Saponin Purification

Abstract

This invention provides a robust method for producing QS-21 and a QS-21 substance with a clear composition. [Solution] A saponin extract is provided that contains at least 93% of the QS-21 main peak and 0.25-3% of the 2018 component, as determined by UV absorbance at 214 nm.

Description

Technical Field

[0001] The present application generally relates to saponin extracts, particularly extracts of Quillaja saponaria Molina, methods for their production and related aspects.

Background Art

[0002] Particularly in the case of subunit vaccines with low immunogenicity, an adjuvant is included in the vaccine to improve the humoral and cellular immune responses. Similar to natural infection by a pathogen, the adjuvant relies on activation of the innate immune system that enhances adaptive immunity over the long term.

[0003] Adjuvant System 01 (AS01) is a liposome-based adjuvant containing two immunostimulants, 3-O-desacyl-4'-monophosphoryl lipid A (3D-MPL) and QS-21 (Garcon and Van Mechelen, 2011; Didierlaurent et al., 2017). 3D-MPL is a non-toxic derivative of lipopolysaccharide derived from Salmonella minnesota, a TLR4 agonist, and QS-21 is a natural saponin extract derived from the bark of the South American tree Quillaja saponaria Molina (Kensil et al., 1991; Ragupathi et al., 2011). AS01 is included in recently developed vaccines against malaria (RTS,S - Mosquirix (registered trademark)) and herpes zoster (HZ / su - Shingrix (registered trademark)), as well as numerous candidate vaccines under development against pathogens such as human immunodeficiency virus and Mycobacterium tuberculosis.

[0004] As a result of injection of AS01, rapid and transient activation of innate immunity occurs in animal models. Neutrophils and monocytes are rapidly replenished in the draining lymph nodes (dLN) during immunization. Furthermore, AS01 is required for MHCII for T cell activation highIt induces dendritic cell (DC) replenishment and activation (Didierlaurent AM et al., 2014). Several data on the mechanism of action of AS01 components are also available. 3D-MPL is transmitted via TLR4 to stimulate NF-κB transcriptional activity and cytokine production, directly activating antigen-presenting cells (APCs) in both humans and mice (De Becker et al., 2000; Ismaili et al., 2002; Martin et al., 2003; Mata-Haro et al., 2007). QS-21 exhibits a high antigen-specific antibody response and CD8 in mice. + It enhances T cell responses (Kensil and Kammer, 1998; Newman et al., 1992; Soltysik et al., 1995) and antigen-specific antibody responses in humans (Livingston et al., 1994). Due to its physical properties, QS-21 may function as a danger signal in vivo (Lambrecht et al., 2009; Li et al., 2008). QS-21 has been shown to activate the ASC-NLRP3 inflammasome and subsequent IL-1β / IL-18 release (Marty-Roix, R. et al., 2016), but the precise molecular pathway involved in the adjuvant effect of saponins has not yet been clearly defined. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] As with all components of products approved for human medicine, the production of QS-21 requires the use of approved manufacturing processes and careful control of the final composition to ensure that the required specifications are met. Modifying existing processes requires costly and time-consuming re-verification, and even then, deviations from specifications result in discarding the product. There remains a need for robust methods for the production of QS-21 and for QS-21 material with a clearly defined composition. [Means for solving the problem]

[0006] The present invention provides a saponin extract containing at least 93% of the QS-21 main peak and 0.25-3% of the 2018 component, as determined by UV absorbance at 214 nm.

[0007] The invention also provides a saponin extract containing at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9, or 2001.9 as determined by negative ion electrospray mass spectrometry, based on UV absorbance at 214 nm, and 0.25-3% of triterpenoid glycosides having an m / z of 2017.9 as determined by UV absorbance at 214 nm.

[0008] Furthermore, the UV absorbance at 214nm indicates at least 93%

[0009] [ka] JPEG2026102610000002.jpg63130 and 0.25~3%

[0010] [ka] A saponin extract containing the following is provided.

[0011] Furthermore, (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by reverse-phase chromatography using polystyrene resin, and (iii) A method for producing a saponin extract is provided, comprising the step of purifying the extract by reverse-phase chromatography using a phenyl resin.

[0012] The use of the saponin extract of the present invention in the manufacture of pharmaceuticals is provided.

[0013] Furthermore, adjuvant compositions and vaccine compositions containing the saponin extract of the present invention are also provided. [Brief explanation of the drawing]

[0014] [Figure 1] HPLC chromatogram of a crude aqueous Yucca schidigera Molina bark extract. [Figure 2] HPLC-UV chromatogram of a crude aqueous Yucca schidigera Molina bark extract. [Figure 3] UPLC-UV chromatogram of a crude aqueous Yucca schidigera Molina bark extract. [Figure 4] UPLC-UV chromatogram of a saponin extract purified with polystyrene. [Figure 5] UPLC-UV / MS chromatogram of a purified Yucca schidigera Molina saponin extract. [Figure 6] Details of the UPLC-UV / MS chromatogram of a purified Yucca schidigera Molina saponin extract. [Figure 7] Mass chromatogram extracted for the 1988 and 2002 molecular weight ions of a purified Yucca schidigera Molina saponin extract. [Figure 8] Combined centroid spectrum of a purified Yucca schidigera Molina saponin extract. [Figure 9] CD4 T cell response on day 21 in mice vaccinated with gE AS01 vaccine. [Figure 10] Antibody response on day 21 in mice vaccinated with gE AS01 vaccine. [Figure 11] Antibody response on day 28 in mice vaccinated with gE AS01 vaccine.

Mode for Carrying Out the Invention

[0015] Brief Explanation of Sequence Identifiers SEQ ID NO: 1: RTS polypeptide sequence Sequence ID 2: polypeptide sequence of Mycobacterium tuberculosis (M. tuberculosis) H37Rv strain Rv1196 Sequence ID 3: Rv0125 polypeptide sequence of Mycobacterium tuberculosis H37Rv strain. Sequence ID 4: M72 fusion polypeptide sequence Sequence ID 5: M72-2his fusion polypeptide sequence Sequence ID 6: Varicella zoster virus truncated gE polypeptide sequence Sequence ID 7: Conformationally restricted RSV PreF antigen polypeptide sequence Sequence ID 8: HIV TV1 gp120 polypeptide sequence Sequence ID 9: HIV 1086.C gp120 polypeptide sequence

[0016] Detailed description of the invention As already mentioned, all components of products approved as human pharmaceuticals must be manufactured using approved processes and their final composition carefully controlled to ensure they meet the required specifications. Deviations from specifications result in discarding the product. However, safety and efficacy studies rely on testing of the specified compositions, and therefore conformity to component specifications introduces risks. Modifying existing processes requires costly and time-consuming re-verification.

[0017] The inventors have found that the composition of crude aqueous extracts of Quillaja saponaria molina varies, particularly with respect to a component referred to herein as component 2018, and that it is difficult to separate the excess component 2018 by applying existing approved manufacturing processes. Therefore, the present invention provides a method for obtaining a consistent purified extract by using a crude aqueous extract with a clear composition.

[0018] The present invention provides a saponin extract containing at least 93% of the QS-21 main peak and 0.25-3% of the 2018 component, as measured by UV absorbance at 214 nm, particularly a saponin extract in which the most abundant chemical species monoisotope is 1987.9 m / z. The saponin extract is preferably composed of at least 98% of the QS-21 group, as measured by UV absorbance at 214 nm. Typically, the saponin extract contains less than 1% of lyo impurities, particularly less than 1% of the largest peak other than the QS-21 group, as measured by UV absorbance at 214 nm.

[0019] Of particular interest is the saponin extract, which, based on UV absorbance at 214 nm, contains at least 98% of the QS-21 group, at least 93% of the QS-21 main peak, 0.25–3% of the 2018 component, and less than 1% of the largest peak other than the QS-21 group, with the most abundant monoisotope of the chemical species being 1987.9 m / z.

[0020] Also provided are saponin extracts containing at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9, or 2001.9 based on UV absorbance at 214 nm, and 0.25 to 3% of triterpenoid glycosides having an m / z of 2017.9, in particular a saponin extract in which the monoisotope of the most abundant chemical species is 1987.9 m / z. Preferably, the saponin extract contains at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9, or 2001.9 based on UV absorbance at 214 nm, excluding B-isomers and lyo impurities, and 0.25 to 3% of triterpenoid glycosides having an m / z of 2017.9, in particular a saponin extract in which the monoisotope of the most abundant chemical species is 1987.9 m / z. The saponin extract should preferably contain at least 98% of triterpenoid glycosides having m / z values ​​of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118, based on UV absorbance at 214 nm. Preferably, the saponin extract should contain at least 98% of triterpenoid glycosides having m / z values ​​of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118, excluding lyo impurities, based on UV absorbance at 214 nm. Typically, the saponin extract contains 1% or less of lyo impurities based on UV absorbance at 214 nm. The saponin extract should preferably contain 1% or less of other peaks based on UV absorbance at 214 nm.

[0021] Of particular interest is the saponin extract, which contains at least 98% of triterpenoid glycosides with m / z values ​​of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118 based on UV absorbance at 214 nm, at least 93% of triterpenoid glycosides with m / z values ​​of 1855.9, 1987.9, or 2001.9, 0.25-3% of triterpenoid glycosides with m / z value of 2017.9, and less than 1% of other peaks, with the most abundant monoisotope being 1987.9 m / z. In particular, the saponin extract is Based on UV absorbance at 214 nm, it contains at least 98% triterpenoid glycosides excluding lyo impurities, having m / z values ​​of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118; at least 93% triterpenoid glycosides excluding B-isomers and lyo impurities, having m / z values ​​of 1855.9, 1987.9, or 2001.9; 0.25-3% triterpenoid glycosides having m / z value of 2017.9; and less than 1% of other peaks, with the most abundant monoisotope being 1987.9 m / z.

[0022] In addition, the UV absorbance at 214nm indicates at least 93%

[0023] [ka] JPEG2026102610000005.jpg62128 and 0.25~3%

[0024] [ka] A saponin extract containing is provided, and in particular, the most abundant monoisotope of the chemical species is 1987.9 m / z. The saponin extract is at least 98%

[0025] [ka] It is appropriate for the JPEG2026102610000008.jpg236130JPEG2026102610000009.jpg46118 to be included. The saponin extract should appropriately contain 98% of the aforementioned components and 2118 components. Typically, the saponin extract should contain less than 1% of the UV absorbance at 214 nm.

[0026] [ka] In particular, it contains other peaks at a concentration of 1% or less.

[0027] Of particular interest is that the UV absorbance at 214 nm indicates at least 98%

[0028] [ka] JPEG2026102610000012.jpg214129 at least 93%

[0029] [ka] JPEG2026102610000014.jpg651290.25~3%

[0030] [ka] This saponin extract contains less than 1% of other peaks, with the most abundant monoisotope of the chemical species being 1987.9 m / z. Of particular interest is that, based on UV absorbance at 214 nm, at least 98%

[0031] [ka] JPEG2026102610000017.jpg232131JPEG2026102610000018.jpg43104 or 2118 components, At least 93%

[0032] [ka] JPEG2026102610000020.jpg611270.25~3%

[0033] [ka] This saponin extract contains less than 1% of other peaks, with the most abundant monoisotope being 1987.9 m / z.

[0034] In some embodiments, the saponin extract contains at least 65%, for example, at least 70%, of the 1988 component, as determined by the UV absorbance and relative ion abundance at 214 nm. In some embodiments, the saponin extract contains 85% or less, for example, 80% or less, of the 1988 component, as determined by the UV absorbance and relative ion abundance at 214 nm.

[0035] In some embodiments, the saponin extract contains 1856 components in an amount of 30% or less, for example, 25% or less, based on UV absorbance and relative ion abundance at 214 nm. In some embodiments, the saponin extract contains 1856 components in an amount of at least 5%, for example, at least 10%, and especially at least 15%, based on UV absorbance and relative ion abundance at 214 nm.

[0036] In some embodiments, the saponin extract contains 6% or less of component 2002, for example 4% or less, based on UV absorbance and relative ion abundance at 214 nm. In some embodiments, the saponin extract contains at least 0.5% or at least 1% of component 2002, based on UV absorbance and relative ion abundance at 214 nm.

[0037] In some embodiments, the saponin extract contains at least 0.5%, for example, at least 1%, or at least 2% of component 2018, based on UV absorbance and relative ion abundance at 214 nm.

[0038] In some embodiments, the saponin extract is determined by UV absorbance and relative ion abundance at 214 nm to be at least 65%, for example, at least 70%.

[0039] [ka] It contains.

[0040] In some embodiments, the saponin extract is defined as having a UV absorbance at 214 nm and relative ion abundance of 30% or less, for example, 25% or less.

[0041] [ka] It contains. In some embodiments, the saponin extract contains at least 5%, for example at least 10%, and especially at least 15%, based on UV absorbance and relative ion abundance at 214 nm.

[0042] [ka] It contains.

[0043] In some embodiments, the saponin extract is present in an amount of 10% or less, for example 5% or less, based on UV absorbance and relative ion abundance at 214 nm.

[0044] [ka] It contains. In some embodiments, the saponin extract is at least 0.5%, for example at least 1%, based on UV absorbance and relative ion abundance at 214 nm.

[0045] [ka] It contains.

[0046] In some embodiments, the saponin extract contains at least 0.5%, for example, at least 1%, or at least 2%, based on UV absorbance and relative ion abundance at 214 nm.

[0047] [ka] It contains.

[0048] Quil A is a saponin preparation isolated from the South American tree Quillaja saponaria molina, and was first described as having adjuvant activity by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archives for the Complete Virus Research, Vol. 44, Springer Verlag, Berlin, pp. 243-254). A purified fraction of Quil A that retains adjuvant activity without the toxicity associated with Quil A has been isolated by HPLC (see, for example, European Patent No. 03622789). Various fractions, such as QS-7, QS-17, QS-18, and QS-21, have been found to possess adjuvant activity, but their toxicity varies considerably.

[0049] As used herein, the term "saponin extract" refers to the extract of Quillaja saponaria molina.

[0050] As used herein, the term “triterpenoid glycoside” means an entity having a triterpenoid core derivatized by sugars linked via glycosidic bonds.

[0051] In this specification, several structures have been determined by MS / MS, and a limitation of this technique in identifying some branched stereochemistry and isomers of sugar species (e.g., apiose and xylose) is that some structures are predicative and based on assumed conserved cores. Therefore, if a predicative structure is incorrect for any reason, it should represent the actual structure of the component as confirmed by other means.

[0052] The term "component 2018" refers to the triterpenoid glycoside identified as the "2018 peak" in Figure 6. In the UPLC-UV / MS method described herein, component 2018 is appropriately identified as the main component of the peak with a retention time of approximately 4.5 minutes and a monoisotopic molecular weight of 2017.9. Component 2018 can also be identified with a retention time of approximately 5.8 minutes in the UPLC-UV method described herein. This main component 2018 has been confirmed by MS / MS to have the following putative structure.

[0053] [ka]

[0054] The term "component 1988" refers to a triterpenoid glycoside identified as part of the main peak of QS-21 in Figure 6, having a monoisotopic molecular weight of 1987.9. In the UPLC-UV / MS method described herein, component 1988 appropriately has a retention time of approximately 4.4 minutes and a monoisotopic molecular weight of 1987.9. This component 1988 is QS-21A V1:

[0055] [ka] and QS-21A V2

[0056] [ka] It can consist of.

[0057] The term "component 1856" refers to a triterpenoid glycoside identified as part of the QS-21 main peak in Figure 6, having a monoisotopic molecular weight of 1855.9. In the ULC-UV / MS method described herein, component 1856 appropriately has a retention time of approximately 4.4 minutes and a monoisotopic molecular weight of 1855.9. This component 1856 is

[0058] [ka] It can consist of.

[0059] The term "component 2002" refers to a triterpenoid glycoside identified as part of the QS-21 main peak in Figure 6, having a monoisotopic molecular weight of 2001.9. In the UPLC-UV / MS method described herein, component 2002 appropriately has a retention time of approximately 4.4 minutes and a monoisotopic molecular weight of 2001.9. The putative structure of this component 2002, determined by MS / MS, is:

[0060] [ka] It has been confirmed that it possesses [this characteristic].

[0061] The term "lyo impurity" refers to the triterpenoid glycoside identified as the "freeze-dried peak" in Figure 6. In the UPLC-UV / MS method described herein, the lyo impurity has a retention time of approximately 4.7 minutes, and the main component of the peak appropriately has a monoisotopic molecular weight of 1855.9. The presumptive structure of this main lyo impurity, determined by MS / MS, is:

[0062] [ka] It has been confirmed that it possesses [this characteristic].

[0063] The term "B-isomer" refers to the triterpenoid glycoside identified as "B-isomer" in Figure 6. In the UPLC-UV / MS method described herein, the B-isomer has a retention time of approximately 4.0 minutes, and the main component of the peak appropriately has a monoisotopic molecular weight of 1987.9. The presumptive structure of this main B-isomer component, determined by MS / MS, is:

[0064] [ka] It has been confirmed that it possesses [this characteristic].

[0065] The term "component 1518" refers to the triterpenoid glycoside identified as the "1518 peak" in Figure 6. In the UPLC-UV / MS method described herein, component 1518 has a retention time of approximately 4.2 minutes, and the main component of the peak appropriately has a monoisotopic molecular weight of 1517.7. The putative structure of this main component 1518, as determined by MS / MS, is:

[0066] [ka] It has been confirmed that it possesses [this characteristic].

[0067] The term "component 1712" refers to the triterpenoid glycoside identified as the "1712 peak" in Figure 6. In the UPLC-UV / MS method described herein, component 1712 has a retention time of approximately 4.6 minutes, and the main component of the peak appropriately has a monoisotopic molecular weight of 1711.8. The presumptive structure of this main component 1712, as determined by MS / MS, is:

[0068] [ka] It has been confirmed that it possesses [this characteristic].

[0069] The term "component 2118" refers to the triterpenoid glycoside identified as "4.607" in Figure 6. In the UPLC-UV / MS method described herein, component 2118 has a retention time of approximately 4.6, and the main component of the peak appropriately has a monoisotopic molecular weight of 2118.

[0070] The term "QS-21 main peak" refers to the triterpenoid glycoside identified as "QS-21" in Figure 6. In the UPLC-UV / MS method described herein, the QS-21 main peak should appropriately have a retention time of approximately 4.4 minutes and molecular weight components of 1855.9, 1987.9, and 2001.9 m / z. This QS-21 main peak corresponds to QS-21A V1:

[0071] [ka] QS-21A V2:

[0072] [ka] 1856 ingredients:

[0073] [ka] and Ingredients 2002:

[0074] [ka] It can consist of.

[0075] The term "QS-21 group" refers to triterpenoid glycosides having major monoisotopic molecular weights of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118, identified by the UPLC-UV / MS method described herein as having a retention time of approximately 4.0 to 4.7 minutes from the B-isomer to the peak preceding the lyo impurity. The QS-21 group includes QS-21A V1:

[0076] [ka] QS-21A V2:

[0077] [ka] 1856 ingredients:

[0078] [ka] Ingredients 2002:

[0079] [ka] Ingredients for 2018:

[0080] [ka] B-isomer:

[0081] [ka] 1518 ingredients:

[0082] [ka] 1712 ingredients:

[0083] [ka] and 2118 ingredients It can consist of.

[0084] The term "largest peak outside of the QS-21 group" refers to the largest UV-detectable peak that is not part of the QS-21 group and is detectable by the UPLC-UV / MS method described herein.

[0085] The term "preceding peak" refers to the peak immediately preceding the QS-21 main peak in the HPLC-UV method described herein (see Figure 2).

[0086] The term "m / z" refers to the mass-to-charge ratio of the monoisotope peak. Unless otherwise specified, "m / z" is determined by negative ion electrospray mass spectrometry.

[0087] The term "ion abundance" refers to the amount of ions at a specified m / z, measured in a sample or, where necessary depending on the circumstances, at a given peak. A mask chromatogram for a specified m / z can be derived from the MS total ion chromatogram in the UPLC-UV / MS method described herein. A mask chromatogram plots signal intensity against time. Ion abundance is measured as the area of ​​the integrated peak. Area at specified m / z / Area at relative reference m / z = Relative abundance.

[0088] The term "UV absorbance at 214 nm" refers to the area of ​​the integrated peak in a UV absorbance chromatogram. The formula is: (Area relative to a specified peak) / (Area of ​​all integrated peaks in the chromatogram) × 100 = Area percentage relative to a specified peak.

[0089] The term "UV absorbance and relative ion abundance at 214 nm" refers to an estimated value relative to the proportion of a given m / z for co-eluting chemical species. The proportion of a given m / z for a given UV peak is calculated as (area percentage relative to the given UV peak) × (relative ion abundance for the m / z of interest in the given peak) / (total relative ion abundance for the given peak). This calculation assumes the relative ion abundance present for all co-eluting chemical species.

[0090] The term "the monoisotope of the most abundant species is at 1988 m / z" means that the monoisotope of the most abundant species, which is the first peak in the group of isotopes with the highest reaction per m / z, is at 1987.9 m / z. The most abundant species can be determined by constructing a binding spectrum across the entire total ion chromatogram using the UPLC-UV / MS method (negative ion electrospray) described herein.

[0091] The term "dried" means that virtually all solvent has been removed. A dried extract typically contains less than 5% (w / w) of solvent (e.g., less than 5% (w / w) of water). A dried extract should ideally contain 100 ppm (w / w) or less of acetonitrile.

[0092] Furthermore, (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by reverse-phase chromatography using polystyrene resin, and (iii) A step of purifying the extract by reverse-phase chromatography using phenyl resin. A method for producing a saponin extract containing the following is provided.

[0093] Ideally, the method would be: (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by polyvinylpyrrolidone adsorption, (iii) A step of purifying the extract by dialysis filtration, ultrafiltration or dialysis, (iv) A step of purifying the extract by reverse-phase chromatography using polystyrene resin, and (v) A step of purifying the extract by reverse-phase chromatography using phenyl resin. Includes, Steps (ii) and (iii) may be performed in the reverse order or simultaneously, but the order is typically given.

[0094] Crude aqueous extracts of Quillaja saponaria molina are obtained by aqueous extraction (however, they do not need to be in aqueous form; they may be subsequently dried, subjected to solvent exchange, or reconstituted in a different solvent). The term "crude aqueous extract of Quillaja saponaria molina having a suitable 2018 component composition" means an extract in which the ratio of 2018 component to the QS-21 main peak is 0.075 or less, particularly 0.064 or less (determined by ULC-UV absorbance at 214 nm). Preferably, the ratio of 2018 component / QS-21 main peak is at least 0.005, e.g., at least 0.01, as measured by UV absorbance at 214 nm.

[0095] The ratio of the preceding peak to the QS-21 main peak should be 0.45 or less, and especially 0.4 or less (determined by the HPLC-UV absorbance at 214 nm). The ratio of the preceding peak to the QS-21 main peak may be 0.05 or more, and especially 0.1 or more (determined by the HPLC-UV absorbance at 214 nm).

[0096] Typically, the crude aqueous extract is a bark extract. The QS-21 main peak content in the aqueous solution of the crude aqueous extract of Quillaja saponaria molina should be at least 1 g / L, for example, at least 2 g / L, especially at least 2.5 g / L, and particularly at least 2.8 g / L (determined, for example, by UV absorbance relative to a control sample of known concentration).

[0097] The step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition should preferably include testing the composition to determine the 2018 component content.

[0098] The step of purifying the extract by polyvinylpyrrolidone adsorption includes treating the extract with a polyvinylpyrrolidone resin. Typically, the extract is stirred together with the polyvinylpyrrolidone resin. The extract may then be separated from the polyvinylpyrrolidone resin, from which impurities have been adsorbed, by filtration. This step of the method generally removes polyphenolic impurities, such as tannins.

[0099] The step of purifying the extract by diafiltration, ultrafiltration, or dialysis is usually best performed by diafiltration using tangential flow. A suitable membrane example is one with a 30 kDa cutoff. This step of the method generally removes salts, sugars, and other low molecular weight substances.

[0100] The step of purifying the extract by reverse-phase chromatography using a polystyrene resin typically uses acetonitrile and water as solvents, usually acidified with a suitable acid, such as acetic acid. An example of a suitable resin is Amberchrom XT20. Chromatography may be carried out under isocratic conditions, but is typically operated under a solvent gradient (continuous, e.g., linear or stepwise), as shown, for example, in the examples. This step of the method generally removes non-saponin substances and enriches the desired saponins. The selected fractions can be pooled to maximize the yield of substances that meet the required criteria (typically %QS-21 ≥ 18%, determined by UV absorbance after HPLC-UV, and 2018 / QS-21 ratio ≤ 0.054, determined by UV absorbance after UPLC-UV). Each polystyrene chromatolane typically contains between 25 and 200 g of QS-21, for example between 50 and 150 g, and especially between 70 and 110 g (the amount is based on the QS-21 main peak content in the material under UV light).

[0101] In the purification of extracts by reverse-phase chromatography using phenyl resin, acetonitrile acidified with a suitable acid, such as acetic acid, and water are typically used as solvents. Chromatography may be performed using a solvent gradient (continuous, e.g., linear or stepwise), but is usually operated under isocratic conditions. This step of the method provides the final purification of the desired saponin. The selected fractions can be pooled to maximize the yield of the substance that meets the required criteria (typically %QS-21 group ≥ 98.5, %QS-21 main peak ≥ 94.5, 2002 / 1988 ≤ 0.027, %2018 ≤ 2.7%, main peaks other than QS-21 group ≤ 1%, determined by UPLC-UV / MS). Each phenyl chromatography run is typically between 4 and 40 g of QS-21, for example between 10 and 30 g, and especially between 13 and 21 g (the amount is based on the QS-21 main peak content in the substance as determined by UV).

[0102] The method may include a further step of removing the solvent to provide a dried saponin extract. Therefore, the present invention is (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by reverse-phase chromatography using polystyrene resin, (iii) A step of purifying the extract by reverse-phase chromatography using phenyl resin, and (iv) step of removing the solvent to provide a dried saponin extract This invention provides a method for producing a saponin extract containing [the specified ingredient].

[0103] The present invention also, (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by polyvinylpyrrolidone adsorption, (iii) A step of purifying the extract by diafiltration, ultrafiltration or dialysis, (iv) A step of purifying the extract by reverse-phase chromatography using polystyrene resin. (v) A step of purifying the extract by reverse-phase chromatography using phenyl resin, and (vi) step of removing the solvent to provide a dried saponin extract The present invention also provides a method for producing a saponin extract, wherein steps (ii) and (iii) may be performed in the reverse order or simultaneously, but are typically in the order described.

[0104] To improve drying efficiency, it is desirable to perform further steps prior to the drying step, such as concentrating the extract by capture and release using an appropriate technique, such as reverse-phase chromatography (e.g., using C8 resin), and / or changing the solvent.

[0105] Also, (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by polyvinylpyrrolidone adsorption, (iii) A step of purifying the extract by diafiltration, ultrafiltration or dialysis, (iv) A step of purifying the extract by reverse-phase chromatography using polystyrene resin. (v) A step of purifying the extract by reverse-phase chromatography using phenyl resin. (vi) Depending on the case, a step of concentrating the extract, (vii) Depending on the case, the step of changing the solvent, (viii) step of removing residual solvent to provide a dried saponin extract. A method for producing a saponin extract, comprising the elements, is also provided, wherein steps (vi) and (vii) may be performed in the reverse order or simultaneously, but are typically in the order described.

[0106] Also, (i) A step of selecting a crude aqueous extract of Quillaja saponaria molina having an appropriate 2018 component composition, (ii) A step of purifying the extract by polyvinylpyrrolidone adsorption, (iii) A step of purifying the extract by diafiltration, ultrafiltration or dialysis, (iv) A step of purifying the extract by reverse-phase chromatography using polystyrene resin. (v) A step of purifying the extract by reverse-phase chromatography using phenyl resin. (vi) A step of concentrating the extract by reverse-phase chromatography using C8 resin. (vii) The step of changing the solvent, (viii) step of removing residual solvent to provide a dried saponin extract. A method for producing a saponin extract, including the above, is also provided.

[0107] The extract can be concentrated using any suitable technique. For example, concentration can be performed using capture and release methods, such as reversed-phase chromatography, particularly using C8 resin. In reversed-phase chromatography, acetonitrile and water, usually acidified with a suitable acid, such as acetic acid, are typically used as solvents. The chromatography is usually operated under a solvent gradient, and the saponin extract is captured in a low-organic solvent and eluted into a high-organic solvent, particularly a stepwise solvent gradient.

[0108] Solvent exchange can be carried out using any suitable technique, particularly diafiltration, ultrafiltration, or dialysis, especially diafiltration. Solvent exchange may be useful, for example, to reduce the acetonitrile content as described in International Publication No. 2014016374. A suitable membrane may be selected to allow solvent exchange while retaining the saponin extract, and may be, for example, a 1 kDa membrane.

[0109] Drying by solvent removal can be carried out by any suitable means, particularly freeze-drying. During drying, degradation of the saponin extract may occur, potentially leading to the formation of lyo impurities. Therefore, it is desirable to dry under conditions that limit the formation of lyo impurities, for example, by limiting the drying temperature and / or drying time. Solvent removal is preferably carried out by a single freeze-drying process. The required degree of drying depends on the properties of the solvent; for example, pharmaceutically unacceptable solvents should be removed to a high degree, while some pharmaceutically acceptable solvents (e.g., water) can be removed to a lesser extent.

[0110] The method of the present invention is suitable for use with QS-21 in quantities between 25 and 1000 g, for example between 50 and 500 g, and particularly between 100 and 500 g (the amount is based on the QS-21 main peak content in the substance as measured by UV).

[0111] Furthermore, a method is provided for identifying a purified saponin extract, for example, a crude aqueous extract of Quillaja saponaria molina for use in the production of the saponin extract of the present invention, and the method is as follows: (i) A step to determine the ratio of the 2018 component to the QS-21 main peak by ULC-UV absorbance at 214 nm. (ii) Selecting a crude extract having a ratio of 0.075 or less for the 2018 component to the QS-21 main peak. Includes.

[0112] In one embodiment, the crude aqueous extract selected in step (ii) has a ratio of 0.064 or less for the 2018 component to the QS-21 main peak.

[0113] The present invention also provides a method for determining the ratio of component 2018 to the QS-21 main peak in a crude aqueous extract of Quillaja saponaria molina, the method being: (i) A step of determining the content of component 2018 in the crude aqueous extract of Quillaja saponaria molina by ULC-UV absorbance at 214 nm. (ii) The step of determining the QS-21 main peak content in the crude aqueous extract of Quillaja saponaria molina by ULC-UV absorbance at 214 nm, and (iii) A step of determining the ratio of the 2018 component to the QS-21 main peak by comparing the 2018 component content with the QS-21 main peak content. Includes.

[0114] The use of the saponin extract of the present invention in the manufacture of pharmaceuticals is provided. Furthermore, the saponin extract of the present invention is provided for use as a pharmaceutical, particularly as an adjuvant. Also provided are adjuvant compositions containing the saponin extract of the present invention.

[0115] The saponin extract of the present invention may be combined with further adjuvants, such as TLR4 agonists, particularly lipopolysaccharide TLR4 agonists, such as lipid A derivatives, especially monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Monophosphoryl lipid A (3D-MPL). 3D-MPL is marketed by GlaxoSmithKline Biologicals NA under the name "MPL" and is referred to as 3D-MPL in the documents. See, for example, U.S. Patents 4,436,727, 4,877,611, 4,866,034 and 4,912,094. 3D-MPL can be produced according to the method described in UK Patent Application Publication No. 220211. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A having 4, 5, or 6 acylated chains.

[0116] Other TLR4 agonists that may be useful in this invention include, for example, International Publication No. 2008 / 153541 or International Publication No. 2009 / 143457 or the literature article Coler RN et al., (2011), Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant., PLoS ONE, 6(1): e16333., doi:10.1371 / journal.pone.0016333 and Arias MA et al., (2012), Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140., PLoS ONE, 7(7): There are glucopyranosyllipid adjuvants (GLAs) described in e41144, doi:10.1371 / journal.pone.0041144. International Publication No. 2008 / 153541 or International Publication No. 2009 / 143457 are incorporated herein by reference for the purpose of specifying TLR4 agonists that may be useful in the present invention.

[0117] A typical adult human dose of the adjuvant contains saponin extract in an amount between 1 and 100 ug per human dose. The saponin extract can be used at a level of approximately 50 ug. Examples of a suitable range are 40–60 ug, preferably 45–55 ug or 49–51 ug, e.g., 50 ug. In a further embodiment, the human dose contains saponin extract at a level of approximately 25 ug. Examples of a lower range are 20–30 ug, preferably 22–28 ug or 24–26 ug, e.g., 25 ug. Human doses intended for children may be reduced compared to those intended for adults (e.g., 50% reduction).

[0118] TLR4 agonists, such as lipopolysaccharides, e.g., 3D-MPL, can be used in amounts between 1 and 100 ug per human dose. 3D-MPL can be used at levels of approximately 50 ug. Examples of suitable ranges include 40-60 ug, preferably 45-55 ug or 49-51 ug, e.g., 50 ug. In further embodiments, human doses include 3D-MPL at levels of approximately 25 ug. Examples of lower ranges include 20-30 ug, preferably 22-28 ug or 24-26 ug, e.g., 25 ug. Human doses intended for children may be reduced compared to those intended for adults (e.g., 50% reduction).

[0119] When both TLR4 agonists and saponin extracts are present in the adjuvant, the weight ratio of TLR4 agonist to saponin should be between 1:5 and 5:1, preferably 1:1. For example, if 3D-MPL is present in an amount of 50 ug or 25 ug, it is also preferable that QS-21 be present in an amount of 50 ug or 25 ug per human dose.

[0120] The adjuvant may also include a suitable carrier, such as an emulsion (e.g., an oil-in-water emulsion) or liposomes.

[0121] Liposomes The term "liposome" is well known in the art and defines a general category of vesicles containing one or more lipid bilayers surrounding an aqueous space. Therefore, liposomes consist of one or more lipid and / or phospholipid bilayers and may contain other molecules, such as proteins or carbohydrates, within their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or contain water-soluble substances, lipid-soluble substances, and / or amphiphilic compounds.

[0122] The size of liposomes can vary from 30 nm to several micrometers depending on the phospholipid composition and the method used for their preparation.

[0123] Liposomes useful in the present invention are preferably those containing DOPC (along with saponins and optionally TLR4 agonists) or essentially composed of DOPC and sterols.

[0124] In this invention, the size of the liposomes is in the range of 50 nm to 200 nm, particularly 60 nm to 180 nm, for example, 70 nm to 165 nm. Optimally, the liposomes should be stable, have a diameter of approximately 100 nm, and be easily sterilized by filtration.

[0125] The structural integrity of liposomes can be evaluated by methods such as dynamic light scattering (DLS) to measure liposome size (mean Z diameter, Zav) and polydispersity, or by electron microscopy for structural analysis of liposomes. In one embodiment, the mean particle size is between 95 and 120 nm, and / or the polydispersity (PdI) index is 0.3 or less (e.g., 0.2 or less).

[0126] Further additives (excipients) In further embodiments, a buffer is added to the composition. The pH of the liquid preparation is adjusted considering the components of the composition and the required suitability for administration to the target. The pH of the liquid mixture is preferably at least 4, at least 5, at least 5.5, at least 5.8, and at least 6. The pH of the liquid mixture may be less than 9, less than 8, less than 7.5, or less than 7. In other embodiments, the pH of the liquid mixture is between 4 and 9, between 5 and 8, for example, between 5.5 and 8. Therefore, the pH is preferably between 6 and 9, for example, between 6.5 and 8.5. In a particularly preferred embodiment, the pH is between 5.8 and 6.4.

[0127] A suitable buffer can be selected from acetates, citrates, histidines, maleates, phosphates, succinates, tartrates, and TRIS. In one embodiment, the buffer is a phosphate buffer, such as Na / Na2PO4, Na / K2PO4, or K / K2PO4.

[0128] The buffering agent may be present in the liquid mixture in an amount of at least 6 mM, at least 10 mM, or at least 40 mM. The buffering agent may be present in the liquid mixture in an amount of less than 100 mM, less than 60 mM, or less than 40 mM.

[0129] As is well known, parenteral administration solutions should have a pharmaceutically acceptable osmolality to avoid cell deformation or lysis. A pharmaceutically acceptable osmolality generally means that the solution has an osmolality that is approximately isotonic or slightly hypertonic. A composition (if presented in dry form, or if reconstituted) is suitable to have an osmolality in the range of 250–750 mOsm / kg, for example, the osmolality may be in the range of 250–550 mOsm / kg, e.g., 280–500 mOsm / kg. In a particularly preferred embodiment, the osmolality may be in the range of 280–310 mOsm / kg. The osmolality can be measured according to techniques known in the art, for example, using a commercially available osmometer, e.g., the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).

[0130] An "isotonic agent" is a compound that is physiologically acceptable and imparts to the formulation a tension suitable for preventing net water flow across cell membranes in contact with the formulation. In some embodiments, the isotonic agent used in the composition is a salt (or mixture of salts), the salt is conveniently sodium chloride, and a concentration of approximately 150 nM is appropriate. However, in other embodiments, the composition contains a nonionic isotonic agent, and the concentration of sodium chloride in the composition is less than 100 mM, e.g., less than 80 mM, e.g., less than 50 mM, e.g., less than 40 mM, less than 30 mM, especially less than 20 mM. The ionic strength in the composition may be less than 100 mM, e.g., less than 80 mM, e.g., less than 50 mM, e.g., less than 40 mM or less than 30 mM.

[0131] In certain embodiments, the nonionic isotonic agent is a polyol, such as sucrose and / or sorbitol. The concentration of sorbitol may be, for example, between about 3% and about 15% (w / v), or between about 4% and about 10% (w / v). An adjuvant comprising an immunologically active saponin fraction and a TLR4 agonist, wherein the isotonic agent is a salt or polyol, is described in International Publication No. 2012 / 080369.

[0132] A suitable human dose volume is between 0.05 ml and 1 ml, for example, between 0.1 and 0.5 ml, particularly a dose volume of about 0.5 ml or 0.7 ml. The volume of the composition used may depend on the delivery route and location, with smaller doses used in the intradermal route. Unit dose containers may contain an excess amount to allow for precise handling of the substance during the administration of the unit dose.

[0133] The saponin:DOPC ratio is typically around 1:50 to 1:10 (w / w), preferably between 1:25 and 1:15 (w / w), and more preferably between 1:22 and 1:18 (w / w), for example, 1:20 (w / w).

[0134] Saponins are best presented as less reactive compositions that can be quenched with exogenous sterols, such as cholesterol. Cholesterol is disclosed in the Merck Index, 13th edition, page 381, as a naturally occurring sterol found in animal fats. Cholesterol is given by formula (C 27 H 46 It contains (O) and is also called (3β)-cholesta-5-en-3-ol.

[0135] The saponin:sterol ratio is typically around 1:100 to 1:1 (w / w), preferably between 1:10 and 1:1 (w / w), and more preferably between 1:5 and 1:1 (w / w). An excess of sterols is appropriate, and the saponin:sterol ratio is at least 1:2 (w / w). In one embodiment, the saponin:sterol ratio is 1:5 (w / w). In one embodiment, the sterol is cholesterol.

[0136] The amount of liposomes (weight of lipids and sterols) is typically in the range of 0.1 mg to 10 mg per human dose of the composition, and more specifically, 0.5 mg to 2 mg per human dose of the composition.

[0137] In particularly suitable embodiments, the liposomes used in the present invention contain DOPC and sterols, especially cholesterol. Therefore, in certain embodiments, the composition used in the present invention comprises a saponin extract in the form of liposomes, the liposomes containing DOPC and sterols, especially cholesterol.

[0138] antigen The adjuvants prepared according to the present invention can be used in combination with immunogens or antigens. In some embodiments, polynucleotides encoding immunogens or antigens are provided.

[0139] The adjuvant may be administered separately from the immunogen or antigen, or it may be combined with the immunogen or antigen as an immunogenic composition for co-administration during or in the process of manufacture.

[0140] Therefore, a method is provided for preparing an immunogenic composition comprising an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen, and the method is as follows: (i) A step of preparing an adjuvant composition containing the saponin extract of the present invention, (ii) The step of mixing an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen, with an adjuvant. include.

[0141] Furthermore, the use of an adjuvant containing the saponin extract of the present invention in the manufacture of pharmaceuticals is also provided. The pharmaceuticals may preferably contain an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

[0142] Furthermore, an adjuvant containing the saponin extract of the present invention for use as a pharmaceutical is provided. The pharmaceutical may preferably contain an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

[0143] The term immunogen refers to a polypeptide capable of eliciting an immune response. An immunogen is appropriately an antigen containing at least one B or T cell epitope. The elicited immune response may be an antigen-specific B cell response producing neutralizing antibodies. The elicited immune response may also be an antigen-specific T cell response, which may be systemic and / or local. An antigen-specific T cell response may include a CD4+ T cell response, for example, a response involving CD4+ T cells expressing multiple cytokines, such as IFN-gamma, TNF-alpha, and / or IL-2. Alternatively, or in addition, an antigen-specific T cell response may include a CD8+ T cell response, for example, a response involving CD8+ T cells expressing multiple cytokines, such as IFN-gamma, TNF-alpha, and / or IL-2.

[0144] Antigens may be derived from (for example, obtained from) human or non-human pathogens, such as bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates, or cancer cells or tumor cells.

[0145] In one embodiment, the antigen is a recombinant protein, such as a recombinant prokaryotic protein.

[0146] In one embodiment, the antigen is derived from a malaria parasite (Plasmodium spp.) (e.g., Plasmodium falciparum), a species of the genus Mycobacterium (e.g., Mycobacterium tuberculosis (TB)), varicella-zoster virus (VZV), human respiratory multinucleated virus, human immunodeficiency virus (HIV), a species of the genus Moraxella (e.g., Moraxella catarrhalis), or nontypable Haemophilus influenzae (ntHi).

[0147] The antigen may include, or may consist of, preparations derived from malaria-causing parasites, such as Plasmodium falciparum or Plasmodium vivax.

[0148] In one embodiment, the antigen may be the parasporozoite perisporozoite (CS) protein of Plasmodium falciparum or a variant thereof. A suitable variant of the CS protein may be a variant in which a portion of the CS protein is in the form of a hybrid protein with the hepatitis B surface antigen S (HBsAg). The CS variant antigen may be in the form of a hybrid protein containing, for example, substantially the entire C-terminal portion of the CS protein, four or more tandem repeat sequences of the immunodominant region of the CS protein, and HBsAg. The hybrid protein may contain at least 160 amino acids and may include a sequence that is substantially homologous to the C-terminal portion of the CS protein but lacks a hydrophobic anchor sequence. The CS protein may also lack the last 12 amino acids of the C-terminus. Furthermore, it may contain four or more, for example, ten or more Asn-Ala-Asn-Pro tetrapeptide (NANP) repeat motifs.

[0149] The hybrid protein used in this invention may be a protein containing a portion of the Plasmodium falciparum CS protein substantially corresponding to amino acids 207-395 of Plasmodium falciparum clone 3D7 from strain NF54, fused in-frame to the N-terminus of HBsAg via a linear linker. The linker may contain a portion of preS2 derived from HBsAg. Suitable CS constructs for use in this invention are outlined in International Publication No. 93 / 10152, which was patented in the United States as U.S. Patents No. 5,928,902 and No. 6,169,171 (both incorporated by reference for the purpose of illustrating suitable proteins for use in this invention).

[0150] The specific hybrid protein used in this invention is a hybrid protein known as RTS (SEQ ID NO: 1, International Publication No. 2015 / 150568, International Publication No. 93 / 10152 (RTS) * This is stated as (and is also mentioned in International Publication No. 98 / 05355), - Methionine residue - 3 amino acid residues Met Ala Pro - A sequence of 189 amino acids representing amino acids 207-395 of the CS protein of Plasmodium falciparum 3D7 strain. - Glycine residue - The four amino acid residues Pro VaI Thr Asn, which represent the four carboxy-terminal residues of the hepatitis B virus (adw serotype) preS2 protein, and - Encoded by nucleotides 1653-2330, a sequence of 226 amino acids that defines the S protein of the hepatitis B virus (ADW serotype). It consists of.

[0151] RTS can be in the form of RTS,S mixed particles. RTS,S particles contain two polypeptides, RTS and S, which can be synthesized simultaneously and spontaneously form a composite particulate structure (RTS,S).

[0152] The antigen may include, or consist of, a species of the genus Mycobacterium, such as Mycobacterium bovis or Mycobacterium tuberculosis, particularly preparations derived from Mycobacterium tuberculosis.

[0153] Rv1196 and Rv0125 are antigens of interest in the field of tuberculosis. Rv1196 (for example, described as Mtb39a in Dillon et al., Infection and Immunity, 1999, 67(6): 2941~2950) is highly conserved with 100% sequence identity across H37Rv, C, Haarlem, CDC1551, 94-M4241A, 98-R604INH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506 strains, and F11 strain with a single point mutation Q30K (most other clinical isolates have more than 90% identity with H37Rv). Rv0125 (for example, described as Mtb32a in Skeiky et al., Infection and Immunity, 1999, 67(8): 3998~4007) is also highly conserved with 100% sequence identity across many strains. The full-length Rv0125 contains an N-terminal signal sequence, which is cleaved to produce a mature protein.

[0154] In one embodiment, the antigen is derived from Rv1196 and comprises, for example, a sequence having at least 70% identity with, for example, SEQ ID NO: 2, for example, at least 80%, particularly at least 90%, especially at least 95%, for example at least 98%, for example at least 99% identity. Typical Rv1196-associated antigens include (for example, comprise) derivatives of SEQ ID NO: 2 having a small number of deletions, insertions and / or substitutions. Examples include deletions of up to 5 residues at positions 0-5, insertions of up to 5 residues at positions 0-5, and substitutions of up to 20 residues. Other derivatives of Rv1196 include (for example, comprise) fragments of SEQ ID NO: 2 having a length of at least 200 amino acids, for example at least 250 amino acids, particularly at least 300 amino acids, especially at least 350 amino acids.

[0155] In one embodiment, the antigen is derived from Rv0125 and comprises, for example, a sequence having at least 70% identity with, for example, SEQ ID NO: 3, e.g., at least 80%, particularly at least 90%, especially at least 95%, e.g., at least 98%, e.g., at least 99% identity. Typical Rv0125-associated antigens comprise (for example, comprise) derivatives of SEQ ID NO: 3 having a small number of deletions, insertions, and / or substitutions. Examples include deletions of up to 5 residues at positions 0-5, insertions of up to 5 residues at 5 positions 0-5, and substitutions of up to 20 residues. Other derivatives of Rv0125 comprise (for example, comprise) fragments of SEQ ID NO: 3 having a length of at least 150 amino acids, e.g., at least 200 amino acids, particularly at least 250 amino acids, especially at least 300 amino acids. Specific derivatives of Rv0125 comprise (for example, comprise) fragments of SEQ ID NO: 3 corresponding to residues 1-195 of SEQ ID NO: 3. Further immunogenic derivatives of Rv0125 include (e.g., consist of) fragments of SEQ ID NO: 3 corresponding to residues 192-323 of SEQ ID NO: 3. Particularly preferred Rv0125-associated antigens are derivatives of SEQ ID NO: 3 in which at least one (e.g., one, two, or even all three) of the three catalytic residues is substituted or deleted, resulting in reduced protease activity and easier protein production, i.e., the catalytic serine residue may be deleted or substituted (e.g., substituted with alanine) and / or the catalytic histidine residue may be deleted or substituted, and the catalytic aspartic acid residue may be deleted or substituted. Derivatives of SEQ ID NO: 3 in which the catalytic serine residue is substituted (e.g., substituted with alanine) are of particular interest.Also of interest are Rv0125-related antigens comprising, for example, a fragment of SEQ ID NO: 3 having at least 70% identity, e.g., at least 80%, particularly at least 90%, especially at least 95%, e.g., at least 98%, e.g., at least 99%, and comprising a sequence in which at least one of the three catalytic residues is substituted or deleted, or a fragment of SEQ ID NO: 3 having a length of at least 150 amino acids, e.g., at least 200 amino acids, particularly at least 250 amino acids, especially at least 300 amino acids, and comprising, for example, a fragment in which at least one of the three catalytic residues is substituted or deleted. Further immunogenic derivatives of Rv0125 include (for example, comprise) a fragment of SEQ ID NO: 3 corresponding to residues 192-323 of SEQ ID NO: 3, in which at least one of the three catalytic residues (e.g., one, two, or even all three) is substituted or deleted. Certain immunogenic derivatives of Rv0125 include (e.g., consist of) a fragment of SEQ ID NO: 3 corresponding to residues 1-195 of SEQ ID NO: 3, wherein the catalyst serine residue (position 176 of SEQ ID NO: 3) is substituted (e.g., substituted with alanine).

[0156] The antigen appropriately comprises, for example, a sequence having at least 70% identity with SEQ ID NO: 4, e.g., at least 80%, particularly at least 90%, especially at least 95%, e.g., at least 98%, e.g., at least 99% identity. A typical M72-associated antigen comprises, for example, a derivative of SEQ ID NO: 4 having a small number of deletions, insertions, and / or substitutions. An example is having the deletion of up to 5 residues at positions 0-5, the insertion of up to 5 residues at 5 positions 0-5, and the substitution of up to 20 residues. Other derivatives of M72 comprise, for example, a fragment of SEQ ID NO: 4 having a length of at least 450 amino acids, e.g., at least 500 amino acids, e.g., at least 550 amino acids, e.g., at least 600 amino acids, e.g., at least 650 amino acids, or at least 700 amino acids. Since M72 is a fusion protein derived from two individual antigens, Rv0125 and Rv1196, at least one of the 450 residue fragments contains multiple epitopes derived from the full-length sequence (Skeiky et al., J. Immunol., 2004, 172:7618~7628; Skeiky, Infect. Immun., 1999, 67(8):3998~4007; Dillon, Infect. Immun., 1999, 67(6):2941~2950).

[0157] The M72-related antigen comprises, for example, a sequence having at least 70% identity with SEQ ID NO: 4, for example at least 80%, particularly at least 90%, especially at least 95%, for example at least 98%, for example at least 99% identity.

[0158] Typical M72-associated antigens include derivatives of SEQ ID NO: 4 with a small number of deletions, insertions, and / or substitutions, for example. Examples include deletions of up to 5 residues at positions 0-5, insertions of up to 5 residues at positions 0-5, and substitutions of up to 20 residues.

[0159] In certain embodiments, the M72-related antigen comprises residues 2-723 of SEQ ID NO: 4, for example, SEQ ID NO: 4, or SEQ ID NO: 5.

[0160] Further antigens that can be used in accordance with the present invention include, for example, the tuberculosis antigen Rv1753 and its variants described in International Publication No. 2010010180, such as Rv1753 sequences selected from SEQ ID NOs. 1 and 2-7 of International Publication No. 2010010180, particularly SEQ ID NO. 1. Another antigen of interest in the field of tuberculosis is, for example, Rv2386 and its variants described in International Publication No. 2010010179, such as Rv2386 sequences selected from SEQ ID NOs. 1 and 2-7 of International Publication No. 2010010179, particularly SEQ ID NO. 1. Other antigens of interest in the field of tuberculosis include, for example, Rv3616 and its variants described in International Publication No. 2011092253, natural Rv3616 sequences or modified Rv3616 sequences selected from, for example, SEQ ID NOs. 1 and 2-7 of International Publication No. 2011092253, such as selected from SEQ ID NOs. 161-169, 179 and 180 of International Publication No. 2011092253, particularly SEQ ID NO. 167. Additional antigens of interest are HBHA, as described in International Publication Nos. 97044463, 03044048 and 2010149657. The aforementioned International Patent Publications No. 2010010180, 2010010179, 2011092253, 97044463, 03044048, and 2010149657 are incorporated herein by reference in their entirety for the purpose of defining antigens that may be useful in the present invention.

[0161] Other antigens of interest include Rv1174, also known as DPV, as described in SEQ ID NO. 8 of International Publication No. 2010010177; Rv1793, also known as MTI or Mtb9.9, as described in SEQ ID NO. 10 of International Publication No. 2010010177; Rv2087, also known as MSL or Mtb9.8, as described in SEQ ID NO. 9 of International Publication No. 2010010177; and HTCC1 or Mtb4, as described in SEQ ID NOs. 1 and 2-7 of International Publication No. 2010010177 or SEQ ID NOs. 161-169, 179, or 180 of International Publication No. 2011092253. Rv3616, also known as 0, and / or Rv3874, also known as CFP10 or Tb38.1, as described in Sequence ID No. 9 of International Publication No. 2010010177, or comprising (or consisting of) an immunogenic portion (e.g., at least 20, 50, 75, or 100 residues derived therefrom) or a variant thereof (e.g., having at least 70%, 80%, 90%, or 95% identity thereto) (International Publication Nos. 2010010177 and International Publication Nos. 2011092253 are incorporated herein by reference in their entirety for the purpose of defining antigens that may be useful in the present invention).

[0162] Tuberculosis antigens are most appropriately utilized in polypeptide form, but they may instead be provided in the form of polynucleotides encoding the polypeptides.

[0163] Further antigens that can be used in accordance with the present invention are derived from varicella-zoster virus (VZV). The VZV antigen used in the present invention may be any suitable VZV antigen or its immunogenic derivative, and purified VZV antigen is suitable.

[0164] In one embodiment, the VZV antigen is the VZV glycoprotein gE (also known as gp1) or an immunogenic derivative thereof. The wild-type or full-length gE protein consists of 623 amino acids, including a signal peptide, a major protein segment, a hydrophobic anchor region (residues 546-558), and a C-terminal tail. In one embodiment, the C-terminal truncated form of gE (also known as truncated gE or gE truncated form) is used, where truncation removes 4-20 percent of the total amino acid residues from the carboxyl terminus. In a further embodiment, the truncated gE lacks the carboxyl-terminal anchor region (approximately amino acids 547-623 of the wild-type sequence is suitable). In a further embodiment, gE is truncated gE having the sequence of Sequence ID No. 6.

[0165] gE antigen, its unanchored derivatives (which are also immunogenic derivatives), and their manufacture are described in European Patent No. 0405867 and its references (see also Vafai A., Antibody binding sites on truncated forms of varicalla-zoster virus gpI(gE) glycoprotein, Vaccine, 1994, 12:1265-9). European Patent No. 192902 also describes gE and its manufacture. Truncated gE is also described in Haumont et al., Virus Research, (1996), Vol. 40, pp. 199-204, which is incorporated herein by reference in its entirety. Adjuvanted VZV gE compositions suitable for use in accordance with the present invention, namely, carboxy-terminated VZV gE combined with adjuvants further containing cholesterol, QS-21, 3D-MPL, and liposomes, are described in International Publication No. 2006 / 094756. Leroux-Roels I. et al. (J. Infect. Dis., 2012, 206: 1280~1290) reported on a Phase I / II clinical trial evaluating an adjuvant-enhanced VZV truncated gE subunit vaccine.

[0166] The antigen may include or consist of preparations derived from human respiratory multinuclear virus (RSV). In some advantageous embodiments, the polypeptide antigen is an RSV-derived F protein polypeptide antigen. Particularly suitable as the polypeptide antigen component in the present invention is a conformationally restricted F polypeptide antigen. Conformationally restricted F proteins have been previously described for both prefusion (PreF) and postfusion (PostF) conformations. Such conformationally restricted F proteins typically comprise an engineered RSV F protein extracellular domain. The F protein extracellular domain polypeptide comprises all or part of the extracellular domain of the RSV F protein and is a portion of the RSV F protein lacking a functional transmembrane domain (e.g., by deletion or substitution), and can be expressed, for example, in a soluble (non-membrane-attached) form in cell culture.

[0167] Representative conformationally restricted F protein antigens in the pre-fusion conformation have already been described in the Art and are disclosed in detail, for example, in U.S. Patent No. 8,563,002 (International Publication No. 2009079796), U.S. Patent Application Publication No. 2012 / 0093847 (International Publication No. 2010 / 149745), U.S. Patent Application Publication No. 2011 / 0305727 (International Publication No. 2011 / 008974), U.S. Patent Application Publication No. 2014 / 0141037, International Publication No. 2012 / 158613, and International Publication No. 2014 / 160463, each incorporated herein by reference for the purpose of describing the pre-fusion F polypeptide (and nucleic acid) and the method for producing the same. Typically, the antigen is in the form of a polypeptide trimer. Additional publications providing examples of F proteins in pre-fusion conformations include McLellan et al., Science, 340: 1113-1117; McLellan et al., Science, 342: 592-598; and Rigter et al., PLOS One, 8: e71072, which can also be used in relation to the immunogenicity combinations disclosed herein.

[0168] For example, a pre-fusion conformationally stabilized F protein polypeptide typically includes an external domain of the F protein (e.g., a soluble F protein polypeptide) that includes at least one modification that stabilizes the pre-fusion conformation of the F protein. For example, the modification can be selected from the addition of a trimerizing domain (usually to the C-terminus), the deletion of one or more furin cleavage sites (at approximately amino acids 105-109 and approximately 133-136), the deletion of the pep27 domain, or the substitution or addition of hydrophilic amino acids in a hydrophobic domain (e.g., HRA and / or HRB). In one embodiment, a conformationally restricted PreF antigen includes the F2 domain (e.g., amino acids 1-105) and F1 domain (e.g., amino acids 137-516) of an RSV F protein polypeptide that does not involve a furin cleavage site, and the polypeptide further includes a heterologous trimerizing domain located C-terminal to the F1 domain. In some cases, PreF antigens include modifications that alter glycosylation (e.g., increase glycosylation), such as the substitution of one or more amino acids at positions corresponding to approximately 500-502 amino acids in the RSV F protein. Trimerized sequences are preferred if oligomerized sequences are available. Suitable oligomerized sequences are well known in the art and include, for example, the coiled-coil of the yeast GCN4 leucine zipper protein, the trimerized sequence from bacteriophage T4 fibrin ("foldon"), and the trimer domain of influenza HA. ​​In addition, or alternatively, the conformationally restricted F polypeptide in the pre-fusion conformation may contain at least two introduced cysteine ​​residues that are close to each other and form a disulfide bond that stabilizes the pre-fusion RSV F polypeptide. For example, the two cysteines may be within approximately 10 Å of each other. For example, the cysteines may be introduced at positions 165 and 296 or positions 155 and 290. A representative PreF antigen is represented by SEQ ID NO: 7.

[0169] The antigen may include, or consist of, preparations derived from HIV. The antigen may be an HIV protein, such as the HIV envelope protein. For example, the antigen may be the HIV envelope gp120 polypeptide or an immunogenic fragment thereof.

[0170] One suitable antigen is the HIV clade B gp120 polypeptide (or an immunogenic fragment of this polypeptide) of Sequence ID No. 8 of the published application, International Publication No. 2008 / 107370. Sequence ID No. 8 of International Publication No. 2008 / 107370 is incorporated into this application by reference.

[0171] Suitable antigens also include polypeptides containing the V1V2 region of SEQ ID NO: 1 of the published application, International Publication No. 2015 / 036061, or immunogenic derivatives or fragments of the V1V2 region of SEQ ID NO: 1. In addition, polypeptides containing the V1V2 region of SEQ ID NO: 5 of International Publication No. 2015 / 036061, or immunogenic derivatives or fragments of the V1V2 region of SEQ ID NO: 5, may be used as suitable antigens. SEQ ID NOs: 1 and 5 of International Publication No. 2015 / 036061 are incorporated by reference.

[0172] In another embodiment, the antigen may comprise two or more different HIV envelope gp120 polypeptide antigens (or immunogenic fragments of these polypeptides). Suitable antigens include HIV clade C gp120 polypeptide antigens, including TV1 gp120 (SEQ ID NO: 8) and 1086.C gp120 (SEQ ID NO: 9).

[0173] Other suitable HIV antigens include the Nef, Gag, and Pol HIV proteins, as well as their immunogenic fragments.

[0174] The compositions include, for example, fusions containing fimbrin protein [(U.S. Patent No. 5,766,608, Ohio State Research Foundation)] and peptides derived therefrom [e.g., LB1(f) peptide fusion, U.S. Patent No. 5,843,464 (OSU) or International Publication No. 99 / 64067], OMP26 [International Publication No. 97 / 01638 (Cortecs)], and P6 [European Patent No. 281673 (State University of New York)]. It may contain an unencapsulated Haemophilus influenzae antigen selected from (York), TbpA and / or TbpB, Hia, Hsf, Hin47, Hif, Hmw1, Hmw2, Hmw3, Hmw4, Hap, D15 (International Publication No. 94 / 12641), Protein D (European Patent No. 594610), P2, and P5 (International Publication No. 94 / 26304), Protein E (International Publication No. 07 / 084053), and / or PilA (International Publication No. 05 / 063802).The compositions include, for example, OMP106 [International Publication No. 97 / 41731 (Antex) and International Publication No. 96 / 34960 (PMC)], OMP21, LbpA and / or LbpB [International Publication No. 98 / 55606 (PMC)], TbpA and / or TbpB [International Publication No. 97 / 13785 and International Publication No. 97 / 32980 (PMC)], CopB [Helminen ME et al., (1993), Infect. Immun., 61:2003~2010], UspA1 and / or UspA2 [International Publication No. 93 / 03761 (University of Texas), OmpCD, HasR (International application PCT / EP99 / 03824), PilQ (International application PCT / EP99 / 03823), OMP85 (International application PCT / EP00 / 01468), lipo06 (UK Patent Application No. 9917977.2), lipo10 (UK Patent Application No. 9918208.1), lipo11 (UK Patent Application No. 9918302.2), It may also contain Moraxella catarrhalis protein antigens selected from lipo18 (UK Patent Application No. 9918038.2), P6 (International Application PCT / EP99 / 03038), D15 (International Application PCT / EP99 / 03822), OmplA1 (International Application PCT / EP99 / 06781), Hly3 (International Application PCT / EP99 / 03257), and OmpE.

[0175] In one embodiment, the composition may contain the protein antigen of non-encapsulated Haemophilus influenzae (NTHi) and / or the protein antigen of Moraxella catarrhalis. The composition may also contain protein D (PD) derived from Haemophilus influenzae. Protein D may be that described in International Publication No. 91 / 18926. The composition may further contain protein E (PE) and / or pyrin A (PilA) derived from Haemophilus influenzae. Protein E and pyrin A may be those described in International Publication No. 2012 / 139225. Protein E and pyrin A may be presented as a fusion protein, for example, LVL735 as described in International Publication No. 2012 / 139225. For example, the composition may contain three NTHi antigens (PD, PE, and PilA, the latter two of which are combined as a PEPilA fusion protein). The composition may further contain UspA2 derived from Moraxella catarrhalis. UspA2 may be the one described in International Publication No. 2015125118, for example, MC-009((M)(UspA2 31-564)(HH)) described in International Publication No. 2015125118. For example, the composition may contain three NTHi antigens (PD, PE, and PilA, the latter two of which are combined as a PEPilA fusion protein) and one Moraxella catarrhalis antigen (UspA2).

[0176] Multiple antigens may be provided. For example, multiple antigens may be provided to enhance the immune response induced (e.g., to ensure robust protection), to expand the immune response (e.g., to ensure protection against a wide range of pathogenic strains or in a large portion of the target population), or to induce a broad immune response against several disorders (therefore simplifying the administration protocol). If multiple antigens are provided, they may be distinct proteins or in the form of one or more fusion proteins.

[0177] The antigen may be supplied in amounts of 0.1 to 100 ug per human dose.

[0178] The present invention may be applied to use in the treatment or prevention of diseases or disorders associated with one or more of the above-mentioned antigens. In one embodiment, the disease or disorder is selected from malaria, tuberculosis, COPD, HIV, and herpes.

[0179] The adjuvant may be administered separately from the immunogen or antigen, or it may be combined with the immunogen or antigen as an immunogenic composition for co-administration during or in the process of manufacture.

[0180] Sterilization (sterilization) Especially in the case of parenteral administration, the composition must be sterile. Sterilization can be carried out by various methods, but filtration through a sterile-grade filter is convenient. Sterilization may be carried out multiple times during the preparation of the adjuvant or immunogenic composition, but is usually done at least at the end of manufacturing.

[0181] A "sterile grade filter" refers to a filter with an effective filtration area of ​​1 cm². 2 1x10 7 This refers to a filter that produces a sterile effluent after microbial loading at the above loading levels. Sterile grade filters are well known to those skilled in the art for the purposes of the present invention, and sterile grade filters have pore sizes between 0.15 and 0.25 μm, preferably between 0.18 and 0.22 μm, for example, 0.2 or 0.22 μm.

[0182] The membranes of sterile-grade filters can be made from any suitable material known to those skilled in the art, for example, but not limited to, cellulose acetate, polyethersulfone (PES), polyvinylidene fluoride (PVDF), and polytetrafluoroethylene (PTFE). In certain embodiments of the present invention, one or more or all of the filter membranes of the present invention comprise polyethersulfone (PES), particularly hydrophilic polyethersulfone. In certain embodiments of the present invention, the filters used in the methods described herein are double-layer filters, particularly sterile filters having a built-in pre-filter having a pore diameter larger than that of the end filter. In one embodiment, the sterile filter is a double-layer filter in which the pre-filter membrane layer has a pore diameter between 0.3 and 0.5 nm, for example, 0.35 or 0.45 nm. According to further embodiments, the filter comprises an asymmetric filter membrane, for example, an asymmetric hydrophilic PES filter membrane. Alternatively, the sterile filter layer may be made of PVDF, for example, in combination with an asymmetric hydrophilic PES pre-filter membrane layer.

[0183] Considering the intended medical use, the material should be pharmaceutical grade (e.g., parenteral grade).

[0184] All teachings in the literature, including pending patent applications and granted patents, are incorporated herein in full by reference. A composition, method, or process defined as "containing" certain elements is understood to encompass (each) a composition, method, or process comprising those elements. As used herein, "essentially consisting of" means that additional components may be present, provided they do not alter the overall nature or function.

[0185] The present invention will be further described with reference to the following non-limiting embodiments. [Examples]

[0186] [Example 1] HPLC of crude aqueous extract of Quillaja saponaria molina Crude bark extracts were separated by reverse-phase HPLC using a C4 column and gradient elution: mobile phase A - water / acetonitrile, 7 / 3 v / v, 0.15% trifluoroacetic acid; mobile phase B - acetonitrile, 0.15% trifluoroacetic acid. UV detection was performed at 214 nm.

[0187] The crude bark extract sample was diluted with purified water as needed. PVPP (60 mg / mL) was added, the mixture was stirred for approximately 30 minutes, and then the PVPP resin was separated from the supernatant by centrifugation.

[0188] Next, the supernatant was analyzed to obtain an HPLC UV chromatogram.

[0189] Figure 1 shows a representative example of an HPLC UV chromatogram. The peak corresponding to the QS-21 fraction is shown.

[0190] [Example 2] Analysis method HPLC-UV device Waters Alliance 2690 / 2695 Isolation Module Waters 2487 UV Detector or 2996 PDA Detector Vydac Protein C4 4.6×250mm 5um column Mobile phase A (MPA) - 0.15% trifluoroacetic acid in water / acetonitrile (70:30 v / v) Mobile phase B (MPB) - 0.15% trifluoroacetic acid in acetonitrile

[0191] [Table 1]

[0192] Inject 10 µl of sample. Set UV detection to 214 nM.

[0193] For reference, blank injection is used and the integration of the peaks in the chromatogram provides the total absorbance. The peak content is determined as a percentage by comparing the peak of interest (e.g., the QS-21 main peak) with the total absorbance.

[0194] UPLC-UV Instrument Waters Acquity UPLC Waters Acquity Tunable UV Detector Waters Acquity BEH C18 2.1×100mm 1.7um column Mobile phase A (MPA) - 0.025% acetic acid in water / acetonitrile (70:30 v / v) Mobile phase B (MPB) - 0.025% trifluoroacetic acid in water / acetonitrile (30:70 v / v)

[0195]

Table 2

[0196] The column temperature is 28°C. 10 μl of the sample is injected. UV detection is set at 214 nM.

[0197] For reference, blank injection is used and the integration of the peaks in the chromatogram provides the total absorbance. The peak content is determined as a percentage by comparing the peak of interest (e.g., the QS-21 main peak) with the total absorbance.

[0198] UPLC-UV / MS Instrument Waters Acquity UPLC Waters Acquity Tunable UV Detector Waters Single-Quadrupole Mass Detector Waters Acquity BEH C18 2.1×100mm 1.7um column Mobile Phase A (MPA) - 0.025% trifluoroacetic acid in water / acetonitrile / isopropyl alcohol (75:20:5 v / v) Mobile Phase B (MPB) - 0.025% trifluoroacetic acid in water / acetonitrile / isopropyl alcohol (10:72:18 v / v)

[0199]

Table 3

[0200] The test sample is prepared as 0.2% acetic acid in water / acetonitrile (70:30 v / v). The column temperature is 55 °C. 10 μl of the sample is injected. UV detection is set at 214 nM.

[0201] The retention times vary slightly between runs, but the QS-21 group is located approximately at 3.8 minutes (B-isomer) to approximately 4.5 minutes (before the lyo impurity).

[0202] Using a reference blank injection, integrate the chromatogram peaks that elute between 0.5 minutes and approximately 5.50 minutes after the solvent front and are not seen in the blank.

[0203] Identify the monoisotope of the most abundant species by combining the TIC across the entire chromatogram to create a binding spectrum.

[0204] The ratio of the 2002 component to the 1988 component is calculated by comparing the ion current associated with the 2002 component to the ion current associated with the 1988 component within the QS-21 main peak.

[0205] Figure 5 shows a representative chromatogram of the saponin extract of the present invention. Figure 6 shows an enlarged detail of the region containing the QS-21 group and the lyo component.

[0206] Figure 7 shows a representative extracted mass chromatogram for the 1988 and 2002 molecular weight ions of the purified Quillaja saponaria Molina saponin extract.

[0207] [Example 3] Purification of crude aqueous extract of Quillaja saponaria molina Crude aqueous extracts of Quillaja saponaria molina having a ratio of 0.064 or less to the QS-21 main peak and a ratio of 0.4 or less to the preceding peak to the QS-21 main peak were treated with PVPP (1 kg of PVPP per liter of crude aqueous extract). After adsorption, the mixture was filtered to separate PVPP and bound impurities from the liquid.

[0208] Figure 2 shows an example of an HPLC-UV chromatogram for a crude aqueous extract of Quillaja saponaria molina (used to determine the ratio of the preceding peak to the QS-21 main peak).

[0209] Figure 3 shows an example of a ULC-UV chromatogram for a crude aqueous extract of Quillaja saponaria molina (used to determine the ratio of 2018 components to the QS-21 main peak).

[0210] The filtered solution was concentrated and further purified by ultrafiltration / dialysis filtration using a 30kD Hellicon membrane.

[0211] [Table 4]

[0212] The fractions were pooled to obtain a saponin extract purified with polystyrene having the following composition: %QS-21 main peak ≥ 18% (by HPLC). and 2018 component / QS-21 main peak ratio ≤ 0.054 (by ULC-UV).

[0213] Figure 4 shows an example of a ULC-UV chromatogram for a pool of saponin extracts purified with polystyrene.

[0214] [Table 5]

[0215] The fractions were pooled to obtain a phenyl-purified saponin extract having the following composition: %QS-21 group ≥ 98.5 %QS-21 main peak ≥ 94.5 %2018 component ≤ 2.7% Main peaks other than the QS-21 group ≤ 1% (by UPLC-UV / MS).

[0216] The combined phenyl-purified saponin extract was concentrated by capture and release using reverse-phase chromatography with a C8 resin (Lichroprep RP8) and the following conditions: Load onto a column conditioned with 24% acetonitrile and 0.20% acetic acid. Elute with 60% acetonitrile and 0.20% acetic acid. 11 cm column Load: 50 - 142 g per injection

[0217] The C8-concentrated saponin extract was subjected to solvent exchange using ultrafiltration / diafiltration and a Pellicon 1 kDa membrane to reduce the acetonitrile content to less than 21%.

[0218] The resulting solvent-exchanged saponin extract was then lyophilized in a single step to obtain the final product.

[0219] A series of process runs resulting in out-of-specification saponin extracts were found to be due to the use of crude bark extracts having an excessive 2018 component content.

[0220] When multiple runs according to the present invention were carried out starting from crude extracts having an appropriate 2018 component composition, the final product was within specifications in each case.

[0221] Using the process described in Example 3, it is possible to consistently provide purified saponin extracts of Quillaja saponaria molina that exhibit a chromatographic profile comparable to the chromatograms shown in Figures 5, 6, and 7, with clear content in terms of the QS-21 main peak and the 2018 component, for example, consistently having at least 93% of the QS-21 main peak and 0.25-3% of 2018.

[0222] [Example 4] Immune response Female C57BL6 mice (5 mice / group) aged 6-8 weeks were administered gE antigen twice at 14-day intervals using adjuvant system AS01, a liposomal formulation containing 3D-MPL and saponin extract prepared according to the present invention. Five control mice were administered gE along with a buffer only.

[0223] Vaccines were prepared from saponin extracts from three different lots. Results are provided for two different dose levels (0.4 and 0.1 ug of 3D-MPL / QS-21 per animal, corresponding to 1 / 125 and 1 / 500 of the human dose, respectively).

[0224] Spleen samples were collected on D21, and serum samples were collected on D21 and D28. T and B cell responses were then analyzed for each sample.

[0225] - ICS (Intracellular Cytokine Staining) The spleen was collected in RPMI medium and dissociated using a Potter-type tissue homogenizer with two back-and-forth strokes. The homogenized sample was transferred to a 50 ml polypropylene tube. Fibrous material was removed by filtration through a 100 μM nylon cell strainer. The cells were then washed and counted, and 10 7 The cells were resuspended in 1 ml.

[0226] ICS is a scientific technology that enables the quantification of antigen-specific T lymphocytes based on cytokine production.

[0227] Lymphocyte-like cells are re-stimulated in vitro overnight (ON) with peptide gE or culture medium in the presence of a protein transport inhibitor (brefeldin A). These cells are then treated with conventional immunofluorescence methods using fluorescent antibodies (extracellular staining: CD4, CD8; intracellular staining: TNF-alpha, IFN-gamma, and IL2).

[0228] The results are expressed as the frequency of cytokine-positive cells within the CD4 cell population after deducting culture medium conditions for each mouse. Data are shown for populations that expressed at least two cytokines (IL2, IFN-alpha, or TNF-alpha). The results are shown in Figure 9.

[0229] - ELISA Anti-gE total IgG was measured by ELISA. Antigen was inoculated into a 96-well plate overnight at 4°C. The plate was then washed and saturated with a saturated buffer at 37°C for 1 hour. Subsequently, 100 μl of diluted mouse serum or a standard or control was added and incubated at 37°C for 1 hour and 30 minutes. After washing, the plate was incubated with 100 μl of biotinylated anti-mouse IgG at 37°C for 1 hour. After washing, the plate was incubated with 100 μl of streptavidin-POD conjugate at 37°C for 30 minutes. After washing, 100 μl of TMB / well was added and the plate was kept in the dark at room temperature for 15 minutes. To stop the reaction, 100 μl of H2SO4 0.4N was added per well. Absorbance was read at wavelengths of 450 / 630 nm using an Elisa plate reader. The results were calculated using Softmax-Pro software. The results are shown in Figures 10 (D21) and 11 (D28).

[0230] conclusion Consistent composition across different production lots of saponin extract resulted in immunological responses with limited variability.

[0231] List of References TIFF2026102610000054.tif21165TIFF2026102610000055.tif244167TIFF2026102610000056.tif84166

[0232] [Sequence Listing] SEQUENCE LISTING <110> GlaxoSmithKline Biologicals S.A. <120> Saponin purification <130> PA25-552 <150> US 62 / 593,581 <151> 2017-12-01 <150> EP 17209780.0 <151> 2017-12-21 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 424 <212> PRT <213> Artificial <220> <223> RTS <400> 1 Met Met Ala Pro Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala 1 5 10 15 Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala 20 25 30 Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala 35 40 45 Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala 50 55 60 Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Lys 65 70 75 80 Asn Asn Gln Gly Asn Gly Gln Gly His Asn Met Pro Asn Asp Pro Asn 85 90 95 Arg Asn Val Asp Glu Asn Ala Asn Ala Asn Ser Ala Val Lys Asn Asn 100 105 110 Asn Asn Glu Glu Pro Ser Asp Lys His Ile Lys Glu Tyr Leu Asn Lys 115 120 125 Ile Gln Asn Ser Leu Ser Thr Glu Trp Ser Pro Cys Ser Val Thr Cys 130 135 140 Gly Asn Gly Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asn Lys Pro 145 150 155 160 Lys Asp Glu Leu Asp Tyr Ala Asn Asp Ile Glu Lys Lys Ile Cys Lys 165 170 175 Met Glu Lys Cys Ser Ser Val Phe Asn Val Val Asn Ser Ser Ile Gly 180 185 190 Leu Gly Pro Val Thr Asn Met Glu Asn Ile Thr Ser Gly Phe Leu Gly 195 200 205 Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu 210 215 220 Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu 225 230 235 240 Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser 245 250 255 Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp 260 265 270 Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys 275 280 285 Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val 290 295 300 Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Asn Thr Gly Pro Cys Lys 305 310 315 320 Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys 325 330 335 Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser 340 345 350 Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe 355 360 365 Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu 370 375 380 Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp Gly 385 390 395 400 Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro Ile 405 410 415 Phe Phe Cys Leu Trp Val Tyr Ile 420 <210> 2 <211> 391 <212> PRT <213> Mycobacterium tuberculosis <400> 2 Met Val Asp Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg Met 1 5 10 15 Tyr Ala Gly Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Gln Met Trp 20 25 30 Asp Ser Val Ala Ser Asp Leu Phe Ser Ala Ala Ser Ala Phe Gln Ser 35 40 45 Val Val Trp Gly Leu Thr Val Gly Ser Trp Ile Gly Ser Ser Ala Gly 50 55 60 Leu Met Val Ala Ala Ala Ser Pro Tyr Val Ala Trp Met Ser Val Thr 65 70 75 80 Ala Gly Gln Ala Glu Leu Thr Ala Ala Gln Val Arg Val Ala Ala Ala 85 90 95 Ala Tyr Glu Thr Ala Tyr Gly Leu Thr Val Pro Pro Pro Val Ile Ala 100 105 110 Glu Asn Arg Ala Glu Leu Met Ile Leu Ile Ala Thr Asn Leu Leu Gly 115 120 125 Gln Asn Thr Pro Ala Ile Ala Val Asn Glu Ala Glu Tyr Gly Glu Met 130 135 140 Trp Ala Gln Asp Ala Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala 145 150 155 160 Thr Ala Thr Ala Thr Leu Leu Pro Phe Glu Glu Ala Pro Glu Met Thr 165 170 175 Ser Ala Gly Gly Leu Leu Glu Gln Ala Ala Ala Val Glu Glu Ala Ser 180 185 190 Asp Thr Ala Ala Ala Asn Gln Leu Met Asn Asn Val Pro Gln Ala Leu 195 200 205 Gln Gln Leu Ala Gln Pro Thr Gln Gly Thr Thr Pro Ser Ser Lys Leu 210 215 220 Gly Gly Leu Trp Lys Thr Val Ser Pro His Arg Ser Pro Ile Ser Asn 225 230 235 240 Met Val Ser Met Ala Asn Asn His Met Ser Met Thr Asn Ser Gly Val 245 250 255 Ser Met Thr Asn Thr Leu Ser Ser Met Leu Lys Gly Phe Ala Pro Ala 260 265 270 Ala Ala Ala Gln Ala Val Gln Thr Ala Ala Gln Asn Gly Val Arg Ala 275 280 285 Met Ser Ser Leu Gly Ser Ser Leu Gly Ser Ser Gly Leu Gly Gly Gly 290 295 300 Val Ala Ala Asn Leu Gly Arg Ala Ala Ser Val Gly Ser Leu Ser Val 305 310 315 320 Pro Gln Ala Trp Ala Ala Ala Asn Gln Ala Val Thr Pro Ala Ala Arg 325 330 335 Ala Leu Pro Leu Thr Ser Leu Thr Ser Ala Ala Glu Arg Gly Pro Gly 340 345 350 Gln Met Leu Gly Gly Leu Pro Val Gly Gln Met Gly Ala Arg Ala Gly 355 360 365 Gly Gly Leu Ser Gly Val Leu Arg Val Pro Pro Arg Pro Tyr Val Met 370 375 380 Pro His Ser Pro Ala Ala Gly 385 390 <210> 3 <211> 323 <212> PRT <213> Mycobacterium tuberculosis <400> 3 Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu 1 5 10 15 Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln Val Val 20 25 30 Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr 35 40 45 Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val 50 55 60 Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln 65 70 75 80 Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val Ala 85 90 95 Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly 100 105 110 Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly 115 120 125 Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu 130 135 140 Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 145 150 155 160 Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp Ser 165 170 175 Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr 180 185 190 Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly Phe Ala 195 200 205 Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg Ser Gly 210 215 220 Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu Gly Leu 225 230 235 240 Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg Val Val 245 250 255 Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp Val Ile 260 265 270 Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met Ala Asp 275 280 285 Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser Val Thr Trp Gln 290 295 300 Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala Glu Gly 305 310 315 320 Pro Pro Ala <210> 4 <211> 723 <212> PRT <213> Artificial <220> <223> M72 <400> 4 Met Thr Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly 1 5 10 15 Phe Ala Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg 20 25 30 Ser Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu 35 40 45 Gly Leu Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg 50 55 60 Val Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp 65 70 75 80 Val Ile Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met 85 90 95 Ala Asp Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser Val Thr 100 105 110 Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala 115 120 125 Glu Gly Pro Pro Ala Glu Phe Met Val Asp Phe Gly Ala Leu Pro Pro 130 135 140 Glu Ile Asn Ser Ala Arg Met Tyr Ala Gly Pro Gly Ser Ala Ser Leu 145 150 155 160 Val Ala Ala Ala Gln Met Trp Asp Ser Val Ala Ser Asp Leu Phe Ser 165 170 175 Ala Ala Ser Ala Phe Gln Ser Val Val Trp Gly Leu Thr Val Gly Ser 180 185 190 Trp Ile Gly Ser Ser Ala Gly Leu Met Val Ala Ala Ala Ser Pro Tyr 195 200 205 Val Ala Trp Met Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala Ala 210 215 220 Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala Tyr Gly Leu Thr 225 230 235 240 Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile Leu 245 250 255 Ile Ala Thr Asn Leu Leu Gly Gln Asn Thr Pro Ala Ile Ala Val Asn 260 265 270 Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala Ala Ala Met Phe 275 280 285 Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro Phe 290 295 300 Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly Leu Leu Glu Gln Ala 305 310 315 320 Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu Met 325 330 335 Asn Asn Val Pro Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr Gln Gly 340 345 350 Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp Lys Thr Val Ser Pro 355 360 365 His Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn His Met 370 375 380 Ser Met Thr Asn Ser Gly Val Ser Met Thr Asn Thr Leu Ser Ser Met 385 390 395 400 Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln Thr Ala 405 410 415 Ala Gln Asn Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly 420 425 430 Ser Ser Gly Leu Gly Gly Gly Val Ala Ala Asn Leu Gly Arg Ala Ala 435 440 445 Ser Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn Gln 450 455 460 Ala Val Thr Pro Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu Thr Ser 465 470 475 480 Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly Leu Pro Val Gly 485 490 495 Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly Val Leu Arg Val 500 505 510 Pro Pro Arg Pro Tyr Val Met Pro His Ser Pro Ala Ala Gly Asp Ile 515 520 525 Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu 530 535 540 Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln Val Val 545 550 555 560 Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr 565 570 575 Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val 580 585 590 Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln 595 600 605 Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val Ala 610 615 620 Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly 625 630 635 640 Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly 645 650 655 Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu 660 665 670 Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 675 680 685 Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp Ala 690 695 700 Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr 705 710 715 720 Ala Ala Ser <210> 5 <211> 725 <212> PRT <213> Artificial <220> <223> M72-2His <400> 5 Met His His Thr Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly 1 5 10 15 Gln Gly Phe Ala Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln 20 25 30 Ile Arg Ser Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala 35 40 45 Phe Leu Gly Leu Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val 50 55 60 Gln Arg Val Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr 65 70 75 80 Gly Asp Val Ile Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr 85 90 95 Ala Met Ala Asp Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser 100 105 110 Val Thr Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr 115 120 125 Leu Ala Glu Gly Pro Pro Ala Glu Phe Met Val Asp Phe Gly Ala Leu 130 135 140 Pro Pro Glu Ile Asn Ser Ala Arg Met Tyr Ala Gly Pro Gly Ser Ala 145 150 155 160 Ser Leu Val Ala Ala Ala Gln Met Trp Asp Ser Val Ala Ser Asp Leu 165 170 175 Phe Ser Ala Ala Ser Ala Phe Gln Ser Val Val Trp Gly Leu Thr Val 180 185 190 Gly Ser Trp Ile Gly Ser Ser Ala Gly Leu Met Val Ala Ala Ala Ser 195 200 205 Pro Tyr Val Ala Trp Met Ser Val Thr Ala Gly Gln Ala Glu Leu Thr 210 215 220 Ala Ala Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala Tyr Gly 225 230 235 240 Leu Thr Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met 245 250 255 Ile Leu Ile Ala Thr Asn Leu Leu Gly Gln Asn Thr Pro Ala Ile Ala 260 265 270 Val Asn Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala Ala Ala 275 280 285 Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu 290 295 300 Pro Phe Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly Leu Leu Glu 305 310 315 320 Gln Ala Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln 325 330 335 Leu Met Asn Asn Val Pro Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr 340 345 350 Gln Gly Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp Lys Thr Val 355 360 365 Ser Pro His Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn 370 375 380 His Met Ser Met Thr Asn Ser Gly Val Ser Met Thr Asn Thr Leu Ser 385 390 395 400 Ser Met Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln 405 410 415 Thr Ala Ala Gln Asn Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser 420 425 430 Leu Gly Ser Ser Gly Leu Gly Gly Gly Val Ala Ala Asn Leu Gly Arg 435 440 445 Ala Ala Ser Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala 450 455 460 Asn Gln Ala Val Thr Pro Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu 465 470 475 480 Thr Ser Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly Leu Pro 485 490 495 Val Gly Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly Val Leu 500 505 510 Arg Val Pro Pro Arg Pro Tyr Val Met Pro His Ser Pro Ala Ala Gly 515 520 525 Asp Ile Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro 530 535 540 Ala Leu Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln 545 550 555 560 Val Val Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala 565 570 575 Gly Thr Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn 580 585 590 His Val Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser 595 600 605 Gly Gln Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp 610 615 620 Val Ala Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala 625 630 635 640 Ile Gly Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn 645 650 655 Ser Gly Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val 660 665 670 Ala Leu Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu 675 680 685 Glu Thr Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly 690 695 700 Asp Ala Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met 705 710 715 720 Asn Thr Ala Ala Ser 725 <210> 6 <211> 546 <212> PRT <213> Varicella zoster <400> 6 Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly 1 5 10 15 Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val 20 25 30 Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn 35 40 45 Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp 50 55 60 Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro 65 70 75 80 Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His 85 90 95 Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu 100 105 110 Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp 115 120 125 Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His 130 135 140 Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly 145 150 155 160 Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val 165 170 175 Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr 180 185 190 Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys 195 200 205 Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr 210 215 220 Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr 225 230 235 240 Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly Lys Lys 245 250 255 Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu Phe Asp 260 265 270 Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val 275 280 285 Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg 290 295 300 Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys 305 310 315 320 Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro 325 330 335 Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser 340 345 350 Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His 355 360 365 Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp 370 375 380 Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro 385 390 395 400 Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr 405 410 415 Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys 420 425 430 Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met 435 440 445 Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys 450 455 460 Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val 465 470 475 480 Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr 485 490 495 Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr 500 505 510 Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val 515 520 525 Asn Pro Gly Thr Ser Pro Leu Ile Arg Tyr Ala Ala Trp Thr Gly Gly 530 535 540 Leu Ala 545 <210> 7 <211> 514 <212> PRT <213> respiratory syncytial virus <400> 7 Met Glu Leu Leu Ile Leu Lys Thr Asn Ala Ile Thr Ala Ile Leu Ala 1 5 10 15 Ala Val Thr Leu Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Ser Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Lys Phe Leu Gly Phe Leu Gln 100 105 110 Gly Val Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu 115 120 125 His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr 130 135 140 Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser 145 150 155 160 Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile 165 170 175 Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu 180 185 190 Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser 195 200 205 Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn 210 215 220 Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln 225 230 235 240 Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr 245 250 255 Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln 260 265 270 Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr 275 280 285 Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu 290 295 300 Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser 305 310 315 320 Phe Phe Pro Leu Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe 325 330 335 Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys 340 345 350 Asn Ile Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser 355 360 365 Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val 370 375 380 Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly 385 390 395 400 Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly 405 410 415 Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln 420 425 430 Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr 435 440 445 Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln 450 455 460 Val Asn Glu Lys Ile Asn Gly Thr Leu Ala Phe Ile Arg Lys Ser Asp 465 470 475 480 Glu Lys Leu His Asn Val Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 485 490 495 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 500 505 510 Glu Ala <210> 8 <211> 488 <212> PRT <213> Human immunodeficiency virus <400> 8 Asn Thr Glu Asp Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 1 5 10 15 Arg Asp Ala Lys Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr 20 25 30 Glu Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr 35 40 45 Asp Pro Asn Pro Gln Glu Ile Val Leu Gly Asn Val Thr Glu Asn Phe 50 55 60 Asn Met Trp Lys Asn Asp Met Ala Asp Gln Met His Glu Asp Val Ile 65 70 75 80 Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu 85 90 95 Cys Val Thr Leu Asn Cys Thr Asp Thr Asn Val Thr Gly Asn Arg Thr 100 105 110 Val Thr Gly Asn Ser Thr Asn Asn Thr Asn Gly Thr Gly Ile Tyr Asn 115 120 125 Ile Glu Glu Met Lys Asn Cys Ser Phe Asn Ala Thr Thr Glu Leu Arg 130 135 140 Asp Lys Lys His Lys Glu Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val 145 150 155 160 Pro Leu Asn Glu Asn Ser Asp Asn Phe Thr Tyr Arg Leu Ile Asn Cys 165 170 175 Asn Thr Ser Thr Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro 180 185 190 Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu Lys Cys 195 200 205 Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys Tyr Asn Val Ser Thr 210 215 220 Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu 225 230 235 240 Leu Asn Gly Ser Leu Ala Glu Glu Gly Ile Ile Ile Arg Ser Glu Asn 245 250 255 Leu Thr Glu Asn Thr Lys Thr Ile Ile Val His Leu Asn Glu Ser Val 260 265 270 Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Val Arg 275 280 285 Ile Gly Pro Gly Gln Ala Phe Tyr Ala Thr Asn Asp Val Ile Gly Asn 290 295 300 Ile Arg Gln Ala His Cys Asn Ile Ser Thr Asp Arg Trp Asn Lys Thr 305 310 315 320 Leu Gln Gln Val Met Lys Lys Leu Gly Glu His Phe Pro Asn Lys Thr 325 330 335 Ile Gln Phe Lys Pro His Ala Gly Gly Asp Leu Glu Ile Thr Met His 340 345 350 Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Asn Leu 355 360 365 Phe Asn Ser Thr Tyr His Ser Asn Asn Gly Thr Tyr Lys Tyr Asn Gly 370 375 380 Asn Ser Ser Ser Pro Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile Val 385 390 395 400 Arg Met Trp Gln Gly Val Gly Gln Ala Thr Tyr Ala Pro Pro Ile Ala 405 410 415 Gly Asn Ile Thr Cys Arg Ser Asn Ile Thr Gly Ile Leu Leu Thr Arg 420 425 430 Asp Gly Gly Phe Asn Thr Thr Asn Asn Thr Glu Thr Phe Arg Pro Gly 435 440 445 Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 450 455 460 Val Val Glu Ile Lys Pro Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg 465 470 475 480 Arg Val Val Gln Arg Glu Lys Arg 485 <210> 9 <211> 469 <212> PRT <213> Human immunodeficiency virus <400> 9 Ser Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys 1 5 10 15 Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val 20 25 30 His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 35 40 45 Gln Glu Met Val Leu Ala Asn Val Thr Glu Asn Phe Asn Met Trp Lys 50 55 60 Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 65 70 75 80 Glu Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 85 90 95 Asn Cys Thr Asn Val Lys Gly Asn Glu Ser Asp Thr Ser Glu Val Met 100 105 110 Lys Asn Cys Ser Phe Lys Ala Thr Thr Glu Leu Lys Asp Lys Lys His 115 120 125 Lys Val His Ala Leu Phe Tyr Lys Leu Asp Val Val Pro Leu Asn Gly 130 135 140 Asn Ser Ser Ser Ser Gly Glu Tyr Arg Leu Ile Asn Cys Asn Thr Ser 145 150 155 160 Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro Ile Pro Leu 165 170 175 His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys 180 185 190 Thr Phe Asn Gly Thr Gly Pro Cys Arg Asn Val Ser Thr Val Gln Cys 195 200 205 Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly 210 215 220 Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu Asn Leu Thr Asn 225 230 235 240 Asn Ala Lys Thr Ile Ile Val His Leu Asn Glu Ser Val Asn Ile Val 245 250 255 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro 260 265 270 Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asn Ile Arg Gln 275 280 285 Ala His Cys Asn Ile Asn Glu Ser Lys Trp Asn Asn Thr Leu Gln Lys 290 295 300 Val Gly Glu Glu Leu Ala Lys His Phe Pro Ser Lys Thr Ile Lys Phe 305 310 315 320 Glu Pro Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn 325 330 335 Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Asp Leu Phe Asn Gly 340 345 350 Thr Tyr Arg Asn Gly Thr Tyr Asn His Thr Gly Arg Ser Ser Asn Gly 355 360 365 Thr Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile Ile Asn Met Trp Gln 370 375 380 Glu Val Gly Arg Ala Ile Tyr Ala Pro Pro Ile Glu Gly Glu Ile Thr 385 390 395 400 Cys Asn Ser Asn Ile Thr Gly Leu Leu Leu Leu Arg Asp Gly Gly Gln 405 410 415 Ser Asn Glu Thr Asn Asp Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp 420 425 430 Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu 435 440 445 Ile Lys Pro Leu Gly Val Ala Pro Thr Glu Ala Lys Arg Arg Val Val 450 455 460 Glu Arg Glu Lys Arg 465

Claims

1. A saponin extract containing at least 93% of the QS-21 main peak and 0.25–3% of the 2018 component, as measured by UV absorbance at 214 nm.

2. The saponin extract according to claim 1, wherein the most abundant monoisotope of the chemical species is 1987.9 m / z.

3. The saponin extract according to claim 1 or 2, which contains at least 98% of the QS-21 group based on UV absorbance at 214 nm.

4. A saponin extract according to any one of claims 1 to 3, which contains 1% or less of lyo impurities based on UV absorbance at 214 nm.

5. The saponin extract according to claim 4, which contains the largest peak other than the QS-21 group with a UV absorbance of 1% or less, as determined by UV absorbance at 214 nm.

6. The saponin extract according to claim 1, wherein the UV absorbance at 214 nm contains at least 98% of the QS-21 group, at least 93% of the QS-21 main peak, 0.25–3% of the 2018 component, and 1% or less of the largest peak other than the QS-21 group, and the monoisotope of the most abundant chemical species is 1987.9 m / z.

7. A saponin extract according to any one of claims 1 to 6, containing at least 65%, for example, at least 70%, of component 1988 based on UV absorbance and relative ion abundance at 214 nm.

8. A saponin extract according to any one of claims 1 to 7, containing 1856 components in an amount of 30% or less, for example, 25% or less, based on UV absorbance and relative ion abundance at 214 nm.

9. A saponin extract according to any one of claims 1 to 8, containing at least 5%, for example, at least 10%, of the 1856 component based on UV absorbance and relative ion abundance at 214 nm.

10. A saponin extract according to any one of claims 1 to 9, containing 10% or less of component 2002, for example, 5% or less, based on UV absorbance and relative ion abundance at 214 nm.

11. A saponin extract according to any one of claims 1 to 10, containing at least 0.5%, for example, at least 1%, of component 2002 based on UV absorbance and relative ion abundance at 214 nm.

12. A saponin extract according to any one of claims 1 to 11, comprising at least 1% of component 2018 based on UV absorbance at 214 nm.

13. A saponin extract containing at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9, or 2001.9 based on UV absorbance at 214 nm, and 0.25–3% of triterpenoid glycosides having an m / z of 2017.

9.

14. The saponin extract according to claim 13, wherein the most abundant monoisotope of the chemical species is 1987.9 m / z.

15. The saponin extract according to claim 13 or 14, comprising at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9, or 2001.9 based on UV absorbance at 214 nm, excluding B-isomers and lyo impurities.

16. A saponin extract according to any one of claims 13 to 15, comprising at least 98% of triterpenoid glycosides having an m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118 based on UV absorbance at 214 nm.

17. A saponin extract according to any one of claims 13 to 16, comprising at least 98% of triterpenoid glycosides, excluding lyo impurities, having m / z values ​​of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118, based on UV absorbance at 214 nm.

18. A saponin extract according to any one of claims 13 to 17, comprising a triterpenoid glycoside having a m / z of lyo impurities of 1% or less, as determined by UV absorbance at 214 nm.

19. The saponin extract according to claim 18, which contains 1% or less of other peaks based on UV absorbance at 214 nm.

20. The saponin extract according to claim 13, comprising at least 98% of triterpenoid glycosides having m / z values ​​of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118 based on UV absorbance at 214 nm, at least 93% of triterpenoid glycosides having m / z values ​​of 1855.9, 1987.9, or 2001.9, 0.25 to 3% of triterpenoid glycosides having m / z values ​​of 2017.9, and 1% or less of other peaks, wherein the most abundant monoisotope of the chemical species is 1987.9 m / z.

21. A saponin extract according to any one of claims 13 to 20, containing at least 65%, for example, at least 70%, of component 1988 based on UV absorbance and relative ion abundance at 214 nm.

22. A saponin extract according to any one of claims 13 to 21, containing 1856 components in an amount of 30% or less, for example, 25% or less, based on UV absorbance and relative ion abundance at 214 nm.

23. A saponin extract according to any one of claims 13 to 22, containing at least 5%, for example, at least 10%, of the 1856 component based on UV absorbance and relative ion abundance at 214 nm.

24. A saponin extract according to any one of claims 13 to 23, containing 10% or less of component 2002, for example, 5% or less, based on UV absorbance and relative ion abundance at 214 nm.

25. A saponin extract according to any one of claims 13 to 24, containing at least 0.5%, for example, at least 1%, of component 2002 based on UV absorbance and relative ion abundance at 214 nm.

26. A saponin extract according to any one of claims 13 to 25, comprising at least 1% of a triterpenoid glycoside having a m / z of 2017.9, as determined by UV absorbance at 214 nm.

27. Based on UV absorbance at 214nm, at least 93% 【Chemistry 1】 【change】 and 0.25-3% 【Chemistry 2】 A saponin extract containing this ingredient.

28. The saponin extract according to claim 27, wherein the most abundant monoisotope of the chemical species is 1987.9 m / z.

29. At least 98% 【Transformation 3】 【change】 【change】 The saponin extract according to claim 27 or 28, comprising the above.

30. At least 98% 【Chemistry 4】 【change】 【change】 A saponin extract according to any one of claims 27 to 29, comprising and component 2118.

31. Less than 1% 【Transformation 5】 A saponin extract according to any one of claims 27 to 30, comprising:

32. The saponin extract according to claim 31, which contains 1% or less of other peaks based on UV absorbance at 214 nm.

33. At least 98% based on UV absorbance at 214nm 【Transformation 6】 【change】 【change】 At least 93% 【Transformation 7】 【change】 0.25-3% 【Transformation 8】 The saponin extract according to claim 27, which contains less than 1% of other peaks and has a monoisotope of the most abundant chemical species at 1987.9 m / z.

34. At least 98% based on UV absorbance at 214nm 【Chemistry 9】 【change】 【change】 Or 2118 ingredients, At least 93% 【Chemistry 10】 【change】 0.25-3% 【Chemistry 11】 The saponin extract according to claim 27, which contains less than 1% of other peaks and has a monoisotope of the most abundant chemical species at 1987.9 m / z.

35. Based on UV absorbance and relative ion abundance at 214 nm, at least 65%, for example, at least 70% 【Chemistry 12】 A saponin extract according to any one of claims 27 to 34, comprising:

36. The UV absorbance and relative ion abundance at 214 nm indicate a value of 30% or less, for example, 25% or less. 【Chemistry 13】 A saponin extract according to any one of claims 27 to 35, comprising:

37. Based on the UV absorbance and relative ion abundance at 214 nm, at least 5%, for example, at least 10% 【Chemistry 14】 A saponin extract according to any one of claims 27 to 36, comprising:

38. The UV absorbance and relative ion abundance at 214 nm indicate a value of 10% or less, for example, 5% or less. 【Chemistry 15】 A saponin extract according to any one of claims 27 to 37, comprising:

39. Based on the UV absorbance and relative ion abundance at 214 nm, at least 0.5%, for example, at least 1% 【Chemistry 16】 A saponin extract according to any one of claims 27 to 38, comprising:

40. UV absorbance at 214nm indicates at least 1% 【Chemistry 17】 A saponin extract according to any one of claims 27 to 39, comprising:

41. A dried saponin extract according to any one of claims 1 to 40.

42. A method for identifying a crude aqueous extract of Quillaja saponaria molina used in the production of a purified saponin extract, (i) A step of determining the ratio of the 2018 component / QS-21 main peak by ULC-UV absorbance at 214 nm, and (ii) Selecting a crude aqueous extract having a 2018 component / QS-21 main peak ratio of 0.075 or less. A method that includes this.

43. The method according to claim 42, wherein the crude aqueous extract selected in step (ii) has a 2018 component / QS-21 main peak ratio of 0.064 or less.

44. The method according to claim 42 or 43, used for producing the saponin extract according to any one of claims 1 to 41.

45. (i) Select a crude aqueous extract of Quillaja saponaria molina having a ratio of 0.075 or less of 2018 component to the QS-21 main peak. (ii) A step of purifying the extract by polyvinylpyrrolidone adsorption, (iii) A step of purifying the extract by diafiltration, ultrafiltration or dialysis, (iv) A step of purifying the extract by reverse-phase chromatography using polystyrene resin, and (v) A step of purifying the extract by reverse-phase chromatography using phenyl resin. A method for producing a purified saponin extract containing [the specified substance].

46. The method according to claim 45, wherein step (iii) uses purification by diafiltration.

47. The method according to claim 45 or 46, wherein step (iv) is used particularly under gradient conditions, the acetonitrile and water.

48. The method according to any one of claims 45 to 47, wherein step (v) uses acetonitrile and water, particularly under isocratic conditions.

49. The method according to any one of claims 45 to 48, comprising the additional step of removing the solvent to provide a dried saponin extract.

50. The method according to claim 49, wherein the step of removing the solvent is performed by lyophilization.

51. The method according to any one of claims 45 to 50, further comprising the additional step of concentrating the extract by reverse-phase chromatography using a C8 resin.

52. The method according to claim 51, wherein the step of concentrating the extract by reverse-phase chromatography using a C8 resin is performed using acetonitrile and water, particularly under stepwise gradient conditions.

53. The method according to claim 51 or 52, comprising the additional step of exchanging the solvent.

54. The method according to claim 53, wherein the step of exchanging the solvent is to use diafiltration, ultrafiltration or dialysis, in particular diafiltration.

55. The method according to any one of claims 45 to 54, wherein the crude aqueous extract of Quillaja saponaria molina has a ratio of 0.064 or less for the 2018 component to the QS-21 main peak.

56. The method according to any one of claims 45 to 55, wherein the crude aqueous extract of Quillaja saponaria molina has a ratio of 0.01 or greater for component 2018 to the QS-21 main peak.

57. The method according to claim 56, wherein the crude aqueous extract of Quillaja saponaria molina has a ratio of 0.005 or more of 2018 component to QS-21 main peak.

58. The method according to any one of claims 45 to 57, wherein the crude aqueous extract of Quillaja saponaria molina has a leading peak to QS-21 main peak ratio of 0.45 or less.

59. The method according to claim 58, wherein the crude aqueous extract of Quillaja saponaria molina has a leading peak to QS-21 main peak ratio of 0.40 or less.

60. The method according to any one of claims 45 to 59, wherein the crude aqueous extract of Quillaja saponaria molina is an aqueous solution containing at least 1 g / L, for example, at least 2 g / L, particularly at least 2.5 g / L, and especially at least 2.8 g / L of the QS-21 main peak.

61. The method according to any one of claims 45 to 60, wherein the crude aqueous extract of Quillaja saponaria molina is a bark extract.

62. The method according to any one of claims 45 to 61, wherein step (i) of selecting a crude aqueous extract of Quillaja saponaria molina comprises testing the crude aqueous extract composition to determine the content of component 2018.

63. Use of a saponin extract according to any one of claims 1 to 41 in the manufacture of a pharmaceutical product, such as an adjuvant or immunogenic composition.

64. A saponin extract according to any one of claims 1 to 41 for use as an adjuvant.

65. An adjuvant composition comprising a saponin extract according to any one of claims 1 to 41.

66. The use, saponin extract or adjuvant composition according to any one of claims 63 to 65, wherein the adjuvant is a liposome formulation.

67. The use, saponin extract, or adjuvant composition according to any one of claims 63 to 66, wherein the adjuvant comprises a TLR4 agonist.

68. The use of a saponin extract or adjuvant composition according to claim 67, wherein the TLR4 agonist is 3D-MPL.

69. An immunogenic composition comprising the adjuvant composition according to any one of claims 65 to 68 and an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

70. The immunogenic composition according to claim 69, wherein the antigen is derived from, for example, human or non-human pathogens including bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates, or cancer cells or tumor cells.

71. The immunogenic composition according to claim 70, wherein the antigen is derived from Plasmodium falciparum or Plasmodium vivax, and is, for example, the antigen of SEQ ID NO:

1.

72. The immunogenic composition according to claim 70, wherein the antigen is derived from a species of the genus Mycobacterium, for example, from any one of Sequence ID Nos. 2 to 5.

73. The immunogenic composition according to claim 70, wherein the antigen is derived from the varicella-zoster virus, for example, the antigen of SEQ ID NO:

6.

74. The immunogenic composition according to claim 70, wherein the antigen is derived from a human respiratory multinuclear virus, for example, the antigen of Sequence ID No.

7.

75. The immunogenic composition according to claim 70, wherein the antigen is derived from HIV, for example, the antigen of SEQ ID NO: 8 or 9.

76. The immunogenic composition according to claim 70, wherein the antigen is derived from non-encapsulated Haemophilus influenzae (NTHi) and / or Moraxella catarrhalis.

77. The immunogenic composition according to any one of claims 69 to 76, wherein the antigen is provided as a polypeptide.

78. An immunogenic composition according to any one of claims 69 to 76, wherein a polynucleotide encoding a polypeptide antigen is provided.

79. A method for determining the ratio of component 2018 / QS-21 main peak in a crude aqueous extract of Quillaja saponaria molina, (i) A step of determining the content of component 2018 in the crude aqueous extract of Quillaja saponaria molina by ULC-UV absorbance at 214 nm. (ii) A step of determining the QS-21 main peak content in the crude aqueous extract of Quillaja saponaria molina by ULC-UV absorbance at 214 nm, and (iii) A step to determine the ratio of 2018 component to QS-21 main peak by comparing the 2018 component content with the QS-21 main peak content. A method that includes this.

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