Saponin Purification

Abstract

A saponin extract containing at least 88% of the QS-21 main peak and greater than 3% to 10% of the 2018 component by UV absorbance at 214 nm, methods for making said extract, its use as a vaccine adjuvant, and related aspects.

Description

[Technical field]

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

[0002] Adjuvants are included in vaccines to improve humoral and cellular immune responses, especially in the case of weakly immunogenic subunit vaccines. As with natural infection with pathogens, adjuvants rely on activation of the innate immune system to promote long-term adaptive immunity.

[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 from Salmonella minnesota, a TLR4 agonist, and QS-21 is a natural saponin extract 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™) and Herpes zoster (HZ / su - Shingrix™), as well as a number of candidate vaccines in development against pathogens such as human immunodeficiency virus and Mycobacterium tuberculosis.

[0004] Injection of AS01 results in rapid and transient activation of innate immunity in animal models. Upon immunization, neutrophils and monocytes are rapidly recruited to the draining lymph nodes (dLNs). Furthermore, AS01 inhibits the MHCII receptor, which is required for T cell activation. highIt induces the recruitment and activation of dendritic cells (DC) (Didierlaurent AM et al., 2014). Some data are also available on the mechanism of action of the components of AS01. 3D-MPL stimulates NF-κB transcriptional activity and cytokine production via TLR4 and directly activates antigen-presenting cells (APC) 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 induces high antigen-specific antibody responses and CD8 + 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, it is thought that QS-21 may function as a danger signal in vivo (Lambrecht et al., 2009; Li et al., 2008). Although QS-21 has been shown to activate the ASC-NLRP3 inflammasome and subsequent IL-1β / IL-18 release (Marty-Roix, R. et al., 2016), the exact molecular pathways involved in the adjuvant effect of saponins remain to be clearly defined. Summary of the Invention [Problem to be solved by the invention]

[0005] As with any ingredient in a product approved for human medicine, production of QS-21 requires the use of approved manufacturing processes and careful control of the final composition to ensure that safety and efficacy specifications are met. Modification of existing processes requires costly and time-consuming revalidation, and even then, deviations from specifications can result in scrapping. There continues to be a need for robust methods for the manufacture of QS-21 and for a QS-21 material of defined composition.

[0006] The QS-21 saponin extract contained in commercially available AS01 currently contains 3% or less of triterpenoid glycoside components known as 2018 components. The inventors have observed that QS-21 saponin extracts with higher percentages of 2018 components exhibit a similar bioactivity profile. [Means for solving the problem]

[0007] The present invention provides a saponin extract that contains (i) at least 88% of the QS-21 main peak and (ii) greater than 3-10% of the 2018 component by ultraviolet (UV) absorbance at 214 nanometers (nm).

[0008] Also provided is a saponin extract containing (i) at least 88% triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9 by UV absorbance at 214 nm, as determined by negative ion electrospray mass spectrometry, and (ii) greater than 3-10% triterpenoid glycosides having an m / z of 2017.9, as determined by UV absorbance at 214 nm.

[0009] In addition, the UV absorbance at 214 nm is determined to be (i) at least 88%

[0010] [ka] TIFF0007640473000002.tif135132 and (ii) more than 3 to 10%

[0011] [ka] A saponin extract containing

[0012] Furthermore, (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by reverse phase chromatography using a polystyrene resin; and (iii) A method for producing a saponin extract is provided, comprising purifying the extract by reverse phase chromatography using a phenyl resin.

[0013] There is provided the use of a saponin extract of the invention in the manufacture of a medicament.

[0014] Also provided are adjuvant and vaccine compositions comprising the saponin extracts of the present invention. [Brief description of the drawings]

[0015] [Figure 1] HPLC chromatogram of crude aqueous Quillaja Saponaria Molina bark extract. [Diagram 2] HPLC-UV chromatogram of crude aqueous Quillaja Saponaria Molina bark extract. [Diagram 3] UPLC-UV chromatogram of crude aqueous Quillaja Saponaria Molina bark extract. [Figure 4] UPLC-UV chromatogram of purified polystyrene Quillaja Saponaria Molina QS-21 saponin extract with low content of 2018 components. [Diagram 5] UPLC-UV / MS chromatogram of purified Quillaja Saponaria Molina QS-21 saponin extract with low content of 2018 components. [Figure 6] Detailed UPLC-UV / MS chromatogram of purified Quillaja Saponaria Molina QS-21 saponin extract with low content of 2018 components. [Figure 7A-7B] Mass chromatograms extracted for the 1988 (Figure 7A) and 2002 (Figure 7B) molecular weight ions of the purified Quillaja Saponaria Molina QS-21 saponin extract with low abundance of the 2018 component. [Figure 8] Combined centroid spectrum of purified Quillaja Saponaria Molina QS-21 saponin extract with low content of 2018 components. [Figure 9] UPLC-UV chromatogram of purified Quillaja Saponaria Molina QS-21 saponin extract with low 2018 component content, which was used to prepare a saponin extract rich in 2018 components. [Figure 10A-10B] 10A and 10B are UPLC-UV chromatograms and peak measurements for batches A (FIG. 10A) and B (FIG. 10B) of purified saponin extract enriched in 2018 components. [Figure 11] 13. TNF-α responses induced by purified QS-21 saponin extracts having increasing percentages of the 2018 component. [Figure 12] TNF-α response ratio (percentage of 2018 component increased relative to 2.2% 2018 component). [Figure 13] IL-8 responses induced by purified QS-21 saponin extracts having increasing percentages of the 2018 component. [Figure 14] IL-8 response ratio (increased percentage of 2018 component relative to 2.2% of 2018 component). [Figure 15] QS-21 group concentration (g / L) and 2018 component content of the phenyl resin fraction. [Figure 16] 4 shows anti-gE antibody titers 21 days after vaccination of mice with gE VZV antigen preparations containing different QS-21 compositions as described in Example 6. [Figure 17] 13 is a geometric mean ratio of anti-gE antibody titers 21 days after vaccination of mice with gE VZV antigen formulations containing different QS-21 compositions as described in Example 6. [Figure 18] Percentage of gE-specific CD4 T cells 21 days after vaccination of mice with gE VZV antigen preparations containing different QS-21 compositions as described in Example 6. [Figure 19] 13 is the geometric mean ratio of gE-specific CD4 T cells 21 days after vaccination of mice with gE VZV antigen formulations containing different QS-21 compositions as described in Example 6. [Figure 20]Percentage of gE-specific CD4 T cells 21 days after vaccination of mice with gE VZV antigen preparations containing different QS-21 compositions as described in Example 7. [Figure 21] 13 is the geometric mean ratio of gE-specific CD4 T cells 21 days after vaccination of mice with gE VZV formulations containing different QS-21 compositions as described in Example 7. [Figure 22] Anti-gE antibody titers 14 and 21 days after vaccination of mice with gE VZV antigen preparations containing different QS-21 compositions as described in Example 7. [Diagram 23] 13 is a geometric mean ratio of anti-gE antibody titers 14 and 21 days after vaccination of mice with gE VZV antigen formulations containing different QS-21 compositions as described in Example 7. [Figure 24] FIG. 1 is a schematic diagram of the vaccination protocol followed in Example 8. [Diagram 25] 13 is a graph of the maximum temperature difference observed in each animal and the mean and 95% confidence interval for all treatment groups after the first (day 8) and second (day 23) immunization in Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Brief description of sequence identifiers SEQ ID NO:1: RTS polypeptide sequence SEQ ID NO:2: M. tuberculosis H37Rv strain Rv1196 polypeptide sequence SEQ ID NO:3: Mycobacterium tuberculosis H37Rv strain Rv0125 polypeptide sequence SEQ ID NO:4: M72 fusion polypeptide sequence SEQ ID NO:5: M72-2his fusion polypeptide sequence SEQ ID NO:6: Varicella zoster virus (VZV) truncated gE polypeptide sequence SEQ ID NO: 7: Conformationally restricted RSV PreF antigen polypeptide sequence SEQ ID NO: 8: HIV TV1 gp120 polypeptide sequence SEQ ID NO: 9: HIV 1086.C gp120 polypeptide sequence

[0017] Detailed Description of the Invention As previously mentioned, any component of a product approved for use as a human medicine is required to use approved manufacturing processes and to carefully control the final composition to ensure that it meets safety and efficacy specifications. Any deviation from specifications during manufacturing will result in disposal.

[0018] The inventors have observed that the composition of the crude aqueous extract of Quillaja Saponaria Molina varies, particularly with respect to components referred to herein as 2018 components. Commercially available AS01 currently contains a QS-21 saponin extract that contains 3% or less of the 2018 components. Thus, current manufacturing methods result in waste if levels of 2018 components greater than 3% are found to be present in the purified QS-21 saponin extract. To address this potential waste, the inventors have observed that a QS-21 saponin extract having a higher percentage of the 2018 components exhibits a similar bioactivity profile, thereby allowing a broader range of Quillaja Saponaria Molina crude aqueous extracts to be used, reducing waste.

[0019] In the context of the present invention, the purified QS-21 saponin extract contained in commercially available AS01, which contains 3% or less of 2018 components, is referred to as a "purified QS-21 saponin extract having a low content of 2018 components", in contrast to the purified QS-21 saponin extract defined according to the present invention, which contains more than 3 to 10% of 2018 components. For clarity, such purified saponin extracts defined according to the present invention are referred to as "purified QS-21 saponin extracts having a high content of 2018 components", where appropriate. In other words, a "purified QS-21 saponin extract having a high content of 2018 components" contains more than 3% but not more than 10% of 2018 components.

[0020] The present invention provides a saponin extract containing (i) at least 88% of the QS-21 main peak and (ii) more than 3-10% of the 2018 component by UV absorbance at 214 nm, in particular the monoisotope of the most abundant species is 1987.9 m / z. The saponin extract suitably contains at least 98% of the QS-21 group by UV absorbance at 214 nm. Typically, the saponin extract contains 1% or less of lyo impurities by UV absorbance at 214 nm, in particular 1% or less of the largest peak other than the QS-21 group. The saponin extract suitably contains at least 90%, for example at least 91%, at least 92% or at least 93% of the QS-21 main peak.

[0021] Of particular interest are saponin extracts that contain at least 98% QS-21 groups by UV absorbance at 214 nm and have (i) at least 88% QS-21 main peak, (ii) greater than 3-10% 2018 components, and (iii) 1% or less of the largest peak other than QS-21 groups, with the monoisotope of the most abundant species being 1987.9 m / z. Suitably, the saponin extract contains at least 90%, for example at least 91%, at least 92% or at least 93% QS-21 main peak.

[0022] Also provided is a saponin extract containing, by UV absorbance at 214 nm, (i) at least 88% triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9, and (ii) greater than 3-10% triterpenoid glycosides having an m / z of 2017.9, particularly wherein the monoisotope of the most abundant species is 1987.9 m / z. Desirably, the saponin extract contains (i) at least 88% of triterpenoid glycosides having m / z of 1855.9, 1987.9, or 2001.9, excluding B-isomers and lyo impurities, by UV absorbance at 214 nm, and (ii) greater than 3-10% of triterpenoid glycosides having m / z of 2017.9, in particular the monoisotope of the most abundant species being 1987.9 m / z. Suitably, the saponin extract contains at least 98% of triterpenoid glycosides having m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9, or 2118, by UV absorbance at 214 nm. Desirably, the saponin extract contains at least 98% triterpenoid glycosides by UV absorbance at 214 nm, excluding lyo impurities, having m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118. Typically, the saponin extract contains 1% or less lyo impurities by UV absorbance at 214 nm. Suitably, the saponin extract contains 1% or less other peaks by UV absorbance at 214 nm. Suitably, the saponin extract contains at least 90%, for example at least 91%, at least 92% or at least 93%, of triterpenoid glycosides having m / z of 1855.9, 1987.9 or 2001.9.

[0023] Of particular interest are saponin extracts containing at least 98% triterpenoid glycosides having m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118 by UV absorbance at 214 nm, (i) at least 88% triterpenoid glycosides having m / z of 1855.9, 1987.9 or 2001.9, (ii) greater than 3-10% triterpenoid glycosides having m / z of 2017.9, and (iii) 1% or less other peaks, the most abundant species being a monoisotope of 1987.9 m / z, and in particular saponin extracts contains at least 98% triterpenoid glycosides having an m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118, excluding lyo impurities, by UV absorbance at 214 nm, and has (i) at least 88% triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9, excluding B-isomers and lyo impurities, (ii) greater than 3-10% triterpenoid glycosides having an m / z of 2017.9, and (iii) 1% or less other peaks, with the most abundant monoisotope being 1987.9 m / z. Suitably the saponin extract contains at least 90%, such as at least 91%, at least 92% or at least 93% triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9.

[0024] Further, the ion exchange reaction is characterized by: (i) a denaturation rate of at least 88%, e.g., at least 90%, at least 91%, at least 92%, or at least 93%, measured by UV absorbance at 214 nm;

[0025] [ka] TIFF0007640473000005.tif64150 and (ii) more than 3 to 10%

[0026] [ka] The saponin extract contains at least 98% of the saponin, and in particular the monoisotope of the most abundant chemical species is 1987.9 m / z.

[0027] [ka] Suitably the saponin extract contains 98% of the above mentioned components and 2118 components. Typically the saponin extract contains less than 1% by UV absorbance at 214 nm.

[0028] [ka] In particular, it contains other peaks at less than 1%.

[0029] Of particular interest is that at least 98% of the samples were analyzed by UV absorbance at 214 nm.

[0030] [ka] TIFF0007640473000012.tif194142TIFF0007640473000013.tif89139, and (i) at least 88%, e.g., at least 90%, at least 91%, at least 92%, or at least 93%

[0031] [ka] TIFF0007640473000015.tif133155(ii)3-10%

[0032] [ka] and (iii) a saponin extract having a monoisotope of 1987.9 m / z as the most abundant species with 1% or less of other peaks. Of particular interest are saponin extracts having at least 98% of the saponin identified by UV absorbance at 214 nm.

[0033] [ka] TIFF0007640473000018.tif218136 or 2118 components (i) at least 88%, e.g., at least 90%, at least 91%, at least 92%, or at least 93%

[0034] [ka] TIFF0007640473000020.tif64142(ii)3-10%

[0035] [ka] and (iii) a saponin extract with less than 1% other peaks and the most abundant species having a monoisotope of 1987.9 m / z.

[0036] For the avoidance of doubt, the term "at least 88% of triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9" means "triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9, or combinations thereof," unless the context excludes. Other similar terms including "or," such as "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," should be construed accordingly and also to include combinations, unless the context excludes.

[0037] In some embodiments, the saponin extract contains at least 40%, such as at least 50%, particularly at least 60%, especially at least 65%, such as at least 70% of the 1988 components as determined by UV absorbance at 214 nm and relative ion abundance. In some embodiments, the saponin extract contains no more than 90%, such as no more than 85%, or no more than 80% of the 1988 components as determined by UV absorbance at 214 nm and relative ion abundance. In some embodiments, the saponin extract contains between 40% and 90% of the 1988 components, such as between 50% and 85%, especially between 70% and 80% of the 1988 components as determined by UV absorbance at 214 nm and relative ion abundance.

[0038] In some embodiments, the saponin extract contains 30% or less, such as 25% or less, of the 1856 components as determined by UV absorbance at 214 nm and by relative ion abundance. In some embodiments, the saponin extract contains at least 5%, such as at least 10%, of the 1856 components as determined by UV absorbance at 214 nm and by relative ion abundance. In some embodiments, the saponin extract contains between 5% and 30%, such as between 10% and 25%, of the 1856 components as determined by UV absorbance at 214 nm and by relative ion abundance.

[0039] In some embodiments, the saponin extract contains 40% or less, such as 30% or less, particularly 20% or less, especially 10% or less, of the 2002 components as determined by UV absorbance at 214 nm and by relative ion abundance. In some embodiments, the saponin extract contains at least 0.5%, such as at least 1%, of the 2002 components as determined by UV absorbance at 214 nm and by relative ion abundance. In some embodiments, the saponin extract contains 0.5% to 40%, such as 1% to 10%, of the 2002 components as determined by UV absorbance at 214 nm and by relative ion abundance.

[0040] In some embodiments, the saponin extract contains at least 3.5%, such as at least 4%, or at least 4.5% of the 2018 component by UV absorbance at 214 nm and by relative ion abundance.

[0041] In some embodiments, the saponin extract contains 9% or less, such as 8% or less, such as 7% or less, of the 2018 component by UV absorbance at 214 nm and by relative ion abundance.

[0042] In some embodiments, the saponin extract contains 4-8%, such as 5-7%, or about 6% (e.g., 5.5-6.5%) of the 2018 component by UV absorbance at 214 nm and by relative ion abundance.

[0043] In some embodiments, the saponin extract has a saponin content of at least 40%, such as at least 50%, particularly at least 60%, especially at least 65%, such as at least 70%, by UV absorbance at 214 nm and by relative ion abundance.

[0044] [ka] Contains TIFF0007640473000023.tif62137.

[0045] In some embodiments, the saponin extract has an average purity of 90% or less, such as 85% or less, or 80% or less by UV absorbance at 214 nm and by relative ion abundance.

[0046] [ka] Contains:

[0047] In some embodiments, the saponin extract has a purity of 30% or less, such as 25% or less, by UV absorbance at 214 nm and by relative ion abundance.

[0048] [ka] In some embodiments, the saponin extract contains at least 5%, for example at least 10%, by UV absorbance at 214 nm and by relative ion abundance.

[0049] [ka] Contains:

[0050] In some embodiments, the saponin extract has a saponin content of 40% or less, such as 30% or less, particularly 20% or less, and especially 10% or less, by UV absorbance at 214 nm and by relative ion abundance.

[0051] [ka] In some embodiments, the saponin extract contains at least 0.5%, such as at least 1%, by ion abundance by UV absorbance at 214 nm and by relative ion abundance.

[0052] [ka] Contains:

[0053] In some embodiments, the saponin extract has a saponin content of at least 3.5%, such as at least 4%, or at least 4.5%, by UV absorbance at 214 nm and by relative ion abundance.

[0054] [ka] Contains:

[0055] In some embodiments, the saponin extract has an average purity of 9% or less, such as 8% or less, such as 7% or less, by UV absorbance at 214 nm and by relative ion abundance.

[0056] [ka] Contains:

[0057] 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", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, pp. 243-254). Purified fractions of Quil A that retain adjuvant activity without the toxicity associated with Quil A have been isolated by high performance liquid chromatography (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 have adjuvant activity, although their toxicity varies considerably.

[0058] The term "saponin extract" as used herein means an extract of Quillaja Saponaria Molina.

[0059] The term "triterpenoid glycoside," as used herein, means an entity having a triterpenoid core derivatized with sugars attached via glycosidic bonds.

[0060] Some structures herein have been determined by tandem mass spectrometry (MS / MS), and limitations of this technique in identifying some branched stereochemical and isomeric sugar species (e.g., apiose and xylose) mean that some structures are putative and based on an assumed conserved core. Putative structures should therefore refer to the actual structures of components that have been confirmed by other methods if the putative structure is for any reason incorrect.

[0061] The term "2018 component" refers to the triterpenoid glycoside identified as "2018 peak" in FIG. 6. The 2018 component has a retention time of approximately 4.5 minutes in the UPLC-UV / MS method described herein and is suitably the major component of a peak having a monoisotopic molecular weight of 2017.9. The 2018 component may also be identified as having a retention time of approximately 5.8 minutes in the UPLC-UV method described herein. This major 2018 component has been confirmed by MS / MS to have the following putative structure:

[0062] [ka]

[0063] The term "1988 component" refers to a triterpenoid glycoside identified in Figure 6 as part of the QS-21 main peak and having a monoisotopic molecular weight of 1987.9. Suitably, the 1988 component in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and a monoisotopic molecular weight of 1987.9. This 1988 component is QS-21A V1:

[0064] [ka] and QS-21A V2

[0065] [ka] It may consist of:

[0066] The term "1856 component" refers to a triterpenoid glycoside identified in Figure 6 as part of the QS-21 main peak and having a monoisotopic molecular weight of 1855.9. Suitably, the 1856 component in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and a monoisotopic molecular weight of 1855.9.

[0067] [ka] It may consist of:

[0068] The term "2002 component" refers to a triterpenoid glycoside identified in Figure 6 as part of the QS-21 main peak and having a monoisotopic molecular weight of 2001.9. Suitably, the 2002 component in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and a monoisotopic molecular weight of 2001.9. The 2002 component has been identified by MS / MS with the putative structure:

[0069] [ka] It has been confirmed that it has

[0070] The term "lyo impurity" refers to a triterpenoid glycoside identified as the "lyophilized peak" in Figure 6. Suitably, the lyo impurity in the UPLC-UV / MS method described herein has a retention time of approximately 4.7 minutes and the major component of the peak has a monoisotopic molecular weight of 1855.9. This major lyo impurity has the putative structure by MS / MS:

[0071] [ka] It has been confirmed that it has

[0072] The term "B-isomer" refers to the triterpenoid glycoside identified as "B-isomer" in Figure 6. Suitably, the B-isomer in the UPLC-UV / MS method described herein has a retention time of approximately 4.0 minutes, with the major component of the peak having a monoisotopic molecular weight of 1987.9. This major B-isomer component has the putative structure:

[0073] [ka] It has been confirmed that it has

[0074] The term "1518 component" refers to the triterpenoid glycoside identified as the "1518 peak" in Figure 6. Suitably, the 1518 component in the UPLC-UV / MS method described herein has a retention time of approximately 4.2 minutes, and the major component of the peak has a monoisotopic molecular weight of 1517.7. This major 1518 component has the putative structure:

[0075] [ka] It has been confirmed that it has

[0076] The term "1712 component" refers to the triterpenoid glycoside identified in Figure 6 as the "1712 peak." Suitably, the 1712 component in the UPLC-UV / MS method described herein has a retention time of approximately 4.6 minutes, and the major component of the peak has a monoisotopic molecular weight of 1711.8. This major 1712 component has the putative structure:

[0077] [ka] It has been confirmed that it has

[0078] The term "2118 component" refers to the triterpenoid glycoside identified as "4.607" in Figure 6. Suitably, the 2118 component in the UPLC-UV / MS method described herein has a retention time of approximately 4.6, with the major component of the peak having a monoisotopic molecular weight of 2118.

[0079] The term "QS-21 main peak" refers to the triterpenoid glycoside identified as "QS-21" in Figure 6. Suitably, the QS-21 main peak in the UPLC-UV / MS method described herein has a retention time of approximately 4.4 minutes and molecular weight components of m / z 1855.9 (1856 component), 1987.9 (1988 component) and 2001.9 (2002 component). This QS-21 main peak is QS-21A V1:

[0080] [ka] QS-21A V2:

[0081] [ka] 1856 Ingredients:

[0082] [ka] and 2002 Ingredients:

[0083] [ka] It may consist of:

[0084] The term "QS-21 group" refers to triterpenoid glycosides identified in the UPLC-UV / MS method described herein as having a retention time of about 4.0 minutes to about 4.7 minutes from the B-isomer to the peak preceding the lyo impurity, and having a major monoisotopic molecular weight of 1517.7 (1518 component), 1711.8 (1712 component), 1855.9 (1856 component), 1987.9 (1988 component), 2001.9 (2002 component), 2017.9 (2018 component) or 2118 (2118 component). The QS-21 group includes QS-21A V1:

[0085] [ka] QS-21A V2:

[0086] [ka] 1856 Ingredients:

[0087] [ka] 2002 Ingredients:

[0088] [ka] 2018 Ingredients:

[0089] [ka] B-isomer:

[0090] [ka] 1518 Ingredients:

[0091] [ka] 1712 Ingredients:

[0092] [ka] and It can consist of 2118 components.

[0093] The term "largest peak outside the QS-21 group" refers to the largest peak by UV detectable by the UPLC-UV / MS method described herein that is not part of the QS-21 group.

[0094] The term "leading peak" refers to the peak preceding the main QS-21 peak immediately prior to the peak in the HPLC-UV method described herein (see FIG. 2).

[0095] The term "m / z" refers to the mass-to-charge ratio of the monoisotopic peak. Unless otherwise specified, "m / z" is determined by negative ion electrospray mass spectrometry.

[0096] The term "ion abundance" refers to the amount of a defined m / z measured in a sample, or a given peak if the context requires. A mass chromatogram for a defined m / z can be derived from the MS total ion chromatogram in the UPLC-UV / MS method described herein. A mass chromatogram is a plot of signal intensity versus time. Ion abundance is measured as the area of ​​the integrated peak. Area for defined m / z / area for relative reference m / z=relative abundance.

[0097] The term "UV absorbance at 214 nm" means the area of ​​the integrated peak in a UV absorbance chromatogram. (Area for a given peak) / (Area of ​​all integrated peaks in a chromatogram) x 100 = Area Percentage for a given peak.

[0098] The term "UV absorbance at 214 nm and relative ion abundance" refers to an estimate for the fraction of a given m / z relative to the co-eluting species: (area percentage for a given UV peak) x (relative ion abundance for the m / z of interest in the given peak) / (sum of all relative ion abundances for the given peak) = fraction of the m / z of interest in the given UV peak assuming the relative ion abundances included for all co-eluting species.

[0099] The term "the monoisotope of the most abundant species is 1988 m / z" means that the monoisotope of the most abundant species that is the first peak in the isotope group with the highest response for m / z is 1987.9. The most abundant species can be determined by generating a combined spectrum across the total ion chromatogram using the UPLC-UV / MS method (negative ion electrospray) described herein.

[0100] The term "dried" means that substantially all of the solvent has been removed. A dried extract typically contains less than 5% (w / w) solvent (e.g., less than 5% (w / w) water). Suitably, the dried extract contains no more than 100 ppm (w / w) of acetonitrile.

[0101] 1. A method for producing a purified saponin extract containing at least 88% of the QS-21 main peak by UV absorbance at 214 nm and greater than 3-10% of a 2018 component (such as those described herein), comprising: (i) selecting a Quillaja Saponaria Molina material having a suitable 2018 ingredient composition; (ii) preparing a crude aqueous extract of Quillaja Saponaria Molina material under suitable conditions; and (iii) purifying the crude aqueous extract to obtain a purified saponin extract A method is provided that includes:

[0102] Those skilled in the art will understand that the composition of the purified saponin extract will depend on the Quillaja Saponaria Molina material used, the conditions applied during the aqueous extraction, and the conditions applied during purification. By appropriately adapting these parameters, a purified saponin extract of the desired composition can be ensured. Step (ii) may include multiple parallel extractions, some or all of the products of which are combined before proceeding to step (iii). Step (iii) may include multiple parallel purifications, some or all of which products are combined to obtain a final purified saponin extract. Optionally, the purified saponin extract obtained from step (iii) can be obtained by a combination of purifying the crude aqueous extract and adding some 2018 components (i.e., "2018 spike", e.g., as described in Example 4).

[0103] Less desirably, a purified saponin extract containing at least 88% of the QS-21 main peak by UV absorbance at 214 nm and greater than 3-10% of the 2018 components (such as those described herein) can be prepared by first obtaining highly purified individual components of QS-21 and then combining such individual components in the required proportions.

[0104] Furthermore, (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by reverse phase chromatography on a polystyrene resin; and (iii) purifying the extract by reverse phase chromatography on phenyl resin; A method for producing a saponin extract is provided, comprising:

[0105] Preferably, the method comprises: (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; and (v) purifying the extract by reverse phase chromatography on phenyl resin; Including, Steps (ii) and (iii) may optionally be performed in the reverse order or simultaneously, but are generally in the order illustrated.

[0106] The crude aqueous extract of Quillaja Saponaria Molina is obtained by aqueous extraction (but need not be in aqueous form, e.g. it may subsequently be 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" refers to an aqueous extract of Quillaja Saponaria Molina material, e.g. an extract of Quillaja Saponaria Molina bark, the extract having a ratio of 2018 components to QS-21 main peak of greater than 0.075 (as determined by UPLC-UV absorbance at 214 nm). Suitably the extract has a ratio of 2018 components to QS-21 main peak of greater than 0.078.

[0107] The term "Quillaja Saponaria Molina material having a suitable 2018 component composition" refers to a Quillaja Saponaria Molina material, such as Quillaja Saponaria Molina bark, capable of providing an extract having a ratio of 2018 components to QS-21 main peak (as determined by UPLC-UV absorbance at 214 nm) of greater than 0.075. Suitably, the extract has a ratio of 2018 components to QS-21 main peak of greater than 0.078.

[0108] The ratio of the preceding peak to the main QS-21 peak is suitably 0.45 or less, in particular 0.4 or less (as determined by HPLC-UV absorbance at 214 nm). The ratio of the preceding peak to the main QS-21 peak may be 0.05 or more, in particular 0.1 or more (as determined by HPLC-UV absorbance at 214 nm).

[0109] Typically the crude aqueous extract is a bark extract. Suitably the QS-21 main peak content in the aqueous solution of the crude aqueous extract of Quillaja Saponaria Molina is at least 1 g / L, for example at least 2 g / L, in particular at least 2.5 g / L, particularly at least 2.8 g / L (as determined, for example, by UV absorbance against a control sample of known concentration).

[0110] Suitably, the step of selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable 2018 ingredient composition comprises testing the composition to determine the 2018 ingredient content.

[0111] The step of purifying the extract by polyvinylpyrrolidone adsorption involves treating the extract with a polyvinylpyrrolidone resin. Typically, the extract is stirred with the polyvinylpyrrolidone resin. The extract may then be separated from the polyvinylpyrrolidone resin with adsorbed impurities by filtration. This step of the process generally removes polyphenolic impurities, such as tannins.

[0112] The step of purifying the extract by diafiltration, ultrafiltration or dialysis is suitable, typically by diafiltration using tangential flow. A suitable example of a membrane is a 30 kDa cut-off. This step of the process generally removes salts, sugars and other low molecular weight substances.

[0113] The step of purifying the extract by reversed-phase chromatography on a polystyrene resin typically uses acetonitrile and water as solvents, usually acidified with a suitable acid, e.g., acetic acid. An example of a suitable resin is Amberchrom XT20. Chromatography may be performed using isocratic conditions, but typically is run under a solvent gradient (continuous, e.g., linear, or stepwise), e.g., as given in the examples. This step of the method typically removes non-saponin material and enriches the desired saponin. Selected fractions may be pooled to maximize the yield of material meeting the required criteria (typically %QS-21≧18%, as determined by UV absorbance after HPLC-UV, and 2018 / QS-21 ratio>0.054, as determined by UV absorbance after UPLC-UV). Each polystyrene chromatography run is typically on the order of between 25-200 g of QS-21, for example between 50-150 g, especially between 70-110 g (amount based on main peak QS-21 content in material by UV).

[0114] Purification of the extract by reversed phase chromatography on a phenyl resin typically uses acetonitrile and water as solvents, usually acidified with a suitable acid, e.g., acetic acid. Chromatography may be performed using a solvent gradient (continuous, e.g., linear, or stepwise), but is typically run under isocratic conditions. This step of the method provides final purification of the desired saponin. Selected fractions may be pooled to maximize the yield of material meeting the required criteria (typically % QS-21 group ≧98.5, % QS-21 main peak ≧94.5, 2002 / 1988 ≦0.027, % 2018>2.7%, main peak outside QS-21 group ≦1%, as determined by UPLC-UV / MS). Each phenyl chromatography run typically scales between 4-40 g QS-21, e.g., between 10-30 g, especially between 13-21 g (amount based on QS-21 main peak content in material by UV).

[0115] The method may include the further step of removing the solvent to provide a dried saponin extract. (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by reverse phase chromatography on a polystyrene resin; (iii) purifying the extract by reverse phase chromatography on a phenyl resin; and (iv) Removing the solvent to provide a dried saponin extract. The present invention provides a method for producing a saponin extract, comprising:

[0116] The present invention also provides (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; (v) purifying the extract by reverse phase chromatography on phenyl resin; and (vi) Removing the solvent to provide a dried saponin extract. wherein steps (ii) and (iii) are optionally carried out in the reverse order or simultaneously, but generally in the order exemplified.

[0117] To improve the efficiency of drying, it may be desirable to carry out a further step prior to the drying step, for example concentrating the extract by a suitable technique, such as capture and release, for example using reverse phase chromatography (e.g. with C8 resin) and / or exchanging the solvent.

[0118] Also, (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; (v) purifying the extract by reverse phase chromatography on phenyl resin; (vi) optionally concentrating the extract; (vii) optionally exchanging the solvent; and (viii) Removing the residual solvent to provide a dried saponin extract. wherein steps (vi) and (vii) are optionally carried out in the reverse order or simultaneously, but generally in the order exemplified.

[0119] Also, (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a suitable component composition; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; (v) purifying the extract by reverse phase chromatography on phenyl resin; (vi) concentrating the extract by reverse phase chromatography using a C8 resin; (vii) exchanging the solvent; and (viii) Removing the residual solvent to provide a dried saponin extract. Also provided is a method for producing a saponin extract, comprising:

[0120] Concentration of the extract can be carried out using any suitable technique. For example, concentration can be carried out using capture and release methods, such as reversed phase chromatography, particularly using C8 resin. Reverse phase chromatography typically uses acetonitrile and water as solvents, usually acidified with a suitable acid, such as acetic acid. Chromatography is typically run under a solvent gradient, where the saponin extract is captured in a low organic solvent and eluted in a high organic solvent, particularly a stepwise solvent gradient.

[0121] Solvent exchange can be performed using any suitable technique, in particular diafiltration, ultrafiltration or dialysis, especially diafiltration. Solvent exchange can be useful, for example, to reduce the acetonitrile content, as described, for example, in WO2014016374. A suitable membrane can be selected to allow solvent exchange while retaining the saponin extract, and can be, for example, a 1 kDa membrane.

[0122] Drying by removing the solvent can be carried out by any suitable means, in particular freeze-drying. During drying, degradation of the saponin extract can occur, which can lead to the formation of lyo impurities. It is therefore desirable to dry under conditions that limit the formation of lyo impurities, for example by limiting the drying temperature and / or drying time. The removal of the solvent is suitably carried out by a single freeze-drying process. The degree of drying required depends on the nature of the solvent, for example pharma- ceutical unacceptable solvents are desirably removed to a high degree, whereas some pharma- ceutical acceptable solvents (e.g. water) can be removed to a lesser extent.

[0123] The process of the invention is suitably carried out on a scale of between 25 and 1000 g of QS-21, for example between 50 and 500 g, especially between 100 and 500 g (amount based on main peak QS-21 content in the material by UV).

[0124] Also provided is a method for identifying a crude aqueous extract of Quillaja Saponaria Molina for use in producing a purified saponin extract, such as the saponin extract of the present invention, comprising the steps of: (i) determining the ratio of 2018 components to the main peak of QS-21 by UPLC-UV absorbance at 214 nm; (ii) selecting crude extracts having a ratio of 2018 components to the main peak of QS-21 greater than 0.075; A method is also provided that includes:

[0125] In one embodiment, the crude aqueous extract selected in step (ii) has a ratio of 2018 components to the main QS-21 peak of greater than 0.078.

[0126] The present invention also provides a method for determining the ratio of 2018 components to the main peak of QS-21 in a crude aqueous extract of Quillaja Saponaria Molina, comprising: (i) determining the 2018 component content in a crude aqueous extract of Quillaja Saponaria Molina by UPLC-UV absorbance at 214 nm; (ii) determining the QS-21 main peak content in the crude aqueous extract of Quillaja Saponaria Molina by UPLC-UV absorbance at 214 nm; and (iii) comparing the 2018 component content with the QS-21 main peak content to determine a ratio of the 2018 component to the QS-21 main peak. A method is also provided that includes:

[0127] There is provided the use of the saponin extract of the invention in the manufacture of a medicament. Further provided is the saponin extract of the invention for use as a medicament, particularly as an adjuvant. Also provided is an adjuvant composition comprising the saponin extract of the invention.

[0128] 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). 3D-MPL is sold by GlaxoSmithKline Biologicals NA under the name "MPL" and is referred to as 3D-MPL throughout the literature. See, for example, U.S. Patent Nos. 4,436,727, 4,877,611, 4,866,034 and 4,912,094. 3D-MPL can be produced according to the method described in GB 2 220 211. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.

[0129] Other TLR4 agonists that may be useful in the present invention include those described, for example, in WO 2008 / 153541 or WO 2009 / 143457 or in the literature articles 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): e41144., doi:10.1371 / journal.pone.0041144. WO 2008 / 153541 or WO 2009 / 143457 are incorporated herein by reference for the purposes of defining TLR4 agonists that may be useful in the present invention.

[0130] Particular alkyl glucosaminide phosphates (AGPs) of interest are described below.

[0131] [ka] CRX601

[0132] Interesting TLR4 agonists include:

[0133] [ka] Contains 3-deacylmonophosphoryl hexa-acyl lipid A.

[0134] Another interesting TLR4 agonist is

[0135] [ka] It is 3-deacylmonophosphoryl lipid A.

[0136] An interesting TLR4 agonist is the following dLOS (as described in Han, 2014).

[0137] [ka]

[0138] A typical adult human dose of the adjuvant contains the saponin extract in an amount between 1-100ug per human dose. The saponin extract can be used at a level of about 50ug. Examples of suitable ranges are 40-60ug, suitably 45-55ug or 49-51ug, for example 50ug. In a further embodiment, a human dose contains the saponin extract at a level of about 25ug. Examples of lower ranges are 20-30ug, suitably 22-28ug or 24-26ug, for example 25ug. Human doses intended for children may be reduced compared to those intended for adults (e.g. reduced by 50%).

[0139] The TLR4 agonist, such as lipopolysaccharide, for example 3D-MPL, may be used in an amount between 1-100ug per human dose. 3D-MPL may be used at a level of about 50ug. Examples of suitable ranges are 40-60ug, suitably 45-55ug or 49-51ug, for example 50ug. In a further embodiment, the human dose comprises 3D-MPL at a level of about 25ug. Examples of lower ranges are 20-30ug, suitably 22-28ug or 24-26ug, for example 25ug. Human doses intended for children may be reduced (for example 50% reduced) compared to those intended for adults.

[0140] When both a TLR4 agonist and a saponin extract are present in the adjuvant, the weight ratio of TLR4 agonist to saponin is suitably between 1:5 and 5:1, suitably 1:1. For example, when 3D-MPL is present in an amount of 50ug or 25ug, QS-21 is suitably also present in an amount of 50ug or 25ug per human dose.

[0141] The adjuvant may also include a suitable carrier, such as an emulsion (eg, an oil-in-water emulsion) or liposomes.

[0142] Liposomes The term "liposome" is well known in the art and defines a general category of vesicles that contain one or more lipid bilayers that surround an aqueous space. Thus, liposomes consist of one or more lipid and / or phospholipid bilayers and can contain other molecules in their structure, such as proteins or carbohydrates. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble substances, lipophilic substances, and / or amphiphilic compounds.

[0143] The size of liposomes can vary from 30 nm to several um depending on the phospholipid composition and the method used for their preparation.

[0144] Liposomes useful in the present invention suitably contain DOPC (together with a saponin and optionally a TLR4 agonist) or consist essentially of DOPC and a sterol.

[0145] In the present invention, the size of the liposomes ranges from 50 nm to 200 nm, particularly from 60 nm to 180 nm, for example from 70 to 165 nm. Optimally, the liposomes should be stable and have a diameter of approximately 100 nm, allowing convenient sterilization by filtration.

[0146] The structural integrity of the liposomes can be assessed by methods such as dynamic light scattering (DLS) to measure the size (Z-average diameter, Zav) and polydispersity of the liposomes, or by electron microscopy to analyze the structure of the liposomes. In one embodiment, the average particle size is between 95-120 nm and / or the polydispersity (PdI) index is 0.3 or less (e.g., 0.2 or less).

[0147] Further additives (excipients) In a further embodiment, a buffering agent is added to the composition. The pH of the liquid preparation is adjusted taking into account the components of the composition and the necessary compatibility for administration to a subject. Suitably, the pH of the liquid mixture is at least 4, at least 5, at least 5.5, at least 5.8, 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. Suitably, therefore, the pH is 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.

[0148] Suitable buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. In one embodiment, the buffer is a phosphate buffer, such as Na / Na2PO4, Na / K2PO4 or K / K2PO4.

[0149] 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.

[0150] As is well known, solutions for parenteral administration should have a pharma- ceutically acceptable osmolality to avoid cell deformation or lysis. By pharma-ceutically acceptable osmolality, it is generally meant that the solution has an approximately isotonic or slightly hypertonic osmolality. The composition (if presented in dry form, when reconstituted) suitably has an osmolality in the range of 250-750 mOsm / kg, for example the osmolality may be in the range of 250-550 mOsm / kg, for example in the range of 280-500 mOsm / kg. In a particularly preferred embodiment, the osmolality may be in the range of 280-310 mOsm / kg. Osmolality can be measured according to techniques known in the art, for example using a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).

[0151] An "isotonicity agent" is a compound that is physiologically tolerated and provides a tonicity to the formulation suitable for preventing net water flow across cell membranes in contact with the formulation. In some embodiments, the isotonicity agent used in the composition is a salt (or a mixture of salts), conveniently the salt is sodium chloride, and a concentration of approximately 150 nM is suitable. However, in other embodiments, the composition comprises a non-ionic isotonicity agent, and the concentration of sodium chloride in the composition is less than 100 mM, such as less than 80 mM, such as less than 50 mM, such as less than 40 mM, less than 30 mM, especially less than 20 mM. The ionic strength in the composition can be less than 100 mM, such as less than 80 mM, such as less than 50 mM, such as less than 40 mM or less than 30 mM.

[0152] In certain embodiments, the non-ionic isotonicity agent is a polyol, such as sucrose and / or sorbitol. The concentration of sorbitol can be, for example, between about 3% and about 15% (w / v), such as between about 4% and about 10% (w / v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist, wherein the isotonicity agent is a salt or a polyol, are described in WO 2012 / 080369.

[0153] A human dose volume 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, is suitable. The volume of composition used may depend on the delivery route and location, with smaller doses being used for intradermal routes. Unit dose containers may contain an overage to allow for precise manipulation of the substance during administration of the unit dose.

[0154] The ratio of saponin:DOPC is typically of the order of 1:50 to 1:10 (w / w), suitably between 1:25 to 1:15 (w / w), preferably 1:22 to 1:18 (w / w), for example 1:20 (w / w).

[0155] Saponins are suitably presented as less reactogenic compositions that are quenched with an exogenous sterol, such as cholesterol. Cholesterol is disclosed in the Merck Index, 13th Edition, p. 381, as a naturally occurring sterol found in tallow. Cholesterol has the formula (C 27 H 46 O) and is also known as (3β)-cholest-5-en-3-ol.

[0156] The saponin:sterol ratio is usually of the order of 1:100 to 1:1 (w / w), suitably between 1:10 and 1:1 (w / w), preferably 1:5 to 1:1 (w / w). Suitably, an excess of sterol is present, the saponin:sterol ratio being 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.

[0157] The amount of liposomes (by weight of lipid and sterol) is typically in the range of 0.1 mg to 10 mg per human dose of the composition, in particular 0.5 mg to 2 mg per human dose of the composition.

[0158] In a particularly suitable embodiment, the liposomes used in the present invention comprise DOPC and a sterol, particularly cholesterol. Thus, in a particular embodiment, the composition used in the present invention comprises a saponin extract in the form of a liposome, said liposome comprising DOPC and a sterol, particularly cholesterol.

[0159] A particular adjuvant of interest features liposomes comprising DOPC and cholesterol together with a TLR4 agonist and a saponin extract of the invention, particularly 3D-MPL and a saponin extract of the invention.

[0160] Another adjuvant of interest features liposomes comprising a TLR4 agonist and a saponin extract of the invention, particularly dLOS and a saponin extract of the invention together with DOTAP and DMPC.

[0161] antigen Adjuvants prepared according to the present invention can be used in conjunction with an immunogen or antigen. In some embodiments, a polynucleotide encoding the immunogen or antigen is provided.

[0162] The adjuvant may be administered to a subject separately from the immunogen or antigen, or the adjuvant may be combined with the immunogen or antigen during manufacture or in situ to provide an immunogenic composition for combined administration.

[0163] As used herein, a subject is a mammal, such as a rodent, non-human primate, or a human.

[0164] Thus, there is provided a method for preparing an immunogenic composition comprising an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen, comprising the steps of: (i) preparing an adjuvant composition comprising the saponin extract of the present invention; (ii) mixing an adjuvant with an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen; A method is provided that includes:

[0165] There is also provided the use of an adjuvant comprising the saponin extract of the invention in the manufacture of a medicament. Suitably the medicament comprises an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

[0166] There is further provided an adjuvant comprising the saponin extract of the invention for use as a medicament. Suitably the medicament comprises an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

[0167] The term immunogen refers to a polypeptide capable of eliciting an immune response. Suitably, the immunogen is an antigen comprising at least one B or T cell epitope. The immune response elicited may be an antigen-specific B cell response that generates neutralizing antibodies. The immune response elicited may be an antigen-specific T cell response that may be a systemic and / or local response. The antigen-specific T cell response may include a CD4+ T cell response, e.g. a response involving CD4+ T cells expressing multiple cytokines, e.g. IFN gamma, TNF alpha and / or IL2. Alternatively or in addition, the antigen-specific T cell response may include a CD8+ T cell response, e.g. a response involving CD8+ T cells expressing multiple cytokines, e.g. IFN gamma, TNF alpha and / or IL2.

[0168] Antigens can be derived from (e.g., obtainable from) human or non-human pathogens, such as bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates, or cancer or tumor cells.

[0169] In one embodiment, the antigen is a recombinant protein, such as a recombinant prokaryotic protein.

[0170] In one embodiment, the antigen is derived from Plasmodium spp. (e.g. Plasmodium falciparum), Mycobacterium spp. (e.g. Mycobacterium tuberculosis (TB)), Varicella-Zoster virus (VZV), human respiratory syncytial virus, human immunodeficiency virus (HIV), Moraxella spp. (e.g. Moraxella catarrhalis) or nontypable Haemophilus influenzae (ntHi).

[0171] The antigen may comprise or consist of a preparation derived from a parasite that causes malaria, such as Plasmodium falciparum or Plasmodium vivax.

[0172] In one embodiment, the antigen may be a Plasmodium falciparum circumsporozoite (CS) protein or a variant thereof. A suitable variant of the CS protein may be one 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, for example, comprising substantially all of the C-terminal portion of the CS protein, four or more tandem repeats of the immunodominant region of the CS protein, and HBsAg. The hybrid protein may comprise a sequence that contains at least 160 amino acids and is substantially homologous to the C-terminal portion of the CS protein, but lacks the hydrophobic anchor sequence. The CS protein may lack the last 12 amino acids at the C-terminus. Additionally, it may contain four or more, for example ten or more, Asn-Ala-Asn-Pro tetrapeptide (NANP) repeat motifs.

[0173] A hybrid protein for use in the present invention may be a protein comprising a portion of the Plasmodium falciparum CS protein substantially corresponding to amino acids 207-395 of P. falciparum clone 3D7 from the NF54 strain fused in-frame to the N-terminus of HBsAg via a linear linker. The linker may comprise a portion of preS2 from HBsAg. CS constructs suitable for use in the present invention are outlined in WO 93 / 10152, patented in the United States as U.S. Pat. Nos. 5,928,902 and 6,169,171, both of which are incorporated by reference for purposes of describing proteins suitable for use in the present invention.

[0174] A particular hybrid protein for use in the present invention is the hybrid protein known as RTS (SEQ ID NO: 1, WO 2015 / 150568, WO 93 / 10152 (RTS * ) and described in WO 98 / 05355), which is - Methionine residue - 3 amino acid residues Met Ala Pro - a stretch of 189 amino acids representing amino acids 207 to 395 of the CS protein of Plasmodium falciparum strain 3D7 - 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 - a stretch of 226 amino acids encoded by nucleotides 1653 to 2330 that defines the S protein of hepatitis B virus (serotype adw) It consists of:

[0175] The RTS may be in the form of RTS,S mixed particles. RTS,S particles contain two polypeptides, RTS and S, which may be synthesized simultaneously and spontaneously form a complex particulate structure (RTS,S).

[0176] The antigen may comprise or consist of a preparation derived from a Mycobacterium species, such as Mycobacterium bovis or Mycobacterium tuberculosis, in particular Mycobacterium tuberculosis.

[0177] Antigens of interest in the field of tuberculosis include Rv1196 and Rv0125. Rv1196 (described, for example, under the name Mtb39a in Dillon et al., Infection and Immunity, 1999, 67(6): 2941-2950) is highly conserved with 100% sequence identity across strains H37Rv, C, Haarlem, CDC1551, 94-M4241A, 98-R604INH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506, and F11 with a single point mutation Q30K (most other clinical isolates share more than 90% identity with H37Rv). Rv0125 (described, for example, under the name Mtb32a in Skeiky et al., Infection and Immunity, 1999, 67(8):3998-4007) is also highly conserved with 100% sequence identity across many strains. Full-length Rv0125 contains the N-terminal signal sequence, which is cleaved to yield the mature protein.

[0178] In one embodiment, the antigen is derived from Rv1196, e.g. comprises, e.g. consists of, a sequence 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% identity to SEQ ID NO: 2. Exemplary Rv1196 related antigens include (e.g. consist of) derivatives of SEQ ID NO: 2 having small numbers of deletions, insertions and / or substitutions. Examples are 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 (e.g. consist of) fragments of SEQ ID NO: 2 that are at least 200 amino acids long, e.g. at least 250 amino acids long, especially at least 300 amino acids long, especially at least 350 amino acids long.

[0179] In one embodiment, the antigen is derived from Rv0125, e.g. comprises, e.g. consists of, a sequence 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% identity to SEQ ID NO: 3. Exemplary Rv0125 related antigens include (e.g. consist of) derivatives of SEQ ID NO: 3 having small numbers of deletions, insertions and / or substitutions. Examples are 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 Rv0125 include (e.g. consist of) fragments of SEQ ID NO: 3 that are at least 150 amino acids long, e.g. at least 200 amino acids long, especially at least 250 amino acids long, especially at least 300 amino acids long. Particular derivatives of Rv0125 include (e.g. consist of) fragments of SEQ ID NO: 3 that correspond to residues 1-195 of SEQ ID NO: 3. Further immunogenic derivatives of Rv0125 include (e.g., consist of) a fragment of SEQ ID NO:3 corresponding to residues 192-323 of SEQ ID NO:3. Particularly preferred Rv0125-related antigens are derivatives of SEQ ID NO:3 in which at least one (e.g., one, two or even all three) of the catalytic triad has been 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. Of particular interest are derivatives of SEQ ID NO:3 in which the catalytic serine residue has been substituted (e.g., substituted with alanine).Also of interest is an Rv0125 related antigen comprising, e.g. consisting of, a sequence having at least 70% identity with SEQ ID NO:3, such as at least 80%, particularly at least 90%, in particular at least 95%, such as at least 98%, for example at least 99% identity, in which at least one of the catalytic triad has been substituted or deleted, or a fragment of SEQ ID NO:3, which is at least 150 amino acids in length, such as at least 200 amino acids in length, in particular at least 250 amino acids in length, in particular at least 300 amino acids in length, in which at least one of the catalytic triad has been substituted or deleted. A further immunogenic derivative of Rv0125 comprises (e.g. consisting of) a fragment of SEQ ID NO:3 corresponding to residues 192-323 of SEQ ID NO:3, in which at least one (e.g. one, two or even all three) of the catalytic triad has been substituted or deleted. A particular immunogenic derivative of Rv0125 comprises (e.g. consists of) a fragment of SEQ ID NO:3 corresponding to residues 1 to 195 of SEQ ID NO:3, in which the catalytic serine residue (position 176 of SEQ ID NO:3) is substituted (e.g. with alanine).

[0180] Suitably the antigen comprises, e.g. consists of, a sequence having at least 70% identity to SEQ ID NO: 4, such as at least 80%, particularly at least 90%, especially at least 95%, such as at least 98%, for example at least 99% identity. Exemplary M72 related antigens include, e.g. consist of, derivatives of SEQ ID NO: 4 having small numbers of deletions, insertions and / or substitutions. Examples are deletions of up to 5 residues at positions 0 to 5, insertions of up to 5 residues at positions 0 to 5 and substitutions of up to 20 residues. Other derivatives of M72 include, e.g. consist of, fragments of SEQ ID NO: 4 that are at least 450 amino acids long, such as at least 500 amino acids long, such as at least 550 amino acids long, such as at least 600 amino acids long, for example at least 650 amino acids long or at least 700 amino acids long. M72 is a fusion protein derived from two individual antigens, Rv0125 and Rv1196, such that any fragment of at least 450 residues 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).

[0181] An M72 related antigen comprises, e.g. consists of, a sequence having at least 70% identity to SEQ ID NO:4, such as at least 80%, particularly at least 90%, especially at least 95%, such as at least 98%, for example at least 99% identity.

[0182] Exemplary M72-related antigens include, for example consist of, derivatives of SEQ ID NO: 4 having small numbers of deletions, insertions and / or substitutions, for example 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.

[0183] In certain embodiments, the M72 related antigen comprises residues 2 to 723 of SEQ ID NO:4, such as comprising (or consisting of) SEQ ID NO:4, or comprising (or consisting of) SEQ ID NO:5.

[0184] Further antigens which can be used according to the invention are the tuberculosis antigen Rv1753 and variants thereof, e.g. as described in WO2010010180, such as the Rv1753 sequences selected from SEQ ID NOs: 1 and 2-7 of WO2010010180, in particular SEQ ID NO: 1. Another antigen of interest in the field of tuberculosis is Rv2386 and variants thereof, e.g. as described in WO2010010179, such as the Rv2386 sequences selected from SEQ ID NOs: 1 and 2-7 of WO2010010179, in particular SEQ ID NO: 1. Other antigens of interest in the field of tuberculosis include Rv3616 and variants thereof, for example as described in WO2011092253, such as the native Rv3616 sequence selected from SEQ ID NOs: 1 and 2-7 of WO2011092253 or modified Rv3616 sequences, for example those selected from SEQ ID NOs: 161-169, 179 and 180 of WO2011092253, in particular SEQ ID NO: 167. An additional antigen of interest is HBHA, as described in WO97044463, WO03044048 and WO2010149657. The above-mentioned patent applications WO2010010180, WO2010010179, WO2011092253, WO97044463, WO03044048 and WO2010149657 are hereby incorporated by reference in their entirety for the purposes of defining antigens that may be useful in the present invention.

[0185] Other antigens of interest include Rv1174, also known as DPV, as set forth in SEQ ID NO:8 of WO2010010177; Rv1793, also known as MTI or Mtb9.9, as set forth in SEQ ID NO:10 of WO2010010177; Rv2087, also known as MSL or Mtb9.8, as set forth in SEQ ID NO:9 of WO2010010177; HTCCl or Mtb1, as set forth in SEQ ID NO:1 and 2-7 of WO2010010177 or SEQ ID NO:161-169, 179 or 180 of WO2011092253. 40, and / or Rv3874, also known as CFP10 or Tb38.1, as set forth in SEQ ID NO:9 in WO2010010177, or an immunogenic portion (e.g. at least 20, 50, 75 or 100 residues derived therefrom) or variant thereof (e.g. having at least 70%, 80%, 90% or 95% identity thereto) (WO2010010177 and WO2011092253 are incorporated by reference in their entireties for the purposes of defining antigens that may be useful in the present invention).

[0186] Tuberculosis antigens are most suitably utilised in the form of a polypeptide, but may alternatively be provided in the form of a polynucleotide encoding said polypeptide.

[0187] A further antigen which may be used in accordance with the present invention is derived from the Varicella Zoster Virus (VZV). The VZV antigen used in the present invention may be any suitable VZV antigen or immunogenic derivative thereof, suitably a purified VZV antigen.

[0188] 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 the signal peptide, the bulk of the protein, the hydrophobic anchor region (residues 546-558) and the C-terminal tail. In one embodiment, a gE C-terminal truncated form (also referred to as truncated gE or gE truncated form) is used, in which truncation removes between 4 and 20 percent of the total amino acid residues from the carboxy terminus. In a further embodiment, the truncated gE lacks the carboxy terminal anchor region (suitably approximately amino acids 547-623 of the wild-type sequence). In a further embodiment, the gE is a truncated gE having the sequence of SEQ ID NO:6.

[0189] The gE antigen, its anchorless derivatives (which are also immunogenic derivatives) and its preparation are described in EP 0405867 and references therein (see also Vafai A., Antibody binding sites on truncated forms of varicalla-zoster virus gpI(gE) glycoprotein, Vaccine, 1994, 12:1265-9). EP 192902 also describes gE and its preparation. 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 according to the present invention, i.e. carboxy-terminally truncated VZV gE in combination with adjuvants including QS-21, 3D-MPL and liposomes further containing cholesterol, are described in WO 2006 / 094756. Leroux-Roels I. et al. (J. Infect. Dis., 2012, 206: 1280-1290) reported on a phase I / II clinical trial evaluating an adjuvanted VZV truncated gE subunit vaccine.

[0190] The antigen may comprise or consist of a preparation derived from human respiratory syncytial virus (RSV). In some advantageous embodiments, the polypeptide antigen is an F protein polypeptide antigen derived from RSV. Particularly suitable as a polypeptide antigen component in the present invention is a conformationally restricted F polypeptide antigen. Conformationally restricted F proteins have been previously described in both prefusion (PreF) and postfusion (PostF) conformations. Such conformationally restricted F proteins typically comprise an engineered RSV F protein ectodomain. The F protein ectodomain polypeptide is a portion of the RSV F protein that contains all or a portion of the extracellular domain of the RSV F protein and lacks 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.

[0191] Exemplary F protein antigens that are conformationally restricted in the pre-fusion conformation have been described in the art and are disclosed in detail, for example, in U.S. Patent No. 8,563,002 (WO 2009079796), U.S. Patent Application Publication No. 2012 / 0093847 (WO 2010 / 149745), U.S. Patent Application Publication No. 2011 / 0305727 (WO 2011 / 008974), U.S. Patent Application Publication No. 2014 / 0141037, WO 2012 / 158613, and WO 2014 / 160463, each of which is incorporated herein by reference for purposes of describing pre-fusion F polypeptides (and nucleic acids) and methods for their production. Typically, the antigens are in the form of trimers of polypeptides. Additional publications providing examples of F proteins in the prefusion conformation include McLellan et al., Science 340:1113-1117; McLellan et al., Science 342:592-598, and Rigter et al., PLOS One 8:e71072, each of which may also be used in connection with the immunogenic combinations disclosed herein.

[0192] For example, an F protein polypeptide with a stabilized pre-fusion conformation typically comprises an ectodomain of an F protein (e.g., a soluble F protein polypeptide) that comprises at least one modification that stabilizes the pre-fusion conformation of the F protein. For example, the modification can be selected from the addition (usually to the C-terminus) of a trimerization domain, the deletion of one or more furin cleavage sites (at about amino acids 105-109 and about 133-136), the deletion of the pep27 domain, the substitution or addition of hydrophilic amino acids in a hydrophobic domain (e.g., HRA and / or HRB). In one embodiment, the conformationally restricted PreF antigen comprises an F2 domain (e.g., amino acids 1-105) and an F1 domain (e.g., amino acids 137-516) of a RSV F protein polypeptide that is not interrupted by a furin cleavage site, and the polypeptide further comprises a heterologous trimerization domain located C-terminal to the F1 domain. Optionally, the PreF antigen also includes a modification that alters glycosylation (e.g., increases glycosylation), such as a substitution of one or more amino acids at a position corresponding to approximately amino acids 500-502 of the RSV F protein. When an oligomerization sequence is present, a trimerization sequence is preferred. Suitable oligomerization sequences are known in the art, and include, for example, the coiled coil of the yeast GCN4 leucine zipper protein, the trimerization sequence from bacteriophage T4 fibritin ("foldon"), and the trimer domain of influenza HA. ​​Additionally or alternatively, the conformationally restricted F polypeptide in the pre-fusion conformation can include at least two introduced cysteine ​​residues that are adjacent to each other and form a disulfide bond that stabilizes the pre-fusion RSV F polypeptide. For example, the two cysteines can be within about 10 Å of each other. For example, cysteines can be introduced at positions 165 and 296 or positions 155 and 290. A representative PreF antigen is represented by SEQ ID NO:7.

[0193] The antigen may comprise or consist of a preparation derived from HIV. The antigen may be an HIV protein, such as an HIV envelope protein. For example, the antigen may be an HIV envelope gp120 polypeptide or an immunogenic fragment thereof.

[0194] One suitable antigen is the HIV clade B gp120 polypeptide of SEQ ID NO:8 of published application WO 2008 / 107370 (or an immunogenic fragment of this polypeptide), SEQ ID NO:8 of WO 2008 / 107370 being incorporated by reference into this application.

[0195] Suitable antigens are also polypeptides comprising the V1V2 region of SEQ ID NO: 1 of published application WO 2015 / 036061, or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 1. In addition, polypeptides comprising the V1V2 region of SEQ ID NO: 5 of WO 2015 / 036061, or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 5 may be used as suitable antigens. SEQ ID NO: 1 and SEQ ID NO: 5 of WO 2015 / 036061 are incorporated by reference.

[0196] 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).

[0197] Other suitable HIV antigens include the Nef, Gag and Pol HIV proteins, and immunogenic fragments thereof.

[0198] Compositions include, for example, fimbrin proteins [(U.S. Pat. No. 5,766,608, Ohio State Research Foundation)] and fusions comprising peptides derived therefrom [e.g., LB1(f) peptide fusions, U.S. Pat. No. 5,843,464 (OSU) or WO 99 / 64067], OMP26 [WO 97 / 01638 (Cortecs)], P6 [European Patent No. 281673 (State University of New York)], In some embodiments, the antigen may comprise a non-capsulated Haemophilus influenzae antigen selected from: Hsf, Hin47, Hif, Hmw1, Hmw2, Hmw3, Hmw4, Hap, D15 (WO 94 / 12641), protein D (EP 594610), P2 and P5 (WO 94 / 26304), protein E (WO 07 / 084053) and / or PilA (WO 05 / 063802).The compositions may be any of the following: OMP106 [WO 97 / 41731 (Antex) and WO 96 / 34960 (PMC)], OMP21, LbpA and / or LbpB [WO 98 / 55606 (PMC)], TbpA and / or TbpB [WO 97 / 13785 and WO 97 / 32980 (PMC)], CopB [Helminen ME et al., (1993), Infect. Immun., 61:2003-2010], UspA1 and / or UspA2 [WO 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), The invention may include a Moraxella catarrhalis protein antigen 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.

[0199] In one embodiment, the composition may comprise a non-capsulated H. influenzae (NTHi) protein antigen and / or a M. catarrhalis protein antigen. The composition may comprise protein D (PD) from H. influenzae. Protein D may be as described in WO 91 / 18926. The composition may further comprise protein E (PE) and / or pilin A (PilA) from H. influenzae. Protein E and pilin A may be as described in WO 2012 / 139225. Protein E and pilin A may be presented as a fusion protein, for example LVL735 as described in WO 2012 / 139225. For example, the composition may comprise three NTHi antigens (PD, PE and PilA, the latter two combined as a PEPilA fusion protein). The composition may further comprise UspA2 from M. catarrhalis. The UspA2 may be as described in WO2015125118, such as MC-009 ((M)(UspA2 31-564)(HH)) as described in WO2015125118. For example, the composition may include three NTHi antigens (PD, PE and PilA, the latter two combined as a PEPilA fusion protein) and one Moraxella catarrhalis antigen (UspA2).

[0200] Multiple antigens may be provided. For example, multiple antigens may be provided to strengthen the immune response induced (e.g. to ensure robust protection), to broaden the immune response (e.g. to ensure protection against a broad range of pathogen strains or in a large proportion of a population of subjects), or to broadly provoke an immune response against several disorders (thereby simplifying administration protocols). When multiple antigens are provided, these may be distinct proteins or may be in the form of one or more fusion proteins.

[0201] The antigen may be provided in an amount of 0.1-100 ug per human dose.

[0202] The present invention may be applied for use in the treatment or prevention of a disease or disorder associated with one or more of the above antigens. In one embodiment, the disease or disorder is selected from malaria, tuberculosis, COPD, HIV and herpes.

[0203] The adjuvant may be administered separately from the immunogen or antigen or may be combined with the immunogen or antigen during manufacture or in situ to provide an immunogenic composition for combined administration.

[0204] Sterilization (sterilization) The compositions must be sterile, particularly for parenteral administration. Sterilization can be accomplished in a variety of ways, but is conveniently accomplished by filtration through a sterile grade filter. Sterilization may be performed any number of times during the preparation of the adjuvant or immunogenic composition, but is usually performed at least at the end of production.

[0205] A "sterile grade filter" is one with an effective filtration area of ​​1 cm 2 1x10 7 It is meant a filter which produces a sterile effluent after challenge with microorganisms at or above the challenge level. Sterile grade filters are known to those skilled in the art for the purposes of the present invention, and have a pore size between 0.15-0.25 um, suitably 0.18-0.22 um, for example 0.2 or 0.22 um.

[0206] The membrane of the sterile grade filter can be made of any suitable material known to those skilled in the art, including but not limited to cellulose acetate, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE). In certain embodiments of the invention, one or more or all of the filter membranes of the invention comprise polyethersulfone (PES), particularly hydrophilic polyethersulfone. In certain embodiments of the invention, the filter used in the methods described herein is a bilayer filter, particularly a sterile filter having a build-in prefilter with a pore size larger than the pore size of the end filter. In one embodiment, the sterile filter is a bilayer filter in which the prefilter membrane layer has a pore size between 0.3 and 0.5 nm, e.g., 0.35 or 0.45 nm. According to further embodiments, the filter comprises an asymmetric filter membrane, e.g., an asymmetric hydrophilic PES filter membrane. Alternatively, the sterile filter layer may be made of PVDF, e.g., in combination with an asymmetric hydrophilic PES prefilter membrane layer.

[0207] Having regard to the intended medical use, the materials should be of pharmaceutical grade (eg, parenteral grade).

[0208] The present invention is exemplified by the following items.

[0209] Item 1. A saponin extract containing at least 88% of the QS-21 main peak and more than 3% to 10% of the 2018 component by UV absorbance at 214 nm.

[0210] Item 2. The saponin extract according to item 1, containing at least 3.5% of the 2018 component.

[0211] Item 3. The saponin extract according to item 2, containing at least 4% of the 2018 component.

[0212] Item 4. The saponin extract according to item 4, containing at least 4.5% of the 2018 component.

[0213] Item 5. The saponin extract according to any one of items 1 to 4, containing 9% or less of the 2018 component.

[0214] Item 6. The saponin extract according to Item 5, containing 8% or less of the 2018 component.

[0215] Item 7. The saponin extract according to Item 6, containing 7% or less of the 2018 component.

[0216] Item 8. The saponin extract according to any one of items 1 to 7, containing at least 90%, such as at least 91%, at least 92% or at least 93% of the QS-21 main peak.

[0217] Item 9. The saponin extract according to any one of items 1 to 8, wherein the monoisotope of the most abundant chemical species is 1987.9 m / z.

[0218] Item 10. The saponin extract of any one of items 1 to 9, containing at least 98%, at least 99%, at least 99.5%, or at least 99.8% QS-21 groups by UV absorbance at 214 nm.

[0219] Item 11. The saponin extract of any one of items 1 to 10, containing less than 1% lyo impurity by UV absorbance at 214 nm.

[0220] Item 12. The saponin extract according to Item 11, containing less than 1% of the largest peak other than the QS-21 group by UV absorbance at 214 nm.

[0221] Item 13. The saponin extract according to Item 1, which contains at least 98% of the QS-21 group by UV absorbance at 214 nm, at least 88% of the main QS-21 peak, greater than 3% to 10% 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.

[0222] Item 14. The saponin extract according to any one of items 1 to 13, containing at least 40%, such as at least 50%, particularly at least 60%, especially at least 65%, such as at least 70% of the 1988 component by UV absorbance at 214 nm and by relative ion abundance.

[0223] Item 15. The saponin extract according to any one of items 1 to 14, containing 90% or less, such as 85% or less, or 80% or less of the 1988 components by UV absorbance at 214 nm and by relative ion abundance.

[0224] Item 16. The saponin extract of any one of items 1 to 15, containing 30% or less, such as 25% or less, of 1856 components by UV absorbance at 214 nm and by relative ion abundance.

[0225] Item 17. The saponin extract of any one of items 1 to 16, containing at least 5%, such as at least 10%, of the 1856 component by UV absorbance at 214 nm and by relative ion abundance.

[0226] Item 18. The saponin extract according to any one of items 1 to 17, containing 40% or less, such as 30% or less, particularly 20% or less, especially 10% or less of the 2002 component by UV absorbance at 214 nm and by relative ion abundance.

[0227] Item 19. The saponin extract of any one of items 1 to 18, containing at least 0.5%, such as at least 1%, of the 2002 component by UV absorbance at 214 nm and by relative ion abundance.

[0228] Item 20. A saponin extract containing at least 88%, for example at least 90%, at least 91%, at least 92% or at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9 by UV absorbance at 214 nm, as well as more than 3% to 10% of triterpenoid glycosides having an m / z of 2017.9.

[0229] Item 21. The saponin extract according to item 20, wherein the monoisotope of the most abundant chemical species is 1987.9 m / z.

[0230] Item 22. The saponin extract according to item 20 or 21, containing at least 88% triterpenoid glycosides having an m / z of 1855.9, 1987.9 or 2001.9, excluding B-isomers and lyo impurities, by UV absorbance at 214 nm.

[0231] Item 23. The saponin extract according to any one of items 20 to 22, containing at least 98% triterpenoid glycosides having an m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118 by UV absorbance at 214 nm.

[0232] Item 24. The saponin extract according to any one of items 20 to 23, containing at least 98% triterpenoid glycosides having an m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118, excluding lyo impurities, by UV absorbance at 214 nm.

[0233] Item 25. The saponin extract according to any one of items 20 to 24, containing triterpenoid glycosides having an m / z of lyo impurity of 1% or less by UV absorbance at 214 nm.

[0234] Item 26. The saponin extract according to Item 25, containing 1% or less of other peaks as determined by UV absorbance at 214 nm.

[0235] Item 27. The saponin extract according to Item 20, which contains at least 98% triterpenoid glycosides having m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 or 2118, at least 88% triterpenoid glycosides having m / z of 1855.9, 1987.9 or 2001.9, more than 3% to 10% triterpenoid glycosides having m / z of 2017.9, and 1% or less other peaks, and the monoisotope of the most abundant chemical species is 1987.9 m / z, by UV absorbance at 214 nm.

[0236] Item 28. The saponin extract according to any one of items 20 to 27, containing at least 40%, such as at least 50%, particularly at least 60%, especially at least 65%, such as at least 70% of the 1988 component by UV absorbance at 214 nm and by relative ion abundance.

[0237] Item 29. The saponin extract of any one of items 20 to 28, containing 90% or less, such as 85% or less, or 80% or less of the 1988 components by UV absorbance at 214 nm and by relative ion abundance.

[0238] Item 30. The saponin extract of any one of items 20 to 29, containing 30% or less, such as 25% or less, of the 1856 components by UV absorbance at 214 nm and by relative ion abundance.

[0239] Item 31. The saponin extract of any one of items 20 to 30, containing at least 5%, such as at least 10%, of the 1856 component by UV absorbance at 214 nm and by relative ion abundance.

[0240] Item 32. The saponin extract of any one of items 20 to 31, containing 10% or less, such as 5% or less, of the 2002 component by UV absorbance at 214 nm and by relative ion abundance.

[0241] Item 33. The saponin extract of any one of items 20 to 32, containing at least 0.5%, such as at least 1%, of the 2002 component by UV absorbance at 214 nm and by relative ion abundance.

[0242] Item 34. The saponin extract according to any one of items 20 to 33, comprising at least 3.5% triterpenoid glycoside having an m / z of 2017.9 by UV absorbance at 214 nm.

[0243] Item 35. The saponin extract according to item 34, containing at least 4% of a triterpenoid glycoside having an m / z of 2017.9 by UV absorbance at 214 nm.

[0244] Item 36. The saponin extract according to item 35, containing at least 4.5% triterpenoid glycoside having an m / z of 2017.9 by UV absorbance at 214 nm.

[0245] Item 37. The saponin extract according to any one of items 20 to 36, containing 9% or less of a triterpenoid glycoside having an m / z of 2017.9 by UV absorbance at 214 nm.

[0246] Item 38. The saponin extract according to item 37, containing 8% or less of a triterpenoid glycoside having an m / z of 2017.9 by UV absorbance at 214 nm.

[0247] Item 39. The saponin extract according to item 38, containing 7% or less of a triterpenoid glycoside having an m / z of 2017.9 by UV absorbance at 214 nm.

[0248] Item 40. At least 88%, for example at least 90%, at least 91%, at least 92% or at least 93% by UV absorbance at 214 nm.

[0249] [ka] and over 3% to 10%

[0250] [ka] A saponin extract containing:

[0251] Item 41. The saponin extract according to item 40, wherein the monoisotope of the most abundant chemical species is 1987.9 m / z.

[0252] Item 42. At least 98%

[0253] [ka] 42. The saponin extract according to item 40 or 41, containing TIFF0007640473000059.tif191132TIFF0007640473000060.tif163139.

[0254] Item 43. At least 98%

[0255] [ka] TIFF0007640473000062.tif226147 and 2118 components are contained in the saponin extract according to any one of items 40 to 42.

[0256] Item 44. Less than 1%

[0257] [ka] 44. The saponin extract according to any one of items 40 to 43, comprising:

[0258] Item 45. The saponin extract according to Item 44, containing 1% or less of other peaks as determined by UV absorbance at 214 nm.

[0259] Item 46. At least 98% of the samples were analyzed by UV absorbance at 214 nm.

[0260] [ka] TIFF0007640473000065.tif251143TIFF0007640473000066.tif88143At least 88%, for example at least 90%, at least 91%, at least 92% or at least 93%

[0261] [ka] TIFF0007640473000068.tif1351353% to 10%

[0262] [ka] 41. The saponin extract according to item 40, containing 1% or less of other peaks and having a monoisotope of 1987.9 m / z as the most abundant chemical species.

[0263] Item 47. At least 98% of the samples were analyzed by UV absorbance at 214 nm.

[0264] [ka] TIFF0007640473000071.tif220140 or 2118 components, At least 88%, for example at least 90%, at least 91%, at least 92% or at least 93%

[0265] [ka] TIFF0007640473000073.tif631403% to 10%

[0266] [ka] 41. The saponin extract according to item 40, containing 1% or less of other peaks and having a monoisotope of 1987.9 m / z as the most abundant chemical species.

[0267] Item 48. At least 40%, for example at least 50%, in particular at least 60%, especially at least 65%, for example at least 70%, by UV absorbance at 214 nm and by relative ion abundance.

[0268] [ka] 48. The saponin extract according to any one of items 40 to 47, containing TIFF0007640473000076.tif60155.

[0269] Item 49. Not more than 30%, e.g. not more than 25%, by UV absorbance at 214 nm and by relative ion abundance

[0270] [ka] 49. The saponin extract according to any one of items 40 to 48, comprising:

[0271] Item 50. At least 5%, e.g. at least 10%, by UV absorbance at 214 nm and by relative ion abundance

[0272] [ka] 50. The saponin extract according to any one of items 40 to 49, comprising:

[0273] Item 51. By UV absorbance at 214 nm and by relative ion abundance, 40% or less, for example 30% or less, in particular 20% or less, especially 10% or less

[0274] [ka] 51. The saponin extract according to any one of items 40 to 50, comprising:

[0275] Item 52. At least 0.5%, e.g. at least 1%, by UV absorbance at 214 nm and by relative ion abundance

[0276] [ka] 52. The saponin extract according to any one of items 40 to 51, comprising:

[0277] Item 53. At least 3.5%, e.g., at least 4% or at least 4.5%, by UV absorbance at 214 nm.

[0278] [ka] 53. The saponin extract according to any one of items 40 to 52, comprising:

[0279] Item 54. UV absorbance at 214 nm is 9% or less, e.g., 8% or less or 7% or less.

[0280] [ka] 54. The saponin extract according to any one of items 40 to 53, comprising:

[0281] Item 55. The saponin extract according to any one of items 1 to 54, which is dried.

[0282] Item 56. A method for identifying a crude aqueous extract of Quillaja Saponaria Molina for use in producing a purified saponin extract, comprising: (i) determining the ratio of 2018 component / QS-21 main peak by UPLC-UV absorbance at 214 nm; and (ii) selecting crude aqueous extracts having a ratio of 2018 components / QS-21 main peak greater than 0.075; The method includes:

[0283] Item 57. The method according to item 56, wherein the crude aqueous extract selected in step (ii) has a ratio of 2018 components / QS-21 main peak of more than 0.078.

[0284] Item 58. The method according to item 56 or 57 for use in producing the saponin extract according to any one of items 1 to 55.

[0285] Item 59. (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a ratio of 2018 components to the main peak of QS-21 of greater than 0.075; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; and (v) purifying the extract by reverse phase chromatography on phenyl resin; A method for producing a purified saponin extract comprising:

[0286] Item 60. The method of item 59, wherein step (iii) uses purification by diafiltration.

[0287] Item 61. The method according to item 59 or 60, wherein step (iv) uses acetonitrile and water, particularly under gradient conditions.

[0288] Item 62. The method according to any one of items 59 to 61, wherein step (v) uses acetonitrile and water, particularly under isocratic conditions.

[0289] Item 63. The method of any one of items 59 to 62, comprising the additional step of removing the solvent to provide a dried saponin extract.

[0290] Item 64. The method of item 63, wherein the step of removing the solvent uses lyophilization.

[0291] Item 65. The method of any one of items 59 to 64, comprising the additional step of concentrating the extract by reverse phase chromatography using a C8 resin.

[0292] Item 66. The method according to item 65, wherein the step of concentrating the extract by reverse phase chromatography using a C8 resin uses acetonitrile and water, particularly under step gradient conditions.

[0293] Item 67. The method of items 65 or 66, comprising the additional step of exchanging the solvent.

[0294] Item 68. The method of item 67, wherein the step of exchanging the solvent uses diafiltration, ultrafiltration or dialysis, especially diafiltration.

[0295] Item 69. The method according to any one of items 59 to 68, wherein the crude aqueous extract of Quillaja Saponaria Molina has a ratio of 2018 components to QS-21 main peak of more than 0.078.

[0296] Item 70. The method according to any one of items 59 to 69, 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, in particular at least 2.5 g / L, particularly at least 2.8 g / L of QS-21 main peak.

[0297] Item 71. The method according to any one of items 59 to 70, wherein the crude aqueous extract of Quillaja Saponaria Molina is a bark extract.

[0298] Item 72. The method according to any one of items 59 to 71, wherein the step (i) of selecting a crude aqueous extract of Quillaja Saponaria Molina comprises testing the crude aqueous extract composition to determine the 2018 component content.

[0299] Item 73. Use of the saponin extract according to any one of items 1 to 55 in the manufacture of a medicament, such as an adjuvant or an immunogenic composition.

[0300] Item 74. The saponin extract according to any one of items 1 to 55 for use as an adjuvant.

[0301] Item 75. An adjuvant composition comprising the saponin extract of any one of items 1 to 55.

[0302] Item 76. The use, saponin extract or adjuvant composition according to any one of items 73 to 75, wherein the adjuvant is a liposomal formulation.

[0303] Item 77. The use, saponin extract or adjuvant composition of any one of items 73 to 76, wherein the adjuvant comprises a TLR4 agonist.

[0304] Item 78. The use, saponin extract or adjuvant composition according to item 77, wherein the TLR4 agonist is 3D-MPL.

[0305] Item 79. An immunogenic composition comprising the adjuvant composition according to any one of items 75 to 78 and an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

[0306] Item 80. The immunogenic composition according to item 79, wherein the antigen is derived from a human or non-human pathogen, including, for example, a bacterium, a fungus, a parasitic microorganism or a multicellular parasite that infects humans and non-human vertebrates, or a cancer cell or a tumor cell.

[0307] Item 81. The immunogenic composition according to item 80, wherein the antigen is derived from Plasmodium falciparum or Plasmodium vivax, for example the antigen of SEQ ID NO: 1.

[0308] Item 82. The immunogenic composition according to item 80, wherein the antigen is derived from a species of the genus Mycobacterium, for example from any one of SEQ ID NOs: 2 to 5.

[0309] Item 83. The immunogenic composition according to item 80, wherein the antigen is derived from the Varicella-Zoster virus, for example the antigen of SEQ ID NO:6.

[0310] Item 84. The immunogenic composition of item 80, wherein the antigen is derived from a human respiratory syncytial virus, such as the antigen of SEQ ID NO: 7.

[0311] Item 85. The immunogenic composition according to item 80, wherein the antigen is derived from HIV, for example an antigen of SEQ ID NO: 8 or 9.

[0312] Item 86. The immunogenic composition of item 80, wherein the antigen is derived from non-encapsulated Haemophilus influenzae (NTHi) and / or Moraxella catarrhalis.

[0313] Item 87. The immunogenic composition of any one of items 79 to 86, wherein the antigen is provided as a polypeptide.

[0314] Item 88. The immunogenic composition of any one of items 79 to 86, wherein a polynucleotide encoding the polypeptide antigen is provided.

[0315] Item 89. A method for determining the ratio of 2018 components / QS-21 main peak in a crude aqueous extract of Quillaja Saponaria Molina, comprising: (i) determining the 2018 component content in the crude aqueous extract of Quillaja Saponaria Molina by UPLC-UV absorbance at 214 nm; (ii) determining the QS-21 main peak content in the crude aqueous extract of Quillaja Saponaria Molina by UPLC-UV absorbance at 214 nm; and (iii) comparing the 2018 component content with the QS-21 main peak content to determine the 2018 component / QS-21 main peak ratio. The method includes:

[0316] Item 90. A method for producing a purified saponin extract containing at least 88% of the QS-21 main peak and more than 3-10% of the 2018 component by UV absorbance at 214 nm, comprising: (i) selecting a Quillaja Saponaria Molina material having a suitable 2018 ingredient composition; (ii) preparing a crude aqueous extract of Quillaja Saponaria Molina material under suitable conditions; and (iii) purifying the crude aqueous extract to obtain a purified saponin extract The method includes:

[0317] Item 91. The method according to item 90, for producing the saponin extract according to any one of items 1 to 55.

[0318] The teachings of all references, including patent applications and allowed patents, contained herein are incorporated herein in their entirety by reference. A composition or method or process defined as "comprising" a certain element is understood to encompass a composition, method, or process (respectively) consisting of that element. As used herein, "consisting essentially of" means that additional components may be present, provided that they do not alter the nature or function of the whole. An embodiment of the present invention will be described below. [1] A saponin extract containing (i) at least 88% of the QS-21 main peak and (ii) greater than 3% to 10% of the 2018 component, as determined by UV absorbance at 214 nm. [2] The saponin extract described in [1] containing at least 4.5% of the 2018 component. [3] A saponin extract according to [1] or [2] containing 9% or less of the 2018 component. [4] The saponin extract according to [1], which contains at least 98% of the QS-21 group, at least 88% of the main QS-21 peak, greater than 3% to 10% of the 2018 component, and 1% or less of the largest peak other than the QS-21 group, as determined by UV absorbance at 214 nm, and the monoisotope of the most abundant chemical species is 1987.9 m / z. [5] A saponin extract containing, by UV absorbance at 214 nm, (i) at least 88%, e.g., at least 90%, at least 91%, at least 92% or at least 93% of triterpenoid glycosides having an m / z of 1855.9, 1987.9 and / or 2001.9, and (ii) greater than 3% to 10% of triterpenoid glycosides having an m / z of 2017.9. [6] The saponin extract according to [5], which contains at least 98% triterpenoid glycosides having m / z of 1517.7, 1711.8, 1855.9, 1987.9, 2001.9, 2017.9 and / or 2118, at least 88% triterpenoid glycosides having m / z of 1855.9, 1987.9 and / or 2001.9, greater than 3% to 10% triterpenoid glycosides having m / z of 2017.9, and 1% or less other peaks, by UV absorbance at 214 nm, and the monoisotope of the most abundant chemical species is 1987.9 m / z. [7] By UV absorbance at 214 nm, (i) at least 88%, e.g., at least 90%, at least 91%, at least 92%, or at least 93% [C1] TIFF0007640473000083.tif247143 and (ii) more than 3% to 10% [C2] TIFF0007640473000084.tif79166 A saponin extract containing: [8] At least 98% [C3] TIFF0007640473000085.tif120133TIFF0007640473000086.tif189140TIFF0007640473000087.tif164132 The saponin extract described in [7] contains: [9] At least 98% of the samples were analyzed by UV absorbance at 214 nm. [C4] TIFF0007640473000088.tif63138TIFF0007640473000089.tif252145TIFF0007640473000090.tif158138 、 At least 88%, for example at least 90%, at least 91%, at least 92% or at least 93% [5] TIFF0007640473000091.tif60139TIFF0007640473000092.tif193140 、 Over 3% to 10% [6] TIFF0007640473000093.tif62135 、 Other peaks below 1% The saponin extract according to [7] or [8], wherein the monoisotope of the most abundant chemical species is 1987.9 m / z.

[10] At least 98% of the samples were analyzed by UV absorbance at 214 nm. [7] TIFF0007640473000094.tif120144TIFF0007640473000095.tif132143TIFF0007640473000096.tif219135 and / or 2118 components, At least 88%, for example at least 90%, at least 91%, at least 92% or at least 93% [8] TIFF0007640473000097.tif242126 、 Over 3% to 10% [9] TIFF0007640473000098.tif63128 、 Other peaks below 1% The saponin extract according to any one of [7] to [9], wherein the monoisotope of the most abundant chemical species is 1987.9 m / z.

[11] At least 3.5%, e.g., at least 4% or at least 4.5%, by UV absorbance at 214 nm. [C10] TIFF0007640473000099.tif63128 The saponin extract according to any one of [7] to

[10] , comprising:

[12] Less than 9% by UV absorbance at 214 nm, e.g., less than 8% or less than 7% [C11] TIFF0007640473000100.tif60140 The saponin extract according to any one of

[10] to

[11] , comprising:

[13] A saponin extract according to any one of [1] to

[12] , containing at least 40%, such as at least 50%, particularly at least 60%, especially at least 65%, such as at least 70% of the 1988 component based on UV absorbance at 214 nm and relative ion abundance.

[14] A saponin extract according to any one of [1] to

[13] , containing 90% or less, for example 85% or less, or 80% or less of the 1988 component based on UV absorbance at 214 nm and relative ion abundance.

[15] A saponin extract according to any one of [1] to

[14] , containing 30% or less, for example 25% or less, of 1856 components based on UV absorbance at 214 nm and relative ion abundance.

[16] A saponin extract according to any one of [1] to

[15] , containing at least 5%, for example at least 10%, of the 1856 component based on UV absorbance at 214 nm and relative ion abundance.

[17] A saponin extract according to any one of [1] to

[16] , containing 10% or less, for example 5% or less, of a 2002 component based on UV absorbance at 214 nm and relative ion abundance.

[18] A saponin extract according to any one of [1] to

[17] , containing at least 0.5%, for example at least 1%, of the 2002 component based on UV absorbance at 214 nm and relative ion abundance.

[19] A method for identifying a crude aqueous extract of Quillaja Saponaria Molina for use in producing a purified saponin extract, comprising: (i) determining the ratio of 2018 component / QS-21 main peak by UPLC-UV absorbance at 214 nm; and (ii) selecting crude aqueous extracts having a ratio of 2018 components / QS-21 main peak greater than 0.075; The method includes:

[20] (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a ratio of 2018 components to a QS-21 main peak of greater than 0.075; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; and (v) purifying the extract by reverse phase chromatography on phenyl resin. A method for producing a purified saponin extract comprising:

[21] Use of the saponin extract according to any one of [1] to

[17] in the manufacture of a medicament, such as an adjuvant or immunogenic composition.

[22] A saponin extract according to any one of [1] to

[17] for use as an adjuvant.

[23] An adjuvant composition comprising the saponin extract described in any one of [1] to

[17] .

[24] The use, saponin extract or adjuvant composition according to any one of

[21] to

[23] , wherein the adjuvant is a liposomal preparation.

[25] The use, saponin extract or adjuvant composition according to any one of

[21] to

[23] , wherein the adjuvant comprises a TLR4 agonist, such as a lipopolysaccharide TLR4 agonist.

[26] The use, saponin extract or adjuvant composition according to

[25] , wherein the TLR4 agonist is 3D-MPL.

[27] An immunogenic composition comprising the adjuvant composition according to any one of

[23] to

[26] and an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

[28] A method for determining the ratio of 2018 component / QS-21 main peak in a crude aqueous extract of Quillaja Saponaria Molina, comprising: (i) determining the 2018 component content in the crude aqueous extract of Quillaja Saponaria Molina by UPLC-UV absorbance at 214 nm; (ii) determining the QS-21 main peak content in the crude aqueous extract of Quillaja Saponaria Molina by UPLC-UV absorbance at 214 nm; and (iii) comparing the 2018 component content with the QS-21 main peak content to determine a 2018 component / QS-21 main peak ratio. The method includes:

[29] A method for producing a purified saponin extract containing at least 88% of the QS-21 main peak and more than 3-10% of the 2018 component by UV absorbance at 214 nm, comprising: (i) selecting a Quillaja Saponaria Molina material having a suitable 2018 ingredient composition; (ii) preparing a crude aqueous extract of said Quillaja Saponaria Molina material under suitable conditions; and (iii) purifying the crude aqueous extract to obtain a purified saponin extract. The method includes:

[0319] The invention will now be further described with reference to the following non-limiting examples. EXAMPLES

[0320] [Example 1] HPLC of the crude aqueous extract of Quillaja Saponaria Molina The crude bark extract was separated by reversed-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 at 214 nm.

[0321] The crude bark extract samples were diluted with purified water as necessary. Polyvinylpolypyrrolidone (PVPP, 60 mg / mL) was added and the mixture was stirred for approximately 30 minutes and then centrifuged to separate the PVPP resin from the supernatant.

[0322] The supernatant was then analyzed to obtain an HPLC UV chromatogram.

[0323] Figure 1 shows a representative HPLC UV chromatogram, with peaks corresponding to the QS-21 fraction indicated.

[0324] [Example 2] Analysis method HPLC-UV device Waters Alliance 2690 / 2695 Separation 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

[0325] TIFF0007640473000101.tif41131

[0326] Inject 40 ul of sample. UV detection is set at 214 nM.

[0327] Using a blank injection as a reference, integration of the peaks in the chromatogram provides the total absorbance. The peak of interest (e.g., the main QS-21 peak) is compared to the total absorbance to determine the peak content as a percentage.

[0328] It is also convenient to use the HPLC-UV method to determine the QS-21 main peak content and the ratio of preceding peak to QS-21 main peak.

[0329] UPLC-UV device 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)

[0330] TIFF0007640473000102.tif49130

[0331] Column temperature 28° C. Inject 10 ul of sample. UV detection is set at 214 nM.

[0332] Using a blank injection as a reference, integration of the chromatogram peaks provides the total absorbance. The peak of interest (e.g., the main QS-21 peak) is compared to the total absorbance to determine the peak content as a percentage.

[0333] It is also convenient to determine the 2018 / QS-21 ratio using the UPLC-UV method.

[0334] UPLC-UV / MS device Waters Acquity UPLC Waters Acquity Tunable UV Detector Waters Single-Quadrupole Mass Detector SQD1 (scan range 1400~2040M / Z) 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)

[0335] TIFF0007640473000103.tif43130

[0336] Test samples are prepared as 0.2% acetic acid in water / acetonitrile (70:30 v / v). Column temperature 55° C. 10 ul of sample is injected. UV detection is set at 214 nM.

[0337] While retention times vary somewhat between runs, the QS-21 group is located at approximately 3.8 minutes (B-isomer) to approximately 4.5 minutes (before the lyo impurity).

[0338] Using a blank injection for reference, perform integration of chromatogram peaks that elute between approximately 0.5 min and 5.50 min after the solvent front but are not seen in the blank.

[0339] The monoisotopes of the most abundant species are identified by combining the TICs across the chromatograms to generate a combined spectrum.

[0340] The ratio of the 2002 to 1988 components is calculated by comparing the ion current associated with the 2002 component to the ion current associated with the 1988 component in the QS-21 main peak.

[0341] Figure 5 shows a chromatogram of a representative saponin extract, and Figure 6 shows an expanded detail of the region containing the QS-21 group and lyo components.

[0342] Figures 7A and 7B show extracted mass chromatograms for the 1988 (Figure 7A) and 2002 (Figure 7B) molecular weight ions of a representative purified Quillaja Saponaria Molinasaponin extract.

[0343] [Example 3] Purification of the crude aqueous extract of Quillaja Saponaria Molina A crude aqueous extract of Quillaja Saponaria Molina material having a ratio of 2018 components to the main QS-21 peak of 0.064 or less and a ratio of preceding peaks to the main QS-21 peak of 0.4 or less was treated with PVPP (1 kg PVPP per liter of crude aqueous extract). After adsorption, the mixture was filtered to separate the PVPP and bound impurities from the liquid.

[0344] FIG. 2 shows an example of an HPLC-UV chromatogram for a crude aqueous extract of Quillaja Saponaria Molina (used for determining the ratio of leading peak to main QS-21 peak and the main QS-21 peak content).

[0345] Figure 3 shows an example of a UPLC-UV chromatogram for a crude aqueous extract of Quillaja Saponaria Molina (used for the determination of the ratio of 2018 components to the QS-21 main peak).

[0346] The filtrate was concentrated and further purified by UF / DF using a 30 kD Helicon membrane.

[0347] TIFF0007640473000104.tif95130

[0348] The fractions were pooled to obtain a polystyrene purified saponin extract with the following composition: % QS-21 main peak ≥ 18% (by HPLC). and The ratio of 2018 components to the main peak of QS-21 was ≦0.054 (by UPLC-UV).

[0349] FIG. 4 shows an example UPLC-UV chromatogram for a pool of polystyrene purified saponin extracts.

[0350] TIFF0007640473000105.tif77134

[0351] The fractions were pooled to give a phenyl-purified saponin extract with the following composition: %QS-21 group ≥98.5 %QS-21 main peak ≧94.5 %2018 ingredients≦2.7% Major peaks other than QS-21 group ≦1% (by UPLC-UV / MS).

[0352] The combined phenyl purified saponin extracts were concentrated by capture and release by reversed phase chromatography using C8 resin (Lichroprep RP8) and the following conditions: Load onto column conditioned with 24% acetonitrile and 0.20% acetic acid. Elute with 60% acetonitrile and 0.20% acetic acid. 11cm column Load: 50-142g per injection

[0353] The C8 concentrated saponin extract was subjected to ultrafiltration / diafiltration and solvent exchange using a Pellicon 1 kDa membrane to reduce the acetonitrile content to less than 21%.

[0354] The resulting solvent-exchanged saponin extract was then freeze-dried in a single step to obtain the purified final saponin extract product.

[0355] Starting with an appropriate crude extract and / or applying component spiking, the process described in Example 3 can be used to consistently provide a purified saponin extract of Quillaja Saponaria Molina having well-defined contents in terms of QS-21 main peak and 2018 components, e.g., consistently at least 88% QS-21 main peak and greater than 3-10% 2018.

[0356] [Example 4] Purified QS-21 saponin extract with high content of 2018 4.1 Preparation of purified saponin extracts enriched in 2018 components Reverse phase chromatography was used to prepare a purified saponin extract enriched in the 2018 components. Starting with a purified QS-21 saponin extract having a low content of 2018 components prepared according to Example 3 (see FIG. 9), fractions corresponding to the 2018 peak were collected over multiple runs to provide Batch A and Batch B of purified saponin extract enriched in the 2018 components (or "2018 concentrate").

[0357] System: UPLC with fraction collector Waters Column: UPLC Waters Acquity BEH C18, 2.1mm x 100mm, 1.7um Mobile phase: A: Water / ACN / IPA (75 / 20 / 5, V / V / V), 0.025%TFA B: Water / ACN / IPA (10 / 72 / 18, V / V / V), 0.025%TFA Flow rate: 0.6mL / min Column oven temperature: 55℃ Injection volume: 10 μl Detector UV: Wavelength 214nm gradient

[0358] TIFF0007640473000106.tif52129

[0359] Batch A was 300 μl with a total concentration of 22.6 μg / ml. Batch B was 750 μl with a total concentration of 66.4 μg / ml. Batches A and B were analyzed by UPLC-UV and then combined to obtain the final 2018 concentrate.

[0360] result The UPLC-UV analysis of batches A and B is shown in Figures 10A (batch A) and 10B (batch B).

[0361] TIFF0007640473000107.tif38157

[0362] 4.2 Spiking of Purified QS-21 Saponin Extracts Having Low Contents of 2018 Components with Increasing Percentages of Purified Saponin Extracts Enriched in 2018 Components The 2018 concentrate obtained in section 4.1 was combined in various proportions with the purified QS-21 saponin extract having a low content of 2018 components (Figure 9) to obtain seven QS-21 saponin extracts having increasing contents of 2018 components, each with a total concentration of 22μg / ml. The percentages of the QS-21 main peak and 2018 components in each of the resulting QS-21 saponin extracts are shown in the table below.

[0363] [Table 1]

[0364] 4.3 Biological activity of saponin extracts rich in 2018 components The biological activity of seven QS-21 saponin extracts prepared according to Sections 4.1 and 4.2 was evaluated by measuring TNF-α and IL-8 cytokine responses in in vitro cell culture assays. The percentages of "QS-21 main peak" and "2018 component" in each extract are shown in Table 1.

[0365] Human THP1 cell line (ATCC) was cultured in 10% FCS-RPMI + supplement medium in T175 flasks. Fresh medium was added twice a week and cells were cultured until they reached PDL13 (population doubling level).

[0366] For testing, cells were transferred to 96-well plates. THP1 cells were stimulated with LPS (3 ng / ml) for 1 hour, then with purified QS-21 saponin extract (2 μg / ml) with low content of 2018 components for 16 hours. Supernatants were then independently tested for cytokine responses.

[0367] After a 16 hour incubation period, the supernatants of the different wells were harvested and frozen at -80°C until testing. Quantification of secreted TNF-α and IL-8 was performed using the CBA kit from BD Biosciences.

[0368] Individual TNF-α and IL-8 concentrations were plotted as a function of the percentage of 2018 components in the purified QS-21 saponin extract. ANOVA models were used to estimate the concentration geometric mean (GMC) and 95% confidence interval (CI), as well as the geometric mean ratio (GMR), for each percentage of 2018 components compared to the reference "purified QS-21 saponin extract with low content of 2018 components" (i.e., containing 2.2% of 2018 components). All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

[0369] result TNF-α response Figure 11 shows the individual TNF-α concentrations measured in the absence of purified QS-21 saponin extract and in the presence of 2 μg / ml of purified QS-21 saponin extract (with increasing percentages of 2018 components), as well as the corresponding GMC + / - CI. In medium containing only LPS (no purified QS-21 saponin extract added), a weak response of less than 220 pg / ml was obtained. Upon addition of purified QS-21 saponin extract with a low content of 2018 components (i.e. 2.2%), a response of 500-700 pg / ml was observed. When purified QS-21 saponin extracts having increasing percentages of the 2018 component (i.e., 5.5%, 8.8%, 15.9%, 29.5%, 56.2% and 70.3%) were added, no significant changes in the TNF-α response were observed for the 2018 component ranging from 5.5% to 15.9%.

[0370] Each purified QS-21 saponin extract with an increasing percentage of the 2018 component was compared to a reference "purified QS-21 saponin extract with a low content of the 2018 component" (i.e., containing 2.2% of the 2018 component). For each comparison, the GMR (GMC ratio) and 95% CI estimated from the ANOVA model are shown in Table 2 and plotted in Figure 12.

[0371] [Table 2]

[0372] IL-8 response FIG. 13 depicts individual Il-8 concentrations measured in the absence of purified QS-21 saponin extract and in the presence of 2 μg / ml purified QS-21 saponin extract (with increasing percentages of 2018 components), as well as the corresponding GMC+ / -CI.

[0373] In media containing only LPS (no purified QS-21 saponin extract added), a weak response of less than 8000 pg / ml was obtained. When purified QS-21 saponin extract with a low content of 2018 components (i.e. 2.2%) was added, a response approaching 22000 pg / ml was observed. When purified QS-21 saponin extract with increasing percentages of 2018 components (i.e. 5.5%, 8.8%, 15.9%, 29.5%, 56.2% and 70.3%) was added, a similar IL-8 response was observed for 2018 components ranging from 5.5% to 15.9%.

[0374] Each purified QS-21 saponin extract with an increasing percentage of the 2018 component was compared to a reference "purified QS-21 saponin extract with a low content of the 2018 component" (i.e., containing 2.2% of the 2018 component). For each comparison, the GMR (GMC ratio) and 95% CI estimated from the ANOVA model are shown in Table 3 and plotted in Figure 14.

[0375] [Table 3]

[0376] [Example 5] Fraction Pooling to Achieve a Purified QS-21 Saponin Extract of a Specific Final Composition A crude aqueous extract of Quillaja Saponaria Molina bark was prepared with a QS-21 concentration of 2.8 g / l, a leading peak to QS-21 main peak ratio of 0.5, and a 2018 / QS-21 main peak ratio of 0.081. The crude extract was then subjected to reversed-phase chromatography using polystyrene resin. Fractions were collected and a subset of fractions was combined to obtain a partially purified extract with a QS-21 purity of 19.5% and a 2018 to 1988 ratio of 0.084.

[0377] The partially purified extract was then subjected to reversed phase chromatography using phenyl resin (Asahi Phenyl column 4.6 mm x 250 mm). A total of 14 fractions in the region of the main QS-21 peak were collected over 30 second intervals and their content was analyzed by UPLC-UV / MS (Figure 15). It was determined that the combination of various fractions promoted a purified phenyl resin extract with the following predicted composition:

[0378] TIFF0007640473000111.tif50162

[0379] Three batches of purified and freeze-dried QS-21 were prepared according to the method described above. The resulting material was analyzed by UPLC-UV / MS and found to have the following compositions:

[0380] TIFF0007640473000112.tif46162

[0381] conclusion Three purified QS-21 saponin extracts with high contents of 2018 components according to the present invention were obtained.

[0382] [Example 6] Immune response The aim of this study was to evaluate whether QS-21 composition changes the affected adjuvant properties. More precisely, the study evaluated the adjuvant properties of three QS-21 lots (QS-21 High Yield, QS-21 High 2002 and QS-21 High 2018) prepared as described in Example 5 and formulated with 3D-MPL and VZV gE antigen and compared to a purified QS-21 saponin extract with a low content of 2018 components (QS-21 Low 2018).

[0383] Liposomal adjuvant formulations of each of the four QS-21 compositions containing QS-21 and 3D-MPL (1:1 w / w) were prepared and mixed with VZV gE antigen to prepare antigen-containing formulations. Three different dose levels of gE antigen were prepared for each QS-21 liposomal formulation: 5 μg, 1 μg, and 0.4 μg gE (corresponding to 1 / 10, 1 / 50, and 1 / 125 of the amount of gE antigen contained in a human dose of Shingrix™).

[0384] Each gE antigen preparation was administered to 6-8 week-old female C57BL6 mice (10 mice / group). The antigen preparation was injected intramuscularly twice with an interval of 14 days. The control group of three mice received only phosphate-buffered saline.

[0385] On day 21, spleens and serum were collected and analyzed for T and antibody responses, respectively.

[0386] - ELISA Anti-VZV gE total IgG was measured by ELISA. Antigen was coated on 96-well plates overnight at 4°C. Coating buffer was removed and plates were saturated with saturation buffer for 1 h at 37°C. Then 100ul of diluted mouse serum or standard or control was added and incubated at 37°C for 1 h 30 min. After washing, plates were incubated with 100ul of biotinylated anti-mouse IgG for 1 h at 37°C. After washing, plates were incubated with 100ul of streptavidin-POD conjugate for 30 min at 37°C. After washing, 100ul of TMB / well was added and plates were kept in the dark at room temperature for 15 min. To stop the reaction, 100ul of H2SO4 0.4N was added per well. Absorbance was read at 450 / 620nm wavelengths by Elisa plate reader. Results were calculated using Softmax-Pro software. Results are shown in Figures 16 and 17.

[0387] - ICS (intracellular cytokine staining) Spleens were collected in RPMI medium and dissociated using two double strokes in a Potter tissue homogenizer. The homogenized samples were transferred to 50 ml polypropylene tubes. Fibrous material was removed by filtration through a 100 uM nylon cell strainer. Cells were then washed, counted, and separated into 10 7 The cells were resuspended at 1000 cells / ml.

[0388] Lymphoid cells were restimulated in vitro for 6 h with gE peptide or medium in the presence of a protein transport inhibitor (Brefeldin A) for the last 4 h, and then processed by conventional immunofluorescence with fluorescent antibodies (extracellular staining: CD4, intracellular staining: TNF-alpha, IFN-gamma, and IL2).

[0389] Results are expressed as the frequency of cytokine positive cells within the CD4 cell population after subtraction of the media conditions for each mouse. Data are shown for populations that showed expression of at least two cytokines (IL2, IFN-alpha or TNF-alpha). Results are shown in Figures 18 and 19.

[0390] conclusion No clinical signs were observed in any animals regardless of the formulation injected, suggesting an acceptable safety profile.

[0391] The CD4+ T cell responses elicited by the extended QS-21 formulations (QS-21 High QS-21 Yield, QS-21 High 2002 and QS-21 High 2018) were shown to be equivalent (by a margin [0.5-2]) to those elicited by a purified QS-21 saponin extract with a lower content of the 2018 component (QS-21 Low 2018) (three doses of each were tested).

[0392] The effect of the extended specification of QS-21 on the antibody response was limited (less than a two-fold difference in the geometric mean ratios was observed), with QS-21 with the extended specification tending to induce higher antibody titers than the purified QS-21 saponin extract with a low content of 2018 components at the lowest dose (0.4 μg) and slightly lower titers than those at the higher doses.

[0393] Taken together, the results suggest that the expansion of QS-21 has no or only a limited effect on the adjuvanticity, for example of liposomal compositions of QS-21 and 3D-MPL.

[0394] [Example 7] Immune Response II The objective of this study was to demonstrate comparability between three QS-21 lots prepared as described in Example 5, formulated with 3D-MPL and VZV gE antigen, and compared to a purified QS-21 saponin extract with a low content of 2018 components (QS-21 Low 2018) for both antibody and CD4+ T cell responses.

[0395] Six to eight week old female C57BL6 mice (16 mice / group) were injected intramuscularly twice at 28 day intervals with 5ug gE antigen formulated using a liposomal formulation containing 3D-MPL and QS-21 (1:1 w / w). A control group of three mice received phosphate buffered saline alone, and a control group of three mice received VZV gE antigen alone.

[0396] Vaccines were prepared with 5ug each of 3D-MPL / QS-21 per animal (corresponding to 1 / 10 of a typical human dose). Injection volume was 50ul.

[0397] Sera were collected on day 42 (2 weeks after the second immunization) and day 49 (3 weeks after the second immunization) and analyzed for antibody responses. Spleens were collected on day 49 and analyzed for CD4+ T cell responses.

[0398] - ICS (intracellular cytokine staining) Spleens were collected in RPMI medium and dissociated using a Potter tissue homogenizer. The homogenized samples were transferred to 50 ml polypropylene tubes. Fibrous material was removed by filtration through a 100 uM nylon cell strainer. Cells were then washed, counted, and separated into 10 7 The cells were resuspended at 1000 cells / ml.

[0399] Lymphoid cells are restimulated in vitro for 6 hours with gE peptide or medium in the presence of a protein transport inhibitor (Brefeldin A) for the last 4 hours. For subsequent flow cytometry analysis, cells were stained with fluorescently labeled antibody extracellular stains (e.g. CD4) and intracellular stains (e.g. TNF-alpha, IFN-gamma and IL2). Results were expressed as the frequency of cytokine positive cells within the CD4+ T cell population after subtraction of the medium conditions for each mouse. Statistical analysis was performed on populations that showed expression of at least two cytokines (IL2, IFN-gamma or TNF-alpha). Results are shown in Figures 20 and 21.

[0400] - ELISA The ELISA method was as described in Example 6.

[0401] The results are shown in Figures 22 and 23.

[0402] - statistical methods Antibody titers and CD4+ frequencies were assumed to be log-normally distributed.

[0403] For the evaluation of antibody responses, an analysis of variance (ANOVA) model was fitted to the log10 titers with test group, time point, and their interaction as fixed effects, and a repeated statement for time point. Equal variances were not assumed between groups. Using this model, geometric means and geometric mean ratios between test groups, as well as their respective 95% confidence intervals, were calculated.

[0404] CD4+ T cell responses were assessed by the frequency of cells expressing at least two of the cytokines IL-2, TNF-alpha, and IFN-gamma following removal of in vitro media stimulation. An analysis of variance (ANOVA) model was then fitted to the log10 frequencies with test group as a fixed effect. Equal variances were not assumed between groups. Using this model, the geometric means and geometric mean ratios between test groups and their respective 95% confidence intervals were calculated.

[0405] conclusion Figure 20 shows the individual percentages of VZV gE-specific CD4+ T cells expressing at least two of the cytokines IL-2, TNF-a, and IFN-g among all CD4+ T cells, as well as their geometric means and 95% confidence intervals. Similar frequencies of VZV gE-specific CD4+ T cells were observed for QS-21 with low 2018 component content (QS-21 Low 2018) and the three other tested compositions (QS-21 High Yield, QS-21 High 2002, QS-21 High 2018). Minimal VZV gE-specific CD4+ T cells (<0.05%) were detected in mice injected with PBS or VZV gE alone (data not shown).

[0406] The geometric mean ratios (and 95% confidence intervals) between the observed percentages of VZV gE-specific CD4+ T cells and QS-21 with low 2018 component content (QS-21 low 2018) are shown in Figure 21. These geometric mean ratios were close to 1 (0.89-1.06) with confidence intervals within the interval [0.5 and 2]. Thus, equivalence in terms of CD4+ T cell responses was demonstrated.

[0407] Anti-VZV gE antibody titers induced by QS-21 with low 2018 component content (QS-21 Low 2018) and the three other tested compositions (QS-21 High Yield, QS-21 High 2002, QS-21 High 2018) were similar on days 42 and 49. Note that low anti-VZV gE antibody titers (<1200 EU / ml) were detected in mice injected with PBS VZV gE alone (data not shown).

[0408] The geometric mean ratios (and 95% confidence intervals) between the observed anti-VZV gE antibody titers compared to QS-21 with low 2018 component content (QS-21 Low 2018) are shown in Figure 23. The observed geometric mean ratios (filled squares) are close to 1, and the 95% confidence intervals (error bars) are within the interval [0.5 and 2]. Thus, equivalence in terms of antibody responses is demonstrated.

[0409] Taken together, these results statistically demonstrated that QS-21 High Yield, QS-21 High 2002, and QS-21 High 2018 elicited potent adaptive immune responses comparable to those observed with QS-21 having low 2018 component content, and therefore did not affect the adjuvant properties, e.g., of the liposomal composition of QS-21 and 3D-MPL.

[0410] [Example 8] Reactogenicity The objective of this study was to demonstrate comparability of reactogenicity between three QS-21 lots prepared as described in Example 5 and formulated with 3D-MPL and VZV gE antigen and compared to a purified QS-21 saponin extract with low content of 2018 components.

[0411] Female OFA rats (8 / group) were injected with VZV gE antigen formulated with PBS or a liposomal formulation containing 3D-MPL and QS-21 (1:1 w / w) at three time points. Vaccines were prepared with 20ug of VZV gE antigen formulated with 20ug each of 3D-MPL / QS-21 per animal (corresponding to 2 / 5 of a typical human dose, 1 / 5 administered per injection site). Injection volume was 100ul per site. PBS was administered into the left and right gastrocnemius on day 1, and vaccine was administered into the left and right quadriceps on days 8 and 22.

[0412] Body temperature was monitored using an implanted temperature probe from day -10 to day 36. Clinical signs, injection site reactions and body weight were monitored from day 1 to day 36. Postmortem microscopic and macroscopic evaluations were performed on days 25 and 36.

[0413] result Clinical signs and local reactions: - No unscheduled deaths occurred during the study. - No treatment-related clinical signs were observed in any of the treated animals. On day 8 (1 to 6 hours), some animals exhibited the expected local reactions with all formulations of VZV gE / AS01 after vaccination, such as edema, erythema, dryness and desquamation. - No significant differences were observed in the occurrence or duration of these transient reactions between the formulation groups, which generally appeared a few hours after injection and resolved within a few days.

[0414] Temperature: - Mean body temperature was similarly elevated (around 1.5°C) after the first and second intramuscular injections compared to the first PBS injection in all groups. - The mean difference in temperature (from baseline) increased and peak temperatures after each intramuscular injection showed a similar pattern profile in all groups with the same proportions. - figure 25 shows the maximum temperature difference observed for each animal for all treatment groups after the first (day 8) and second (day 23) immunization as well as the mean and 95% confidence interval.

[0415] body weight: - Transient and slight mean weight loss was observed in all groups on the day after vaccination. No differences in weight change were observed between groups.

[0416] Postmortem microscopic and macroscopic evaluation: - The three formulations containing QS-21 with increased 2018 induced a non-detrimental, slight to moderate, partially reversible subacute inflammation at the injection site that was similar to the reference formulation containing QS-21 with low 2018 content. This inflammation was primarily characterized by mixed inflammatory cell infiltration and fibroproliferation / fibrosis. - Macroscopic enlargement of the draining lymph nodes, correlating with increased lymphoid cellularity, was seen for all formulations. This observation at day 25 (3dPII) was partially or completely reversed by day 36 (14dPII).

[0417] References TIFF0007640473000113.tif227170TIFF0007640473000114.tif138169

Claims

1. The following Figure 6: A saponin extract derived from Quillaja Saponaria Molina containing (i) at least 88% of a main QS-21 peak and (ii) greater than 3% to 10% of a 2018 component, defined as the "QS-21" and "2018" peaks, respectively, in a chromatogram of the above.

2. 2. The saponin extract of claim 1, containing at least 4.5% of the 2018 component.

3. 3. The saponin extract of claim 1 or 2, containing 9% or less of the 2018 component.

4. A saponin extract derived from Quillaja Saponaria Molina containing (i) at least 88% triterpenoid glycosides having an m / z of 1855.9, 1987.9 and / or 2001.9, as measured by UV absorbance at 214 nm having a retention time of approximately 4.4 minutes using UPLC-UV / MS, and (ii) greater than 3% to 10% triterpenoid glycosides having an m / z of 2017.9, as measured by UV absorbance at 214 nm having a retention time of approximately 4.5 minutes using UPLC-UV / MS.

5. Measured by UV absorbance at 214 nm and by relative ion abundance using UPLC-UV / MS, and by MS / MS. (i) At least 88 percent 【Chemistry 1】 and (ii) more than 3% to 10% 【Chemistry 2】 A saponin extract derived from Quillaja Saponaria Molina containing:

6. The saponin extract of any one of claims 1 to 5, containing at least 40% of the 1988 component, defined as a triterpenoid glycoside having an m / z of 1987.9, as measured by UV absorbance at 214 nm and relative ion abundance with a retention time of approximately 4.4 minutes using UPLC-UV / MS.

7. The saponin extract of any one of claims 1 to 6, containing 90% or less of the 1988 component, defined as a triterpenoid glycoside having an m / z of 1987.9, as determined by UV absorbance at 214 nm and relative ion abundance with a retention time of approximately 4.4 minutes using UPLC-UV / MS.

8. (i) selecting a crude aqueous extract of Quillaja Saponaria Molina having a ratio of 2018 components to QS-21 main peak of greater than 0.075; (ii) purifying the extract by polyvinylpyrrolidone adsorption; (iii) purifying the extract by diafiltration, ultrafiltration or dialysis; (iv) purifying the extract by reverse phase chromatography on a polystyrene resin; and (v) purifying the extract by reverse phase chromatography on phenyl resin.

2. A method for producing the purified saponin extract of claim 1, comprising:

9. The saponin extract according to any one of claims 1 to 7 for use as an adjuvant.

10. An adjuvant composition comprising the saponin extract according to any one of claims 1 to 7.

11. 11. The saponin extract or adjuvant composition of claim 9 or 10, wherein the adjuvant is a liposomal formulation.

12. 12. The saponin extract or adjuvant composition of any one of claims 9 to 11, wherein the adjuvant comprises a TLR4 agonist.

13. 13. The saponin extract or adjuvant composition of claim 12, wherein the TLR4 agonist is 3D-MPL.

14. An immunogenic composition comprising the adjuvant composition according to any one of claims 10 to 13 and an immunogen or antigen, or a polynucleotide encoding an immunogen or antigen.

15. 13. A method for producing a purified saponin extract of claim 1, comprising: (i) selecting a Quillaja Saponaria Molina material having a suitable 2018 ingredient composition; (ii) preparing a crude aqueous extract of said Quillaja Saponaria Molina material under suitable conditions; and (iii) purifying the crude aqueous extract to obtain a purified saponin extract. The method comprising:

Images