyano or cyanopropyl. In some embodiments, the packed column chemistry comprises a phenyl moiety. In some embodiments, the phenyl moiety chemistry comprises phenyl, diphenyl, biphenyl, phenylpropyl, or phenyl-hexyl. In some embodiments, the packed column chemistry comprises fluorophenyl. In some embodiments, the fluorophenyl moiety chemistry comprises fluorophenyl or pentafluorophenyl. See Plachka K, et al. Columns in analytical-scale supercritical fluid chromatography: From traditional to unconventional chemistries. J Sep Sci. 2023 Sep;46(18):e2300431, which is hereby incorporated herein for all purposes.

[0108] In certain aspects, the methods described herein involve applying one or more mobile phases to a SFC stationary phase, such as to apply a saponin source composition to the SFC stationary phase or elute fraction therefrom. SFC uses supercritical fluids, which is a substance which exists above its critical temperature and critical pressure therefore having a range of physical properties that are intermediate between gases and liquids, as mobile phases.

[0109] In some embodiments, the applying the saponin source composition, such as a Quillaja saponin source composition, to a SFC stationary phase comprises use of a loading mobile phase. In some embodiments, the methods described herein comprise admixing a saponin source composition, such as a Quillaja saponin source composition, and a SFC mobile phase, such as a loading mobile phase, prior to applying to a SFC stationary phase. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, is introduced to a fluid path comprising a SFC mobile phase such that the saponin source composition is applied to a SFC stationary phase. In some embodiments, the method provided herein comprise performing a gradient method on a SFC system. Thus, in some embodiments, the methods described herein comprise adjusting the mobile phase applied to a SFC stationary phase over the course of a purification. In some embodiments, the method provided herein comprises performing an isocratic method on a SFC system (z.e., the mobile phase is not adjusted during a purification step of the method). In some embodiments, the isocratic method comprises stacking two or more injections such as to improve purification throughput of a SFC stationary phase.

[0110] In some embodiments, the mobile phase comprises CO2. In some embodiments, the mobile phase comprises a fluoroform. In some embodiments, the mobile phase comprises an organic modifier and / or a pH adjuster. Organic modifiers modify the compressibility of the SFC system and impact the retention and selectivity of the chromatographic process. pH also impacts retention and selectivity by determining the ionization state of analytes. In some embodiments, the organic modifier is methanol acetonitrile ethanol isopropanol (IPA) butanoltetrahydrofuran (THF), hexane, toluene, 1 -propanol, 2-propanol, acetone, dioxane, or dimethylsulfoxide (DMSO), or a combination thereof. In some embodiments, the pH adjuster is formic acid (FA), trifluoroacetic acid (TFA), diethylamine (DEA), dimethylethanolamine (DMEA), triethylamine (TEA), ammonia (NH3), acetic acid, citric acid, or acetate buffers, or a combination thereof. In some embodiments, the mobile phase comprises one or more additional modifiers, such as a base (e.g., triethylamine), water, ion-pair reagent (e.g., alkylbenzoic sulfonate salts or tetrabutyl ammonium salts), or volatile salt (e.g., ammonium formate or ammonium acetate).

[0111] In some embodiments, the mobile phase comprises CO2and methanol. In some embodiments, the mobile phase comprises less than about 10% (v / v) water, such as less than about any of 9% (v / v) water, 8% (v / v) water, 7% (v / v) water, 6% (v / v) water, 5% (v / v) water, 4% (v / v) water, 3% (v / v) water, or 2% (v / v) water. In some embodiments, the mobile phase comprises CO2, methanol, and formic acid. In some embodiments, the mobile phase comprises CO2, methanol, and formic acid. In some embodiments, the mobile phase comprises less than about 2% (v / v) water, such as less than about any of 1.9% (v / v) water, 1.8% (v / v) water, 1.7% (v / v) water, 1.6% (v / v) water, 1.5% (v / v) water, 1.4% (v / v) water, 1.3% (v / v) water, 1.2% (v / v) water, 1.1% (v / v) water, 1.0% (v / v) water, 0.9% (v / v) water, 0.8% (v / v) water, 0.7% (v / v) water, 0.6% (v / v) water, 0.5% (v / v) water, 0.4% (v / v) water, 0.3% (v / v) water, 0.2% (v / v) water, or 0.1% (v / v) water. In some embodiments, the mobile phase does not comprise a substantial amount of water or does not have any added water.

[0112] In some embodiments, the mobile phase comprises a ratio of a first mobile phase comprising CO2to a second mobile phase comprising methanol and formic acid (such as formic acid at 0.1% (v / v)). In some embodiments, the mobile phase gradient applied to a SFC stationary phase may comprise a ratio that can be adjusted (such as a ratio of the first mobile phase and the second mobile phase noted above in this paragraph).C. SFC conditions and systems

[0113] The methods of performing SFC to produce purified saponin compositions described herein may be performed using known SFC systems operated using conventional SFC conditions, such as commercially available from Waters and Shimadzu Scientific Instruments.

[0114] SFC systems comprise numerous pumps, valves, sensors, and regulators to fine-tune the conditions optimal for purification of a particular analyte. In some embodiments, the SFC system comprises an automatic back pressure regulator capable of maintaining a constant back pressure. In some embodiments, the backpressure is between about 500 psi and about 6500 psi. In some embodiments, the backpressure is between about 1000 psi and about 2500 psi. In some embodiments, the backpressure is between about 1500 psi and about 2000 psi. In some embodiments, the backpressure is about 1750 psi. In some embodiments, the SFC system is operated at a flow rate of about 1 mL / min to about 250 mL / min. In certain embodiments, the flow rate is about 1.25 mL / min. In some embodiments, the applying a saponin source composition, such as a Quillaja saponin source composition, to a supercritical fluid chromatography (SFC) stationary phase of a SFC system comprises injecting a Quillaja saponin source composition volume into the SFC system. In certain embodiments, the Quillaja saponin source composition volume is at least about 0.1 pL. In some embodiments, up to about 1 gram of saponin source composition may be injected in a single injection, such as any of 0.1 grams, 0.2 grams, 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 grams, and 1 gram. In some embodiments, the methods described herein are configured to run about 2 to about 20 chromatography runs in 1 hour, e.g., a single run can be completed in less than about 30 minutes, such as less than about any of 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, or 3 minutes.

[0115] The methods of producing a purified saponin composition described herein may be practiced from analytical to commercial scale. For example, in some embodiments described herein, the methods of producing a purified saponin composition produces at least about 0.001, 0.01, 0.1, 1, 5, 10, 50, 100, 500, 1000, 5000, or 10000 grams of a purified saponin composition, such as a purified saponin composition comprising QS-21, from a single run.

[0116] In some embodiments, the SFC system comprises means to regulate temperature of the SFC stationary phase. In certain embodiments, the temperature of the SFC stationary phase is between about 25 °C and 45 °C. In certain embodiments, the temperature of the SFC stationary phase is between about 30°C and 40°C. In certain embodiments, the temperature of the SFC stationary phase is between about 33°C and 37°C. In certain embodiments, the temperature of the SFC stationary phase is about 35°C.

[0117] In some embodiments, the steps of applying a saponin source composition, such as a Quillaja saponin source composition, to a supercritical fluid chromatography (SFC) stationary phase of a SFC system; and collecting at least one fraction from the SFC stationary phase to produce the purified saponin composition, such as a purified saponin composition comprising QS-21, (i.e., representing a single run) occur within about 120 minutes, such as within about any of 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 19 minutes, 18 minutes, 17 minutes, 16 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, or 3 minutes.D. Purified saponin compositions

[0118] The SFC purification methods taught herein provide a diverse array of desired purified saponin compositions. In some embodiments, the purified saponin composition comprises a triterpene saponin. For example, in some embodiments described herein, the purified saponin composition comprises one or more of QS-21, QS-7, QS-17, or QS-18. In some embodiments, the purified saponin composition comprises QS-21. QS-21 is an immuno stimulatory triterpene saponin extracted from the bark of the South American soap tree, Quillaja saponaria. QS-21 has gained a reputation as a premier adjuvant enabling protein subunit vaccines. QS-21 is found in the approved vaccines Shingrix® (herpes zoster), Mosquirix® (malaria), and Arexvy® (RSV) all from Glaxo Smith Kline (GSK). QS-21 is highly polar with a calculated log P of -6.95 and in aqueous conditions is more stable at acidic pH 5.5 than at neutral or basic conditions. QS-21 has an absolute ESi positive mass of 1987.897 and an ESi nominal mass of 1988.

[0119] In some embodiments, the purified saponin composition comprises one or more QS- 21 isomers with a central triterpene core, to which a branched trisaccharide is attached at the terpene C3 oxygen functionality, a linear tetrasaccharide is linked to the triterpene C28 carboxylate group, and a glycosylated pseudo-dimeric acyl chain attached to the fucose moiety via a hydrolytically labile ester linkage. See, e.g., Ragupathi et al. (2013) Expert Rev Vaccines 10(4): 463 -470, the entire contents of which are incorporated herein by reference. In certainembodiments, the QS-21 isomers differ in the terminal sugar residue within the linear tetrasaccharide segment. In certain embodiments, the QS-21 isomers differ in the incorporation of an apiose or a xylose carbohydrate in the terminal sugar residue of the tetrasaccharide segment. In certain embodiments, QS-21-apiose is considered the major constituent and QS-21- xylose is considered the minor constituent. See, e.g., Deng, et al. (2008) Angew Chem Int Ed Engl. 47(34): 6395-6398, and Ragupathi et al. (2010) Vaccine 28(26): 4260-4267, the entire contents of which are incorporated herein by reference.

[0120] As described herein, QS-21 can comprise a mixture of isomers, such as QS-21 -apiose and QS-21 -xylose, and the purified saponin compositions provided herein may comprise QS-21 - apiose and QS-21-xylose at a ratio of about 1:20 to about 20:1. In some embodiments, QS-21 can comprise a mixture of isomers, QS-21-apiose and QS-21-xylose, at a ratio of about 1:19, about 1:18, about 1:17, about 1:16, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, or about 19:1. In some embodiments, the methods provided herein comprise obtaining two or more purified saponin compositions, such as from one or more runs of a purification method taught herein, and combining at least two of the two or more purified saponin compositions to obtain a desired composition (such as a composition comprising a mixture of saponins, including a mixture of saponin isomers at a specific ratio).

[0121] Encompassed in the methods provided herein are also methods of purifying intermediate compounds in the synthesis of a saponin source composition. Although such intermediates may not be found in a plant source, one of ordinary skill will readily understand the scope of such additional methods as the intermediate will be used to produce (directly or indirection) a saponin source composition described herein. For example, in some embodiments, the method comprises: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system and applying an elution condition to the SFC stationary phase to produce the purified saponin composition, wherein the purified saponin composition comprises a Quillaja synthesis intermediate. SFC can also be applied in such situations, especially when the intermediates have similar properties (such as via structural similarity) with a saponin of a saponin source composition described herein. In someembodiments, the purified saponin composition comprises prosapogenin, a triterpene and branched trisaccharide. From this molecule, the remainder of QS-21 can be synthesized, resulting in a semi-synthetic source material. In some embodiments, the further synthesized composition comprising QS-21 can be subjected to a method described herein comprising subjecting the saponin source composition to a SFC stationary phase.

[0122] In some embodiments, the method comprises processing a starting material to produce a saponin source composition, such as a Quillaja saponin source composition. In some embodiments, the starting material may be Quillaja-derived materials, such as Quillaja tree bark, lab grown Quillaja-derived materials, such as callus, cell and plantlets, transgenic organisms expressing genes for the production of Quillaja saponins, chemically Quillaja synthesized saponins, and the like. In some embodiments, the saponin source composition, such as a Quillaja saponin source composition, is Quil-A. In some embodiments, the processing the starting material does not comprise a step of reversed-phase liquid chromatography.The purified saponin compositions, such as a purified Quillaja saponin composition, encompassed by the present application include a diverse array of compositions. In some embodiments, the one or more saponins (including all saponins being targeted for purification) in the purified composition comprise at least 3 sugar moieties, including at least any of 4, 5, 6, or 7 sugar moieties. In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a structure containing aglycone and R groups: RO, Rl, R2, R3, and R4. In some embodiments, the aglycone structure is comprised of quillaic acid, quillaic acid with 22β-OH, phytolaccinic acid, or echynocystic acid. In some embodiments, a quillaic acid moiety is in one or more (including all) saponins in the source composition. In some embodiments, the RO group is comprised of xylose, hydroxyl, or rhamnose. In some embodiments, the Rl group is comprised of apiose-xylose, xylose-apiose, xylose-xylose, xylose, or hydroxyl. In some embodiments, the R2 group is comprised of glucopyranose, hydroxyl, or 2-methylbutanoyl. In some embodiments, the R3 group is comprised of rhamnose, glucopyranose, glucopyranose-acetyl, acetyl, hydroxyl, or Fa-Ara- rhamnose. In some embodiments, the R4 group is comprised of Fa- Ara, hydroxyl, 2- methylbutanoyl, 3-hydroxy-4-methylhexanoyl, or acetyl. Fleck et al. (2019) Molecules 24(1): 171, the entire contents of which are hereby incorporated herein by reference. In someembodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins from Table 1.

[0123] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is apiose-xylose, R2 is hydroxyl, and R3 is hydroxyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is apiose-xylose, R2 is hydroxyl, and R3 is glucopyranose. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid and RO is hydroxyl.

[0124] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid and RO is rhamnose. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid and RO is xylose. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is hydroxyl, R1 is xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl.

[0125] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranose, and R4 is acetyl.

[0126] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is hydroxyl, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is hydroxyl, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranoseacetyl, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is glucopyranose-acetyl, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose-acetyl, and R4 is acetyl.

[0127] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose-acetyl, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglyconestructure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is acetyl, and R4 is Fa-Ara. In some embodiments, the purified saponin composition comprises saponins whereinthe aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara.

[0128] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is rhamnose, R1 is xylose-xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is hydroxyl, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is hydroxyl, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is Fa-Ara- Rhamnose, and R4 is hydroxyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is rhamnose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is hydroxyl, and R4 is methylbutanoyl. In some embodiments, the purifiedsaponin composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is rhamnose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is 3-hydroxy-4-methylhexanoyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid with 22β-OH, RO is xylose, R1 is xylose-apiose, R2 is glucopyranose, R3 is rhamnose, and R4 is 3-hydroxy-4- methylhexanoyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is phytolaccinic acid, RO is hydroxyl, R1 is hydroxyl, R2 is hydroxyl, R3 is glucopyranose, and R4 is methylbutanoyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is echynocystic acid, RO is xylose, R1 is xylose, R2 is glucopyranose, R3 is glucopyranose, and R4 is acetyl. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is phytolaccinic acid- acetyl, RO is hydroxyl, R1 is hydroxyl, R2 is methylbutanoyl, R3 is glucopyranose, and R4 is methylbutanoyl.

[0129] In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-xylose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa- Ara. In some embodiments, the purified saponin composition comprises saponins wherein the aglycone structure is quillaic acid, RO is xylose, R1 is xylose-apiose, R2 is hydroxyl, R3 is hydroxyl, and R4 is Fa-Ara. See, e.g., Fleck et al. (2019) Molecules 24(1): 171, the entire contents of which are herein incorporated by reference.

[0130] In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a molecular weight of about any of 1512 Da, 1674 Da, 824 Da, 970 Da, 956 Da, 1436.6 Da, 1582.7 Da, 1568.7 Da, 1714.7 Da,1700.7 Da, 1714.7 Da, 1700.7 Da, 1598.7 Da, 1584.7 Da, 1628.7 Da, 1614.7 Da, 1730.7 Da,1716.7 Da, 1760.8 Da, 1746.7 Da, 1640.7 Da, 1626.7 Da, 1802.8 Da, 1788.7 Da, 1744.8 Da,1730.7 Da, 1876.8 Da, 1862.8 Da, 1870.9 Da, 1856.9 Da, 2002.9 Da, 1988.9 Da, 2002.9 Da,1988.9 Da, 1912.9 Da, 1898.9 Da, 2045 Da, 2030.9 Da, 2045 Da, 2030.9 Da, 2033 Da, 2018.9Da, 2165 Da, 2151 Da, 2165 Da, 2151 Da, 1886.9 Da, 2018.9 Da, 2297 Da, 1656.7 Da, 1642.7 Da, 1788.8 Da, 1774.8 Da, 1978.9 Da, 1964.9 Da, 1392.7 Da, 1732.8 Da, or 1560.7 Da.

[0131] In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a molecular weight of at least about 1000 Da at least about 1100 Da at least about 1200 Da at least about 1300 Da at least about1400 Da, at least about 1500 Da, at least about 1600 Da, at least about 1700 Da, or at least about 1800 Da. In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a molecular weight of about 1000 Da to about 2500 Da, about 1100 Da to about 2500 Da, about 1200 Da to about 2500 Da, about 1300 Da to about 2500 Da, about 1400 Da to about 2500 Da, about 1500 Da to about 2500 Da, about 1600 Da to about 2500 Da, about 1700 Da to about 2500 Da, about 1800 Da to about 2500 Da, about 1850 Da to about 2500 Da. In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a molecular weight of about 1988 Da.

[0132] Hydrophilicity of compounds can be quantified as a LogP value, wherein P = the concentration of a compound that dissolves in the organic phase divided by the concentration of a compound that dissolves in the aqueous phase of an immiscible biphasic system of lipid and water. A negative value for LogP means the compound has a higher affinity for the aqueous phase (it is more hydrophilic); when LogP = 0 the compound is equally partitioned between the lipid and aqueous phases; a positive value for LogP denotes a higher concentration in the lipid phase (i.e., the compound is more lipophilic). LogP = 1 means there is a 10:1 partitioning in Organic:Aqueous phases. In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a LogP value less than about -4.00, less than about -5.00, less than about -6.00, or less than about -6.50. In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises one or more saponins with a LogP value of about -6.92.

[0133] In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises a synthetic saponin such as one or more of synthetic QS-21, synthetic QS1-0101, synthetic QS1-0102, or synthetic QS1-0103. In certain embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises a synthetic QS in which the labile ester groups within the acyl chain are replaced by more hydrolytically robust amide linkages. In certain embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises a synthetic QS in which the lipophilic acyl chains are glycosylated or nonglycosylated. See, e.g., Ragupathi et al. (2013) Expert Rev Vaccines 10(4): 463 -470, the entire contents of which are incorporated herein by reference.

[0134] In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises a semi-synthetic QS saponin variant. In some embodiments, the semi-synthetic QS saponin variant is an amide-based acyl chain variant. In some embodiments, the semi-synthetic QS saponin variant does not contain the branched trisaccharide domain. See, e.g., Fernandez-Tejada, et al. Nat Chem. (2015) 6(7): 635-643, and Fernandez-Tejada, et al. Bioorg Med Chem. (2014) 22(21 ): 5917-5923, the entire contents of which are incorporated herein by reference.

[0135] In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises triterpene glycoside saponin analogues and intermediates, such as those described in the art (see, e.g., Zeng, et al. (2020) Org Lett. 22(21): 8613-8617 and U.S. Pat. No. 10,906,926, the entire contents of which are herein incorporated by reference). In certain embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises truncated triterpene saponin analogues, which are described in further detail in, e.g., US 11,274,116, the entire contents of which are hereby incorporated herein by reference,

[0136] In some embodiments, the purified saponin composition, such as a purified Quillaja saponin composition, comprises QB-80 or QB-90 derived from Q. brasiliensis . See, e.g., Cibulski, et al. (2018) Vaccine 36( l):55-65, and Wallace, et al. (2019) Phytochem Anal. 30(6):644-652, the entire contents of which are incorporated herein by reference.

[0137] Techniques are known in the art for assessing compositions to determine whether it is a purified saponin composition as described herein, e.g., by identifying a saponin known or discovered to be from a tree in the Quillajacae family. For example, mass spectrometry methods, such as those demonstrated in the examples of this application, may be used to assess a source composition to determine a saponin therein.

[0138] In certain embodiments, the purified saponin compositions are combined with other immuno stimulants comprising nanoparticles, immune- stimulating complexes (ISCOMs), ISCOM matrix, and lipsosomes. See, e.g., Zhu and Tuo, (2016) Nat Prod Chem Res. 3(4): el 13, the entire contents of which are herein incorporated by reference. In certain embodiments, a purified saponin composition may comprise one or more desired saponins.

[0139] In some embodiments, the methods provided herein enable production of a purified saponin composition comprising a desired saponin(s) at a purity of at least about 70%, such as at least about any of 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, the methods provided herein enable production of a purified saponin composition comprising a desired saponin(s) at a purity of about 71% to 99%, 72% to 99%, 73% to 99%, 74% to 99%, 75% to 99%, 76% to 99%, 77% to 99%, 78% to 99%, 79% to 99%, 80% to 99%, 81% to 99%, 82% to 99%, 83% to 99%, 84% to 99%, 85% to 99%, 86% to 99%, 87% to 99%, 88% to 99%, 89% to 99%, 90% to 99%, 91% to 99%, 92% to 99%, 93% to 99%, 94% to 99%, 95% to 99%, 96% to 99%, or about 97% to 99%. In some embodiments, purity is assessed relative to a known composition, such as a standard. In some embodiments, purity is assessed relative to other Quillaja saponins present in the initial Quillaja saponin source composition.

[0140] In certain aspects, provided herein is a method of producing a saponin composition with a purity of at least about 70% (such as at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system; and applying an elution condition to the SFC stationary phase to produce a saponin composition with a purity of at least about 70%.

[0141] In some embodiments, when two or more desired saponins are present in a purified saponin composition, the two or more desired saponins can be summed to assess purity. In some embodiments, purity is assessed relative to other saponins that may still be present in a purified saponin composition, e.g., for QS-21 that would include other compositions with a molecular weight of about 1985 Da to about 1995 Da. In some embodiments, the purified saponin composition comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% QS-21 in the purified saponin composition with a molecular weight of about 1985 Da to about 1995 Da. In some embodiments, the purified saponin composition comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% QS-21 in the purified saponin composition with a molecular weight of about 1988 Da. In some embodiments, purity is assessed relative to a known composition, such as a standard. In some embodiments, purity is assessed relative to other Quillaja saponins present in the initial Quillaja saponin sourcecomposition. In some embodiments, the purified saponin composition comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% QS-21 compared to other Quillaja saponins present in the initial Quillaja saponin source composition.

[0142] In some embodiments, the purified saponin composition comprises a SFC mobile phase. As described in more detail herein, in some embodiments, the purified saponin composition is substantially free of a SFC mobile phase or at least one component thereof, e.g., as degassing allows for the volatile compounds in a SFC mobile phase to escape to air thereby leaving behind the other components of a purified saponin composition, such as one or more saponins. In some embodiments, the purified saponin composition is a dried composition.E. Downstream processing and analysis

[0143] Post-SFC purification, mobile phase components must be removed from the a purified saponin composition. For example, CO2, methanol, and formic acid are all volatile at room temperature and will evaporate. If water is present in the mobile phase, the energy intensive and rate limiting process of lyophilization must be used to further purify the analyte. In some embodiments, the methods of producing a purified saponin composition described herein further comprise degassing SFC mobile phase. In some embodiments, the methods of producing a purified saponin composition do not comprise a lyophilization step.

[0144] In some embodiments, the methods of producing a purified saponin composition further comprise analyzing the purified saponin composition. In certain embodiments, the analysis is performed using a mass spectrometry technique. In some embodiments, the mass spectrometry technique comprises using a quadrupole mass spectrometer. In some embodiments, the mass spectrometer is a nominal mass spectrometer.F. Example methods

[0145] In some aspects, provided herein is a method of producing a purified saponin composition comprising QS-21, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system, wherein the SFC stationary phase comprises inorganic-organic ethylene -bridged hybridparticles; wherein the Quillaja saponin source composition is applied using a mobile phase comprising CO2and an organic modifier and / or a pH adjuster; and applying an elution condition to the SFC stationary phase to produce the purified saponin composition comprising QS-21. In some embodiments, the mobile phase comprises CO2, methanol, and formic acid (such as formic acid at a concentration of 0.05% (v / v) to about 0.2% (v / v), such as 0.1% (v / v)). In some embodiments, the method comprises use of a gradient SFC technique, such as the elution condition is applied to the SFC stationary phase. In some embodiments, the Quillaja saponin source composition is Quil-A. In some embodiments, the Quillaja saponin source composition is from a culture technique for Quillaja. In some embodiments, the Quillaja saponin source composition is a synthetic Quillaja saponin source composition, such as a synthetic QS-21 source composition.

[0146] In some aspects, provided herein is a method of producing a purified saponin composition comprising QS-21, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system, wherein the SFC stationary phase comprises inorganic-organic ethylene -bridged hybrid particles; wherein the Quillaja saponin source composition is applied using a mobile phase comprising CO2and an organic modifier and / or a pH adjuster; and running a gradient SFC technique such that the organic modifier is increased in concentration to elute the purified saponin composition. In some embodiments, the gradient SFC technique comprises using a ratio of a CO2mobile phase and an organic mobile phase including a pH adjuster. In some embodiments, the mobile phase comprises CO2, methanol, and formic acid (such as formic acid at a concentration of 0.05% (v / v) to about 0.2% (v / v), such as 0.1% (v / v)). In some embodiments, the method comprises use of a gradient SFC technique, such as the elution condition is applied to the SFC stationary phase. In some embodiments, the Quillaja saponin source composition is Quil-A. In some embodiments, the Quillaja saponin source composition is from a culture technique for Quillaja. In some embodiments, the Quillaja saponin source composition is a synthetic Quillaja saponin source composition, such as a synthetic QS-21 source composition.

[0147] In some aspects, provided herein is a method of producing a purified saponin composition, such as a purified saponin composition comprising QS-21, the method comprising: applying a Quillaja saponin source composition to a first supercritical fluid chromatography(SFC) stationary phase of a SFC system, and applying one or more fractions from the first SFC stationary phase to a second SFC stationary phase of the SFC system. In some embodiments, the first SFC stationary phase and the second SFC stationary phase are the same type of station phase. In some embodiments, the first SFC stationary phase and the second SFC stationary phase are different types of station phase. In some embodiments, the first SFC stationary phase is a diol column, such as a YMC- Actus Triart Diol column. In some embodiments, the second SFC stationary phase is an amino-phenyl column, such as an ES Industries GreenSep Amino Phenyl column. In some embodiments, the SFC is performed online, e.g., the one or more fractions from the first SFC stationary phase are eluted and directed to the second SFC stationary phase within a single system. In some embodiments, the SFC in performed offline, e.g., the one or more fractions from the first SFC stationary phase are eluted and captured, such as in a vial, and then the one or more fractions are applied to the second SFC stationary phase such as on a second SFC system. In some embodiments, the method comprises one or more elution steps, such as applying an elution condition to the first SFC stationary phrase or applying an elution condition to the second SFC stationary phase. In some embodiments, the one or more fractions applied to the second SFC stationary phase comprise a portion of a peak representing the desired saponin, such as a portion situated approximately to capture the center of the peak. In some embodiments, the method produces a Quillaja saponin composition with a purity of at least about 70%. In some embodiments, the Quillaja saponin source composition is applied using a mobile phase comprising CO2and an organic modifier and / or a pH adjuster; and running a gradient SFC technique such that the organic modifier is increased in concentration to elute the purified saponin composition. In some embodiments, the gradient SFC technique comprises using a ratio of a CO2mobile phase and an organic mobile phase including a pH adjuster. In some embodiments, the mobile phase comprises CO2, methanol, and formic acid (such as formic acid at a concentration of 0.05% (v / v) to about 0.2% (v / v), such as 0.1% (v / v)). In some embodiments, the method comprises use of a gradient SFC technique, such as the elution condition is applied to the SFC stationary phase. In some embodiments, the Quillaja saponin source composition is Quil-A. In some embodiments, the Quillaja saponin source composition is from a culture technique for Quillaja. In some embodiments, the Quillaja saponin source composition is a synthetic Quillaja saponin source composition, such as a synthetic QS-21 source composition. In some embodiments, the purified saponin composition comprises QS-21, such as at a purity of at least about 80%, including about any of 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (e.g., relative to other saponins in the purified saponin composition). In some embodiments, the purified saponin composition comprises QS-18, such as at a purity of at least about 80%, including about any of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (e.g., relative to other saponins in the purified saponin composition).III. Compositions and systems

[0148] In certain aspects, provided herein are compositions obtained from the SFC purification techniques described herein. In some embodiments, provided is a purified saponin composition comprising QS-21. In some embodiments, provided is a purified Quillaja saponin composition produced by a method described herein. In some embodiments, provided is a purified Quillaja saponin composition produced by a method described herein, wherein the purified Quillaja saponin composition comprises QS-21.

[0149] In certain aspects, provided herein are compositions comprising the purified saponin compositions, or derivatives thereof, provided by the techniques taught herein. For example, in some embodiments, provided herein is a vaccine comprising a purified saponin composition, such as a purified saponin composition comprising QS-21. Exemplary formulations for adjuvants comprising QS-21 are described in PCT App. No. PCT / EP2018 / 057488. For example, typically QS-21 is at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. QS-21 can be used at amounts between 1 and 100 pg per human dose. QS-21 may be used at a level of about 50 pg. Examples of suitable ranges are 40-60 pg, 45-55 pg, or 49-51 pg, such as 50 pg. A human dose can include QS-21 at a level of about 25 pg. Examples of lower ranges include 20-30 pg, 22-28 pg, or 24-26 pg, such as 25 pg. Human doses intended for children may be reduced compared to those intended for an adult (e.g., reduction by 50%).

[0150] In certain aspects, provided herein are systems useful for the methods taught herein. For example, in some embodiments, provided herein is a system for producing a purified saponin composition, the system comprising a SFC system comprising a SFC stationary phase, and a saponin source composition, such as a Quillaja saponin source composition. In some embodiments, the system further comprises components useful for the purification methods taught herein, such as a computer and software for performing a purification method taughtherein, and instructions for a gradient or isocratic SFC technique according the to the methods taught herein.EXAMPLESExample 1: Profiling a commercial QS-21 standard using supercritical fluid chromatography and mass spectrometry

[0151] This example describes the use of supercritical fluid chromatography (SFC) in the purification of a commercial QS-21 standard. The purified product was profiled via single ion recording (SIR) and total ion current (TIC) chromatogram.Purification and profiling by supercritical fluid chromatography (SFC) and mass spectrometry

[0152] A supercritical fluid chromatography system (Waters Corporation UPC2 SFC system) was connected to a nominal mass machine (Waters Corporation Xevo-TQ-S micro single quadrupole mass spectrometer) to purify and profile the extract. An ethylene bridged hybrid column with a 1.7 um particle size was used (Viridis BEH 3 / 0 X 100 mm). The mobile phase contained carbon dioxide (CO2), polar solvent methanol, and formic acid (0.1%). The automatic back pressure regulator was set at 1750 pound per square inch (psi). The column flow rate was 1.25 mL / minute, and the column temperature was 35 °C. Both TIC and SIR chromatograms were obtained. The mass spectrometer conditions used include a capillary voltage at 3.5 kV, a cone voltage of negative 30 V, a source temperature of 150 °C, a desolvation temperature of 600 °C, and a desolvation fast flow of 1000 L / hour. Both the full scan mode and a single ion recording (SIR) mode with the known mass / charge of QS-21 1988 m / z = [M-H]-ion were used.

[0153] FIG. 1 depicts TIC and SIR chromatograms of a commercial QS-21 standard. The negative TIC chromatogram shows the QS-21 standard has a peak at 4.72 minutes, and the two QS-21 isomers have a peak at 4.54 and 4.81 minutes. There are also several minor contaminants observed in the general TIC chromatogram. The more specific SIR chromatogram shows the peaks belonging to the two QS-21 isomers at 4.54 and 4.81 minutes. QS-21-apiose is the major isomer with the fatty acyl portion attached at the 4-hydroxyl group and the minor isoform, QS- 21-xylose, has the fatty acyl portion attached at the 3-hydroxyl group. These data cannot be used to distinguish between QS-21 -apiose or QS-21 -xylose. The retention time of the observed peaksis consistent with QS-21 literature. The mass spectra of the two isomer peaks detected in the QS- 21 standard shows a clean profile (FIG. 2).

[0154] These data show that SFC can be used to purify QS-21. The profiles from a full and a specific scan are consistent with a QS-21 standard, which includes the QS21-A and QS-21- xylose isomers.Example 2: Purification of QS-21 from Quil-A saponins by supercritical fluid chromatography

[0155] This example demonstrates the use of supercritical fluid chromatography (SFC) to purify QS-21 from the crude preparation of Quillaja saponaria known as Quil-A. The quality of the purified product was confirmed by mass spectrometry analysis, which includes single ion recording (SIR) and total ion current (TIC) chromatograms. Importantly, the purified QS-21 extract was compared to a commercial QS-21 standard.

[0156] A crude preparation of Quillaja saponaria containing a heterogenous mixture of saponin components was obtained (Fisher / Croda) to purify QS-21. The SFC and mass spectrometry protocol described in Example 1 was followed to extract QS-21 from Quil-A.

[0157] While many peaks are seen, the QS-21 -apiose and QS-21 -xylose peaks are clearly seen in the mass spectrometry analysis TIC and SIR chromatograms from Quil-A (FIG. 3). The ratio of observed QS-21 isomers is different than the ratio in the commercial QS-21 standard.

[0158] The QS-21 standard was used as a spike-in control. The TIC and SIR chromatograms show peaks at 4.54 and 4.81 minutes, corresponding to the two QS-21 isomers (FIG. 4). A comparison of TIC chromatograms of a QS-21 standard, Quil-A, and Quil-A with a spike-in QS- 21 control shows the expected QS-21 isomer peaks at 4.54 and 4.81 minutes (FIG. 5). However, a difference in the isomer peaks due to the addition of a spike-in control cannot be resolved.

[0159] The SIR chromatograms of a QS-21 standard, Quil-A, and Quil-A spiked with QS-21 show a product with an expected retention time at m / z 1988 (FIG. 6). Additionally, the Quil-A with a spike-in control shows an increase in the QS-21 isomer peaks compared to Quil-A alone.

[0160] These data demonstrate that QS-21 can be purified by using supercritical fluid chromatography, which eliminates the use of environmentally harmful organic chemicals. These data also show that SFC can be used to purify QS-21 from a crude preparation of saponins. Thedetected purified product has the same time and mass spectra as a QS-21 standard and the addition of a QS-21 spike-in control further confirms the isolation of the correct product.Example 3: Supercritical fluid extraction (SFE) extraction of QS-21 from Quillaja plant material

[0161] This example describes the attempted use of supercritical fluid extraction (SFE) to extract QS-21 from Quillaja plant material.

[0162] A modified Thar Prep 80 system was used for the SFE. Modifications included a Shimadzu LC8A pump replacing a P50 co-solvent pump and replacing the column with a 175 mL extract bomb with isolation valves pre and post bomb. For the initial experiment, 50 g of plant material was loaded into the bomb, and the bomb was pressurized 3500 psi with 10% methanol / 90% CO2and held for 30 minutes at pressure. After 30 minutes, the bomb was cleared by pumping 250 mL 25% methanol / 75% CO2through the bomb. Additional SFE conditions used increasing steps of methanol from 10% to 25%, 50% and 100% methanol. However, only trace amounts (0.03 wt / wt%) of QS-21 were detected throughout the fractions, despite the fractions being visibly green. In contrast, liquid extracts of MeOH, MeOH with 0.1% formic acid, and 1:1 MeOH:water resulted in 0.07, 0.08, and 0.13 wt / wt% QS-21, respectively. The data suggested that the solubility of QS-21 is highly sensitive to the polarity of the solvent. The trend showed that as the polarity slightly decreased, the solubility of QS-21 sharply decreased. For example, changing from methanol to ethanol, the solubility of QS-21 dropped by 80%.Supercritical CO2is considered a non-polar solvent, comparable to hexane. Thus, SFE with CO2likely failed given the need of solvent polarity in the extraction of QS-21.Example 4: HPLC and UPLC analysis of QS-21 from Quillaja plant material

[0163] This example describes the use of reverse phase (RP) HPLC and UPLC to analyze QS-21 from Quillaja plant material.

[0164] Water extraction of dried and ground Quillaja saponaria plant material was used, and two fractions of enriched saponins were prepared by multiple filtration steps, spray drying, and flash chromatography. One fraction, Flash21, is enriched in QS-21. A second fraction, Flash7, is enriched in QS-7.

[0165] We have developed an RP-HPLC method and an RP-UPLC method to analyze Flash21, shown in FIG. 7 and FIG. 8. The RP-HPLC method used a Waters Acquity XBridgeBEH C18, 5pm, 4.6x250mm column. Mobile phase A was water with 0.15% formic acid, and mobile phase B was acetonitrile with 0.15% formic acid. The sample diluent was methanol, the column temperature was 25 °C, and the flow rate was 1.2 mL / minute. At time zero, %A was 65 and %B was 35%. At 30 minutes, %A was 45 and %B was 55. At 35 minutes, the conditions were returned to those at time zero. Analysis comprised UV at 210nm and mass spectrometry (MS). As shown in FIG. 7, the blue bar eluting at 19.43 minutes was QS-21.

[0166] An RP-UPLC method was developed using a Water Acquity BEHC18, 1.7 pm, 2.1x150mm column. Mobile phase A was water with 0.05% trifluoroacetic acid (TFA), and Mobile Phase B was acetonitrile with 0.05% TFA. Time zero was 35% Mobile Phase A and 65% Mobile Phase B. A linear gradient to 35% Mobile Phase A and 65% Mobile Phase B was performed in 10 minutes with a return to time zero conditions in 0.1 minutes with a hold of 2.9 minutes. The flow rate was 0.3 mL / minute, the column temperature was 40°C, and the sample diluent was methanol. The results of practicing this RP-UPLC method on Flash 21 is shown in FIG. 8. The blue outlined peak eluting at 7.83 minutes was QS-21. The RP-HPLC method and RP-UPLC methods were largely comparable, but the RP-UPLC method took considerably less time per run and provided more resolution.Example 5: Supercritical fluid chromatography (SFC) extraction of QS-21 from Quillaja plant material

[0167] This example describes the use of SFC to extract QS-21 from Quillaja plant material. Unexpectedly, even though SFE did not work in the extraction of QS-21 (Example 3), as described herein, SFC efficiently purifies saponins from Quillaja saponin source compositions.

[0168] First, a generic analytical SFC method was developed to screen different columns and conditions. The columns were variable, Mobile Phase A was liquid CO2and Mobile Phase B was CO2with a variable co-solvent. The gradient was 35-75%B over 4 minutes, holding 75% B for 1 minute, returning to 35% B in 0.1 minute, and holding at 35% B for 0.9 minutes. The flow rate was 4.0 mL / minute, the system back pressure was 100 bar, the column temperature was 25°C, and the sample diluent was methanol. UV at 210nm was monitored and specific peaks were analyzed by mass spectrometry (MS). Multiple columns were tested. Following initial screening for removal of specific contaminants, a diol column (FIG. 9) and an amino-phenyl column (FIG. 10) were selected for further analysis. The diol column dimensions were 4 6x100mm and the amino phenyl column was 4 6x100mm

[0169] For preparative scale SFC, a 2.0 x 25.0 cm YMC- Actus Triart Diol column by YMC (Allentown, PA) was used. Mobile Phase A was CO2and Mobile Phase B was methanol with 5% water. This was an isocratic method with 55% Mobile Phase B with a flow rate of 75 g / minute. The back pressure was 100 bar, the column temperature was 25°C, and the sample diluent was methanol. The sample concentration was 20 mg / mL. The cycle time was 3.25 minutes. Detection was performed by UV at 210nm. FIG. 11 shows the profile of loading 40 mg of Flash21. Fraction F2 contained QS-21. Loading 60 and 100 mg of Flash21 on the same column resulted in substantially the same profile (not shown). The lavender fraction is QS-21 and labeled F2.

[0170] The collected fractions were then loaded onto a second column which was a 2.0 x 25.0 cm GreenSep Amino Phenyl column from ES Industries (West Berlin, NJ). Mobile Phase A was CO2and Mobile Phase B was methanol and 5% water. This was an isocratic method with 55% Mobile Phase B at a flow rate of 80g / minute. The back pressure was 100 bar, the column temperature was 25°C, the sample diluent was methanol, the load on the column was 20mg, the UV detector was at 210nm, and the cycle time was 9.8 minutes. Results are shown in FIG. 12. Regions 2 and 3 contained the QS-21B. FIG. 13 shows 400mg of the lyophilized product QS- 21.Example 6: Analysis of QS-21 extracted from Quillaja plant material via Supercritical fluid chromatography (SFC)

[0171] The purified QS-21 was analyzed by UPLC using a 2.1x150mm Waters Acuity BEH C18, 1.7um column (Medford, MA). Mobile phase A was 0.1% trifloroacetic acid in water, and the Mobile Phase B was acetonitrile. A gradient was used of 35-65% Mobile Phase B for 10 minutes, holding at 65% B for 2 minutes, returning to 35% B in 0.1 minute, and holding at 35% B for 2.9 minutes. The flow rate was 0.3mL / minute, the column temperature was 40°C, and the sample diluent was methanol.

[0172] The SFC-purified QS-21 was compared to different QS-21 preparations (FIG. 14). The Desert King QS-21 standard was purified from bark of the tree Quillaja saponaria. The InvivoGen QS-21 standard was purified from in vitro cell culture growth of Quillaja saponaria. Our HPLC QS-21 was purified from in vitro plant growth of Quillaja saponaria plant material and purified by preparative HPLC. Our SFC QS-21 was purified from in vitro plant growth of Quillaja saponaria and purified by preparative SFC

[0173] As shown in FIG. 14, the Desert King QS-21 purified by HPLC was 99.07% pure, the InvivoGen QS-21 purified by HPLC was 96.66% pure, the plant material-derived QS-21 purified by HPLC was 97.45% pure, and the plant material-derived QS-21 purified by SFC was 97.17% pure. Thus, the plant material-derived QS-21 purified by SFC was substantially equivalent to the other forms of purified QS-21. However, there are different minor impurities in the four samples described in FIG. 14. These are shown in Table 2 below. Using relative retention times (RTT), all four samples contained a contaminant at RTT 0.88. However, the RRT 0.92 contaminant was only found in plant material-derived QS-21 purified by SFC; the RRT 0.93, RRT 0.94 and RRT 1.03 contaminants were only found in the InVivoGen cell culture-derived QS-21; and the RRT 0.96 contaminant was found in the plant material-derived QS-21. Notably, the RRT 0.92 and 0.96 contaminants were unique to plant material-derived QS- 21, and both had the same m / z of 1987.3 (the same molecular weight of QS-21), indicating the contaminants were isomers of QS-21. FIG. 15 shows the high resolution of the common RRT 0.88 ESI- contaminant. FIG. 16 shows the contaminant at RRT 0.92, unique to plant material- derived QS-21 purified by SFC. FIG. 17 shows the RRT 0.96, common to both plant material- derived QS-21 materials purified by either HPLC or SFC.Table 2Example 7: Supercritical fluid chromatography (SFC) extraction of QS-21 and QS-18 from a Quil-A source

[0174] This Example tests if our SFC methods for purifying QS-21 could be applied to other Quillaja saponaria saponins, such as QS-18, and if the same SFC methods could be applied to purifying saponins from other Quillaja saponaria source materials.

[0175] Currently, only bark from older Quillaja saponaria trees is used as the source of QS- 21. Thus, bark-derived plant material is used to produce the QS-21 found in the current approved vaccines Mosquirix® (malaria), Shingrix® (shingles), AREXY® (respiratory syncytial virus) and the Novavax CO VID- 19 vaccine (SARS-CoV2) vaccines, as well as many other vaccines in development including notably a vaccine for tuberculosis. Quil-A™ is a commercially available saponin enriched crude fraction from the bark of Quillaja saponaria.

[0176] Quil-A™ was obtained from Croda Pharma. Quil-A was not immediately soluble in the SFC diluent of methanol, and a significant amount of water was required to dissolve the material. Notably, compared a crude extract from the ground Quillaja saponaria plant material, which contains chlorophyll and is green, Quil-A is derived from the cambium layer of tree bark and is not green. The Quil-A material was processed by flash chromatography using a WATERS OBD BEH Cl 8, 2.5mm, 30-50mm column. Mobile phase A was water and mobile phase B was methanol. A gradient of 60-80% B in 10 minutes, hold 80% B for 2.5 minutes, 80-100% B in 10 minutes, hold 100% B 2.5 minutes, and flow rate 40mL / minute at room temperature was used. Dried fractions containing QS-18 and QS-21 were readily soluble in methanol and moved onto SFC purification. The two-column method of SFC using a diol column followed by an aminophenyl column previously described was used. Collected fractions from the SFC were analyzed by HPEC also as previously described.

[0177] FIG. 18 shows the HPEC trace at UV 205nm of a sample of QS-21 that was previously isolated from our prepared Flash21 using SFC. The SFC-purified QS-21 from Quillaja plant material Flash21 had an observed area% of 98.5%.

[0178] QS-21 isolated by SFC from Quil-A is shown in FIG. 19. This QS-21 material had an observed purity by UV area% of 93.3%. This showed that the SFC methods described herein can isolate QS-21 from bark biomass materials as well as our Quillaja plant materials.

[0179] FIG. 20 shows an HPLC profile of SFC-purified QS-18 from Quil-A (top) compared to SFC-purified QS-18 from Flash21 (bottom). The observed purity area% of QS-18 purified by SFC from Quil-A was 94.0%, whereas the QS-18 isolated by SFC from our Flash21 plant material showed a purity area% of 89.1%. Thus, the SFC methods described herein can purify multiple saponins from both tree bark and our laboratory grown sustainable Quillaja plant materials.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a purified saponin composition, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system; and applying an elution condition to the SFC stationary phase to produce the purified saponin composition.

2. The method of claim 1, wherein the Quillaja saponin source composition is from Quillaja saponaria.

3. The method of claim 1, wherein the Quillaja saponin source composition is from Quillaja brasiliensis.

4. The method of claim 2 or 3, further comprising processing a plant material to produce the Quillaja saponin source composition.

5. The method of claim 4, wherein the plant material is selected from the group consisting of bark, cells, callus, leaf, root, plantlet, and laboratory-derived or bioreactor-derived plant material.

6. The method of claim 2 or 3, further comprising processing spent plant growth media derived from a Quillaja saponin source plant material.

7. The method of claim 1, wherein the Quillaja saponin source composition is a synthetic Quillaja saponin source composition.

8. The method of any of claims 1-7, further comprising collecting at least one elution fraction from the SFC stationary phase.

9. The method of any of claims 1-8, wherein the purified saponin composition comprises one or more of QS-21, QS-7, QS-17, or QS-18.

10. The method of any one of claims 1-9, wherein the purified saponin composition comprises QS-21.

11. The method of claim 10, wherein QS-21 is QS-21-apiose and / or QS-21-xylose.

12. The method of claim 11, wherein QS-21 is a mixture of QS-21-apiose and QS-21-xylose at a ratio of about 1:20 to about 20:1.

13. The method of any one of claims 1-12, wherein the purified saponin composition comprises at least about 90% QS-21 relative to components in the purified saponin composition with a molecular weight of about 1985 Da to about 1995 Da.

14. The method of any one of claims 1-13, wherein the SFC stationary phase is a polar stationary phase.

15. The method of any one of claims 1-14, wherein the SFC stationary phase comprises silica, polysaccharide, zirconia, polystyrene, divinylbenzene, or porous graphitic carbon.

16. The method of any one of claims 1-15, wherein the SFC stationary phase comprises particles.

17. The method of claim 16, wherein the particles have a particle size of about 0.5 pm to about 5 pm.

18. The method of claim 16 or 17, wherein the particles have an average pore size of about 50 A to about 250 A.

19. The method of any one of claims 16-18, wherein the SFC stationary phase comprises Ethylene Bridged Hybrid particles, Charged Surface Hybrid particles, High Strength Silica particles, or Solid-Core particles.

20. The method of any one of claims 16-18, wherein the SFC stationary phase is a Viridis BEH column, Viridis BEH 2-ethylpyridine column, Torus DEA column, Torus 2-PIC column, Torus Diol compound, Torus 1-AA column, or Viridis HSS Cl 8 SB column.

21. The method of any one of claims 1-20, wherein the applying the Quillaja saponin source composition to the SFC stationary phase comprises use of a mobile phase comprising CO2.

22. The method of claim 21, wherein the mobile phase comprises an organic modifier and / or a pH adjuster.

23. The method of claim 22, wherein the organic modifier is methanol, acetonitrile, ethanol, isopropanol (IPA), butanol, tetrahydrofuran (THF), or dimethylsulfoxide (DMSO), or a combination thereof.

24. The method of claim 22 or 23, wherein the pH adjuster is formic acid (FA), trifluoroacetic acid (TFA), diethylamine (DEA), dimethylethanolamine (DMEA), triethylamine (TEA), ammonia (NH3), or acetate buffers, or a combination thereof.

25. The method of any one of claims 1-24, wherein the applying the elution condition to the SFC stationary phase comprises subjecting the SFC station phase to a gradient-based elution.

26. The method of claim 25, wherein the gradient-based elution comprises increasing a percentage of an organic modifier in a mobile phase.

27. The method of claim 26, wherein the organic modifier is methanol.

28. The method of any one of claims 1-24, wherein the applying the elution condition to the SFC stationary phase comprises subjecting the SFC station phase to an isocratic -based elution.

29. The method of any one of claims 21-28, wherein the mobile phase comprises CO2, methanol, and formic acid.

30. The method of claim 29, wherein the formic acid is present at a concentration of about 0.01% to about 1% v / v.

31. The method of any one of claims 21-30, wherein the mobile phase used in the applying a Quillaja saponin source composition to the SFC stationary phase comprises less than about 2% v / v water.

32. The method of claim 31, wherein the mobile phase comprises less than about 0.1% v / v water.

33. The method of any one of claims 1-32, wherein the SFC system comprises an automatic back pressure regulator capable of maintaining a constant back pressure.

34. The method of any one of claims 1-33, wherein the SFC system exhibits a column flow rate between 1-2 mL / min.

35. The method of claim 34, wherein the column flow rate is about 1.25 mL / min.

36. The method of any one of claims 1-35, further comprising degassing the elute from the SFC stationary phase.

37. The method of any one of claims 1-36, further comprising analyzing the purified saponin composition.

38. The method of claim 37, wherein the analysis is performed using a mass spectrometry technique.

39. A method of producing a purified saponin composition comprising QS-21, the method comprising: applying a Quillaja saponin source composition to a supercritical fluid chromatography (SFC) stationary phase of a SFC system, wherein the SFC stationary phase comprises inorganic-organic ethylene- bridged hybrid particles, wherein the Quillaja saponin source composition is applied using a mobile phase comprising CO2and an organic modifier and / or a pH adjuster; and applying an elution condition to the SFC stationary phase to produce the purified saponin composition comprising QS-21.

40. A system for producing a purified saponin composition, the system comprising a SFC system comprising a SFC stationary phase, a SFC mobile phase, and a Quillaja saponin source composition.

41. A purified Quillaja saponin composition produced by the method of any one of claims 1- 39.

42. The purified Quillaja saponin composition of claim 41, wherein the purified Quillaja saponin composition comprises QS-21.

43. A vaccine comprising the purified Quillaja saponin composition of claim 41 or 42.

44. The purified Quillaja saponin composition of claim 41 or 42, wherein the purity of the purified Quillaja saponin composition is at least about 70%.

45. The purified Quillaja saponin composition of claim 44, wherein the purity of the purified Quillaja saponin composition is at least about 90%.

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