Method for quantifying saponin present in particles containing saponin and lipid

Abstract

A method for quantifying the amount of saponin present in particles containing saponin and lipids is disclosed. The method includes the steps of: (1) isolating the saponin from the lipids of the particles by reversed-phase solid-phase extraction of the saponin from a predetermined amount of the particles; (2) lyophilizing the isolated saponin; (3) preparing a solution of the lyophilized saponin and a predetermined amount of an internal standard compound in deuterated methanol; (4) generating a quantitative proton NMR spectrum of the solution containing signals from the three protons of the C26 methyl group of the triterpene core of the saponin and signals from one or more protons of the internal standard compound; and (5) comparing the signals from the three protons of the C26 methyl group of the triterpene core with signals from one or more protons of the internal standard compound.

Description

[Technical Field]

[0001] The present invention relates generally to a method for quantifying the amount of saponin present in a particle comprising saponin and a lipid, and more particularly to a method for quantifying the amount of saponin present in a particle comprising saponin and a lipid, wherein the saponin in the particle comprises a triterpene core comprising a C26 methyl group containing three protons. [Background technology]

[0002] Saponins are a large group of glycoconjugates that share a triterpene structure containing various glycosidic side chains and may possess potent immunostimulatory properties. Saponins extracted from the bark of the South American soap tree Quillaja saponaria Molina contain a complex, heterogeneous mixture of closely related saponins with structurally distinct glycosylation or acylation patterns that affect their biological activity. In their naturally occurring forms, Quillaja saponaria Molina saponins can have a high degree of glycosyl O-acylation, a low degree of glycosyl O-acylation, or even no glycosyl O-acylation. Saponins can also be chemically modified, for example, by partial or complete deacylation or degradation.

[0003] Saponins from Quillaja saponaria molina in particular can have potent adjuvant activity, but are also chemically unstable, can exhibit hemolytic activity, and can be associated with immediate pain at the injection site. Saponin preparations based on the defined composition of purified saponin fractions from Quillaja saponaria molina are described, for example, in PCT / AU1995 / 000670 (WO96011711) by Cox et al.

[0004] Incorporation of Quillaja Saponaria Molina saponins into particles containing saponins and lipids can reduce chemical instability, hemolysis, and immediate pain when injected in combination with the saponins. Examples of particles containing saponins and lipids include iscom matrix particles, iscom antigen-presenting particles, liposome-based adjuvant system 01 particles, and Army Liposome formulation Q particles, as taught, for example, by Stertman et al., Human Vaccines & Immunotherapeutics, 2023, 19(1):2189885.

[0005] Iscom matrix particles, also called iscom matrix or matrix, are discrete, stable nanostructures made from saponin, phospholipids such as phosphatidylcholine, and cholesterol. Iscom matrix particles are described, for example, in Morein et al.'s PCT / SE1989 / 000528 (WO9003184) and Morein et al.'s PCT / SE2003 / 001180 (WO2004004762). Iscom matrix particles also exhibit potent adjuvant activity relative to saponin, but when injected in combination with saponin, they reduce chemical instability, hemolysis, and immediate pain.

[0006] Iscom antigen-presenting particles, also called iscom particles or ISCOMs, are stable nanostructures produced by co-formulation of antigens with saponin, phospholipids, and cholesterol. Iscom antigen-presenting particles are described, for example, in Morein et al.'s PCT / SE1986 / 000480 (WO87002250) and Morein et al.'s PCT / SE2003 / 001180 (WO2004004762). Iscom antigen-presenting particles contain multiple copies of an antigen physically incorporated into a matrix of saponin, phospholipids, and cholesterol. Similar to iscom matrix particles, iscom antigen-presenting particles also exhibit potent adjuvant activity, but when injected in combination with saponin, they are less susceptible to chemical instability, hemolysis, and immediate pain.

[0007] Liposomal Adjuvant System 01 particles are liposomal vaccine adjuvants containing Quillaja Saponaria Molina saponin QS-21 and 3-O-deacylated-4'-monophosphoryl lipid A, as described, for example, in Didierlaurent et al., Expert Review of Vaccines, 2017, 16(1):55-63. According to Didierlaurent et al. (2017), the incorporation of QS-21 saponin into Liposomal Adjuvant System 01 particles overcomes the hemolytic effect of the saponin.

[0008] Army Liposomal Q particles are liposomal vaccine adjuvants containing Quillaja Saponaria Molina saponin QS-21, saturated phospholipids, cholesterol, and monophosphoryl lipid A, as described, for example, in Alving et al., Expert Review of Vaccines, 2020, 19(3):279-292. According to Alving et al. (2020), incorporation of QS-21 saponin into Army Liposomal Q particles inactivates the hemolytic activity of the saponin on red blood cells.

[0009] As noted, saponin- and lipid-containing particles, such as iscom matrix particles, iscom antigen-presenting particles, liposome-based adjuvant system 01 particles, and Army liposome formulation Q particles, are composed of saponin and other components. During product development, it is often necessary to determine the exact saponin concentration in the particles. For most analytical techniques, quantification of saponin concentration involves the use of a suitable saponin reference standard. Unfortunately, in most cases, such standards are not available, limiting the availability of analytical techniques for quantifying saponin in saponin- and lipid-containing particles.

[0010] Nuclear magnetic resonance (NMR) spectroscopy techniques, particularly quantitative proton NMR (also qNMR, proton NMR, or 1H-NMR) techniques, can allow for the measurement of saponin concentrations in liquid compositions relative to a universal reference standard, most often named and used as an internal standard. Throughout this disclosure, "proton" in the context of NMR refers to the 1H nucleus.

[0011] Quantitative proton NMR spectroscopy involves repeated single-pulse NMR experiments with proton detection, where sample and test conditions are adjusted to ensure a fully quantitative signal response. Critical parameters include, for example, the selection of signal, relaxation delay, and digital resolution. Spectral processing procedures must be robust. Signal response is preferably measured as peak area, although peak height may be used in special cases. The resulting signal response is easily converted to molar ratio and / or concentration.

[0012] Therefore, quantitative proton NMR spectroscopy can be used as an analytical methodology for the quantification of proton-containing compounds. Because the signal is directly proportional to the number of protons with the same resonant frequency, quantification by proton NMR is based on signal comparison. The ratio of the areas of the signals observed in the NMR spectrum is proportional to the ratio of the number of nuclei at each molecular location. The ratio of the intensities of the observed signals can also be used as an alternative, although this is less preferred. In absolute quantification, one signal of the analyte is compared to the signal from an internal reference standard. Furthermore, standardization procedures can be applied by comparing two or more signals in a mixture of components, with each signal giving the molar amount of the molecular structure it represents.

[0013] However, several factors complicate the use of quantitative proton NMR spectroscopy to determine the concentration of saponin in a liquid composition containing particles containing saponin and lipid. Adding particles containing saponin and lipid to a liquid, such as a saline solution, results in the formation of a dispersion of the particles in the liquid, rather than a solution. Furthermore, the slow molecular tumbling and rapid NMR relaxation caused by the relatively large size of dispersed particles makes solution NMR spectroscopy unsuitable. Furthermore, the presence of components other than saponin in the particles and residual compositions complicates direct measurement of the sample.

[0014] Therefore, there is a need for analytical techniques for quantifying the amount of saponin present in particles comprising saponin and lipid, and in particular for quantitative proton NMR spectroscopy techniques for doing so. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] Cox et al., PCT / AU1995 / 000670 (WO96011711) [Patent Document 2] Morein et al., PCT / SE1989 / 000528 (WO9003184) [Patent Document 3] Morein et al., PCT / SE2003 / 001180 (WO2004004762) [Patent Document 4] Morein et al., PCT / SE1986 / 000480 (WO87002250) [Non-patent literature]

[0016] [Non-Patent Document 1] Stertman et al., Human Vaccines & Immunotherapeutics, 2023, 19(1):2189885 [Non-patent document 2] Didierlaurent et al., Expert Review of Vaccines, 2017, 16(1):55-63 [Non-patent document 3] Alving et al., Expert Review of Vaccines, 2020, 19(3):279-292 [Non-patent document 4] Fleck et al., Molecules 2019, 24(1), 171; doi.org / 10.3390 / molecules24010171 Summary of the Invention

[0017] A method for quantifying the amount of saponin present in a particle comprising saponin and lipid is disclosed. The saponin in the particle comprises a triterpene core containing a C26 methyl group containing three protons. The method comprises: (1) isolating saponin from lipids of particles comprising saponin and lipids by reversed-phase solid-phase extraction of saponin from a predetermined amount of particles comprising saponin and lipids, thereby obtaining isolated saponin; (2) freeze-drying the isolated saponin, thereby obtaining freeze-dried saponin; (3) preparing a solution of a predetermined amount of the lyophilized saponin and an internal standard compound containing one or more protons in deuterated methanol; (4) generating a quantitative proton NMR spectrum of the lyophilized saponin and internal standard compound solution, the signal comprising the three protons of the C26 methyl group of the triterpene core of the saponin and one or more proton signals of the internal standard compound; (5) A step of comparing the signals of the three protons of the C26 methyl group of the triterpene core of the saponin with the signals of one or more protons of the internal standard compound. Includes:

[0018] In some embodiments, step (1) comprises: (1.1) conditioning a reversed-phase solid-phase extraction sorbent with a polar organic solvent; (1.2) equilibrating a reversed-phase solid-phase extraction sorbent with a mixture comprising a polar organic solvent and water; (1.3) loading particles containing saponin and lipid onto a reversed-phase solid-phase extraction sorbent; (1.4) washing the reversed-phase solid-phase extraction sorbent with a mixture comprising a polar organic solvent and water; (1.5) eluting the saponin from the reversed-phase solid-phase extraction sorbent with a polar organic solvent, thereby obtaining the isolated saponin in a mixture comprising the isolated saponin and the polar organic solvent. Includes:

[0019] In some of these embodiments, the polar organic solvent comprises methanol.

[0020] Also, in some of these embodiments, the mixture comprising a polar organic solvent and water in one or more steps (1.2) or (1.4) comprises methanol and water in a ratio of 1:99 to 20:80 (V:V), 2:98 to 10:90 (V:V), 3:97 to 7:93 (V:V), or about 5:95 (V:V).

[0021] Also in some of these embodiments, during step (1.3), the particles comprising saponin and lipid are loaded onto the reversed-phase solid-phase extraction sorbent at a saponin content of 0.1 mg to 2 mg, a saponin content of 0.2 mg to 1 mg, a saponin content of 0.3 mg to 0.7 mg, a saponin content of 0.4 mg to 0.6 mg, or about 0.5 mg per 500 mg bed weight of the reversed-phase solid-phase extraction sorbent.

[0022] Also in some of these embodiments, the reversed-phase solid-phase extraction sorbent comprises an octadecyl sorbent active group.

[0023] In some of these embodiments, step (1) further comprises (1.6) adding water to the mixture comprising the isolated saponin and the polar organic solvent to a final water content of between 5% and 20% by volume, between 7% and 15% by volume, between 9% and 12% by volume, or about 10% by volume.

[0024] In some embodiments, the predetermined amount of particles comprising saponin and lipid is the saponin content of the formulation used to make the particles comprising saponin and lipid.

[0025] In some embodiments, the internal standard compound comprises maleic acid, and one or more protons of the internal standard compound comprise the two magnetically equivalent olefinic protons of maleic acid.

[0026] In some embodiments, the quantitative proton NMR spectrum is determined using the following parameters: (a) Pulse flip angle of 30°; (b) a relaxation delay of at least 10 seconds; and (c) a collection time of at least 2.7 seconds is generated according to

[0027] In some of these embodiments, the quantitative proton NMR spectrum is determined using the following parameters: (d) temperature between 12 and 30°C; (e) a magnetic field strength of at least 400 MHz or at least 600 MHz; (f) a probe containing a proton channel with a probe diameter of 1 mm to 10 mm, e.g., 5 mm; (g) at least 64 scans, or at least 128 scans, or at least 256 scans; (h) a sweep width of at least 12 ppm or at least 16 ppm; or (i) a line broadening function of at least 0.3 Hz or 1 Hz; is further generated according to one or more of:

[0028] In some embodiments, step (5) comprises manual phase correction, automatic polynomial baseline correction, calibration of the chemical shift scale, manual polynomial baseline correction, and integration of the quantitative proton NMR spectrum signal.

[0029] In some of these embodiments, the one or more triterpene cores of the saponin further comprise a C23 aldehyde group.

[0030] In some of these embodiments, the calibration of the chemical shift scale includes setting the main peak of the C23 aldehyde group to 9.45 ppm.

[0031] Also in some of these embodiments, (i) the manual polynomial baseline correction comprises correcting the signals of the three protons of the C26 methyl group of the triterpene core of the saponin in a chemical shift range of 0.60 to 0.85 ppm; and / or (ii) the signal integration comprises applying integration limits to the signals of the three protons of the C26 methyl group of the triterpene core of the saponin based on a chemical shift range of 0.73 to 0.85 ppm.

[0032] In some embodiments, step (5) comprises performing area normalization of the signals of the three protons of the C26 methyl group of the triterpene core of the saponin and the signals of one or more protons of the internal standard compound to account for differences in the number of magnetically equivalent protons therebetween.

[0033] In some embodiments, the triterpene core of the saponin further comprises the C3 and C28 positions, and the saponin further comprises a disaccharide or trisaccharide group attached to the C3 position of the triterpene core and an oligosaccharide group attached to the C28 position of the triterpene core.

[0034] In some of these embodiments, the triterpene core comprises one or more of a quilacic acid triterpene core, a quilacic acid 22β-OH triterpene core, a phytolaccagenic acid triterpene core, a phytolaccagenic acid 23-OAc triterpene core, a gypsogenin triterpene core, or an echinocystic acid triterpene core.

[0035] Also, in some of these embodiments, the oligosaccharide group comprises a fucosyl group comprising the O-3 and O-4 positions.

[0036] Additionally, in some of these embodiments, one or more of the saponins further comprises a fatty acyl group and / or an acyl II group attached to the O-3 or O-4 position of the fucosyl group.

[0037] Also, in some of these embodiments, the proton NMR spectrum of the lyophilized saponin and internal standard solution further comprises signals for one or more protons of the fatty acyl group attached to the O-3 or O-4 position of the fucosyl group, and the method further comprises a step (6) of comparing the signals for one or more protons of the fatty acyl group attached to the O-3 or O-4 position of the fucosyl group to one or more signals for the three protons of the C26 methyl group of the triterpene core of the saponin or to the signals for one or more protons of the internal standard compound.

[0038] In some embodiments, the saponin comprises saponin extracted from the bark of the South American soap tree Quillaja Saponaria Molina.

[0039] In some embodiments, the particles comprising saponin and lipid comprise or consist of one or more of iscom matrix particles, iscom antigen-presenting particles, liposomal adjuvant system 01 particles, or Army liposomal formulation Q particles.

[0040] In some embodiments, the lipids include one or more phospholipids and cholesterol.

[0041] In some of these embodiments, the one or more phospholipids comprise one or more phosphatidylcholines.

[0042] In some embodiments, the particle comprising saponin and lipid is an iscom matrix particle consisting essentially of saponin, one or more phospholipids and cholesterol.

[0043] In some embodiments, the particle comprising saponin and lipid is an iscom antigen-presenting particle consisting essentially of saponin, one or more phospholipids, cholesterol, and one or more antigens. [Brief explanation of the drawings]

[0044] [Figure 1] The consensus structure of Quillaja saponins is shown. The asterisks indicate the locations of the Fa-2 and Fa-2' protons. [Figure 2] Figure 1 shows the proton NMR spectrum of a chromatographic fraction of the saponin from Quillaja Saponaria Molina designated as saponin type II. The geminal proton pair, Fa-2 and Fa-2', and the methyl Sap26 peaks are indicated. [Figure 3A-C] Baseline correction of the proton NMR spectrum of saponin from a sample of iscom matrix particles is shown. First, as shown in Figure 3A, the spectrum is corrected in the range of 0-10 ppm. Second, as shown in Figure 3B, the signal at 0.8 ppm (Sap26) is corrected. Finally, the signal at 6.35 ppm (maleic acid) is corrected. [Figure 4] Quantitative NMR integrals corresponding to (A) the integral of the Sap26 signal (0.0022) and (B) the integral of the maleic acid signal (0.0045) are shown. [Figure 5] 1 shows the proton NMR spectrum of the first matrix type I sample. [Figure 6] 1 shows the proton NMR spectrum of a second matrix type I sample. [Figure 7] 1 shows the proton NMR spectrum of a third matrix type I sample. [Figure 8] 1 shows the proton NMR spectrum of the first matrix type II sample. [Figure 9] 1 shows the proton NMR spectrum of a second matrix type II sample. [Figure 10] 1 shows the proton NMR spectrum of the third matrix type II sample. DETAILED DESCRIPTION OF THE INVENTION

[0045] Surprisingly, the inventors have determined that the saponin present in particles comprising saponin and lipids can be quantified by a method comprising the steps of: (1) isolating the saponin from the lipids of the particles by reversed-phase solid-phase extraction of the saponin from a predetermined amount of the particles; (2) lyophilizing the isolated saponin; (3) preparing a solution of the lyophilized saponin and a predetermined amount of an internal standard compound in deuterated methanol; (4) generating a quantitative proton NMR spectrum of the solution of the lyophilized saponin and the internal standard compound, the spectrum comprising signals for the three protons of the C26 methyl group of the triterpene core of the saponin and signals for one or more protons of the internal standard compound; and (5) comparing the signals for the three protons of the C26 methyl group of the triterpene core with signals for one or more protons of the internal standard compound.

[0046] Focusing specifically on iscom matrix particles, the inventors have determined that a pretreatment step involving subjecting a predetermined amount of iscom matrix particles in a sample of a known volume of dispersion of iscom matrix particles in an aqueous salt solution to reversed-phase solid-phase extraction causes the iscom matrix particles to disintegrate during the reversed-phase extraction, allowing for isolation of the saponin from other components present in the sample sufficiently to allow quantification of the isolated saponin by qNMR. The inventors have also determined that by subjecting a predetermined amount of iscom matrix particles in a sample of known volume, including a predetermined amount determined for the saponin content of the formulation used to first produce the iscom matrix particles, to reversed-phase solid-phase extraction, the qNMR results of the isolated saponin can be used to quantify the saponin present in the iscom matrix particles of the sample.

[0047] Specifically, we tested several solvent systems and reversed-phase solid-phase extraction sorbents, resulting in a method involving a methanol-water system and an octadecyl sorbent with an active group (also called C18). We observed reasonable saponin recoveries and good repeatability, and other components, particularly lipids and salts, were either retained by the sorbent in the case of lipids or washed away in the case of salts. We quantitatively collected the eluted saponin, added water to prevent degradation, and lyophilized the resulting solution.

[0048] We then redissolved the dried extract in perdeuterated methanol along with the internal standard previously dissolved in perdeuterated water. We determined appropriate conditions for quantitative proton NMR spectroscopy in terms of peak selection, digital resolution, relaxation (cycle) delay, and signal-to-noise ratio. Furthermore, we determined appropriate data processing conditions, including phase and baseline correction and integration limits. We calculated the molar ratios from the peak integrals of the selected saponin and internal standard peaks in the resulting NMR spectrum after correction for the number of protons contained in each peak.

[0049] Based on our approach, we determined that since the amount of internal standard was known, we could calculate the molar amount of saponin in an NMR sample. Furthermore, by subjecting a given amount of iscom matrix particles in a known volume of sample to reversed-phase solid-phase extraction, we were able to relate the molar amount of saponin in the NMR sample to the molar amount of saponin in the iscom matrix sample.

[0050] Our method should also be applicable to other particles containing saponin and lipid, such as iscom antigen-presenting particles, liposomal adjuvant system 01 particles, or Army liposome formulation Q particles, based on the use of reversed-phase solid-phase extraction to disrupt other particles and isolation of saponin from the disrupted particles.

[0051] Considering iscom antigen-presenting particles specifically, iscom antigen-presenting particles are typically prepared from a protein antigen solution containing the nonionic surfactant polysorbate 80 (PS-80), Triton, or NP-40 to stabilize the antigen and form the iscom antigen-presenting particle. Like the lipids of iscom matrix particles, the lipids and nonionic surfactants (e.g., polysorbate 80) of the iscom antigen-presenting particle are retained by the sorbent. Minor impurities and degradants of the nonionic surfactant (e.g., polysorbate 80) may co-elute with saponin but are not expected to interfere with NMR signal integration.

[0052] Thus, a method for quantifying the amount of saponin present in a particle comprising saponin and lipid is disclosed.

[0053] The particles comprise a saponin and a lipid and can comprise or consist of, for example, one or more of an iscom matrix particle, an iscom antigen-presenting particle, a liposomal adjuvant system 01 particle, or an Army liposomal formulation Q particle, among other saponin- and lipid-containing particles.

[0054] Iscom matrix particles can be prepared, for example, as described in Morein et al., PCT / SE1989 / 000528 and Morein et al., PCT / SE2003 / 001180. As taught by Morein et al., PCT / SE2003 / 001180, iscom matrix particles are composed of quillaja saponin, cholesterol, and phospholipids. These particles can be present in a mixture with an antigen, but are not associated with the antigen.

[0055] Iscom antigen-presenting particles can be produced, for example, as described in Morein et al., PCT / SE1989 / 000528 and Morein et al., PCT / SE2003 / 001180. As taught by Morein et al., PCT / SE2003 / 001180, iscom antigen-presenting particles are nanoparticle complexes comprising quillaja saponin, cholesterol, and phospholipids into which vaccine antigens are incorporated.

[0056] The liposome-based adjuvant system 01 particles is described, for example, in Didierlaurent et al., Expert Rev Vaccines 2017, 16(1):55-63.

[0057] Army liposomal formulation Q particles are described, for example, in Alving et al., Expert Rev Vaccines 2020, 19(3):279-292.

[0058] The lipids of the particles can include, for example, one or more phospholipids and cholesterol. The one or more phospholipids can include, for example, one or more phosphatidylcholines. These are the lipids of iscom matrix particles and iscom antigen-presenting particles.

[0059] Thus, in some embodiments, the particles are iscom matrix particles consisting essentially of saponin, one or more phospholipids and cholesterol, which are the components that give the iscom matrix particle its structure and function.

[0060] Additionally, in some embodiments, the particles are iscom antigen-presenting particles consisting essentially of saponin, one or more phospholipids, cholesterol, and one or more antigens, which are the components that provide the structure and function of the iscom antigen-presenting particle.

[0061] Saponins contain a triterpene core containing a C26 methyl group containing three protons, such as those extracted from the bark of the South American soap tree Quillaja Saponaria Molina.

[0062] Quillaja Saponaria Molina saponins generally comprise three major moieties: (1) a triterpene core, typically a quillaric acid triterpene core containing a C26 methyl group (also called the Sap26 methyl group), C3, and C28 positions, but also containing some quillaric acid 22β-OH, phytolaccagenic acid, phytolaccagenic acid 23-OAc, gypsogenin, or echinocystic acid triterpene core; (2) a di- or trisaccharide attached to the C3 position of the triterpene core; and (3) an oligosaccharide attached to the C28 position of the triterpene core. The triterpene core can optionally further contain a C23 aldehyde group (also called the Sap23 aldehyde group). The quillaric acid, gypsogenin, and echinocystic acid triterpene cores contain a C23 aldehyde group. The oligosaccharide can optionally contain an acyl group.

[0063] See Figure 1 and Table 1 for the consensus structure of Quillaja saponins.

[0064] [Table 1]

[0065] Saponin materials may be classified into different categories with respect to the acyl group in TABLE 1. Saponin Type I may contain small amounts of acyl I and no fatty acyl or acyl II, while saponin Type II may contain large amounts of fatty acyl and / or acyl II and only small amounts of acyl I. A third type, saponin Type III, may contain all types of acyl groups.

[0066] The degree of fatty acylation is the percentage of fatty acyl and / or acyl II groups linked to O-4 or O-3 of the fucosyl residue per triterpene residue.

[0067] See Figure 2 for the expected signal.

[0068] The method includes the steps of (1) isolating saponin from lipids of particles comprising saponin and lipid by reversed-phase solid-phase extraction of the saponin from a predetermined amount of the particles, thereby obtaining isolated saponin.

[0069] The predetermined amount of particles containing saponin and lipid can be, for example, the saponin content of the formulation used to produce the particles. According to this approach, the predetermined amount of particles is predetermined based on the amount of saponin contained in the formulation used to produce the particles, assuming 100% incorporation of saponin in the formulation into the particles. Thus, for example, when applied to iscom matrix particles, the predetermined amount of iscom matrix particles can be the saponin content of the formulation used to produce the iscom matrix particles, assuming 100% incorporation of saponin into the iscom matrix particles. Similarly, when applied to iscom antigen-presenting particles, the predetermined amount of iscom antigen-presenting particles can be the saponin content of the formulation used to produce the iscom antigen-presenting particles, assuming 100% incorporation of saponin into the iscom antigen-presenting particles. This approach can also be applied to liposome-based adjuvant system 01 particles and Army liposome formulation Q particles, among other particles containing saponin and lipid.

[0070] As described above, by subjecting a predetermined amount of iscom matrix particles in a known volume of sample to reversed phase solid phase extraction, along with a determined amount of the saponin content of the formulation used to produce the iscom matrix particles, the qNMR results of the isolated saponin can be used to quantify the saponin present in the iscom matrix particles of the sample.

[0071] According to this approach, in practice, the desired sample volume (mL) to be loaded onto the reversed-phase solid-phase extraction sorbent in step (1) can be calculated, for example, as follows: The amount of particles containing saponin and lipid in the sample is defined as the mass (mg) of saponin content of the formulation used to produce the particles in the sample, assuming 100% incorporation of saponin into the particles, and this is known. The volume (mL) of the sample is also known. Therefore, the saponin content concentration (mg / mL) can be calculated by dividing the saponin content mass by the sample volume, also assuming 100% incorporation of saponin into the particles. The desired sample volume to be loaded is then calculated by dividing the desired saponin content for loading, for example, approximately 0.5 mg of saponin content, by the concentration of saponin content of the sample, for example, 5 mg / mL, in this example, a loading volume of 0.1 mL.

[0072] As will be appreciated, the methods disclosed herein can be used to determine the actual percent incorporation of saponin into particles comprising saponin and lipid, among other things, during formulation of the particles.

[0073] In some embodiments, step (1) comprises: (1.1) conditioning a reversed-phase solid-phase extraction sorbent with a polar organic solvent; (1.2) equilibrating a reversed-phase solid-phase extraction sorbent with a mixture comprising a polar organic solvent and water; (1.3) loading a reversed-phase solid-phase extraction sorbent with particles comprising saponin and lipid; (1.4) washing the reversed-phase solid-phase extraction sorbent with a mixture comprising a polar organic solvent and water; (1.5) eluting the saponin from the reversed-phase solid-phase extraction sorbent with a polar organic solvent, thereby obtaining the isolated saponin in a mixture comprising the isolated saponin and the polar organic solvent. Includes:

[0074] In some of these embodiments, the polar organic solvent comprises methanol. Methanol is useful for conditioning a reversed-phase solid-phase extraction sorbent in preparation for isolating the saponin from particles comprising the saponin and lipids. A mixture of methanol and water is useful for equilibrating a reversed-phase solid-phase extraction sorbent. A mixture of methanol and water is also useful for washing a reversed-phase solid-phase extraction sorbent to remove salts, particularly from a sample of a dispersion of particles comprising the saponin and lipids in an aqueous salt solution that is loaded onto the reversed-phase solid-phase extraction sorbent. Methanol is also useful for achieving disintegration of particles comprising the saponin and lipids in the initial portion of the stationary phase of a solid-phase extraction following loading of the particles comprising the saponin and lipids. Methanol is also useful for eluting the saponin from a reversed-phase solid-phase extraction sorbent without eluting lipids such as phospholipids and cholesterol, thereby providing the isolated saponin in a mixture comprising the isolated saponin and methanol, while the lipids are retained on the reversed-phase solid-phase extraction sorbent.

[0075] The mixture comprising the polar organic solvent and water in one or more of steps (1.2) or (1.4) can comprise methanol and water in a ratio of, for example, 1:99 to 20:80 (V:V), 2:98 to 10:90 (V:V), 3:97 to 7:93 (V:V), or about 5:95 (V:V).

[0076] As will be appreciated, polar organic solvents other than methanol may also be used for one or more of conditioning, effecting disintegration of particles comprising saponin and lipids, and eluting saponin without eluting lipids. Such polar organic solvents may also be used in combination with methanol.

[0077] Furthermore, mixtures of other polar organic solvents, with or without methanol, and water may be used for equilibration and washing, and may include other polar organic solvents and water in ratios of, for example, 1:99 to 20:80 (V:V), 2:98 to 10:90 (V:V), 3:97 to 7:93 (V:V), or about 5:95 (V:V), among other ratios.

[0078] The saponin- and lipid-containing particles can be loaded onto the reversed-phase solid-phase extraction sorbent in step (1.3) as a liquid dispersion of particles in a saline solution. This is a common approach for preparing and testing iscom matrix particles and iscom antigen-presenting particles, for example. Advantageously, this approach also works well for loading reversed-phase solid-phase extraction sorbents.

[0079] As described above, the predetermined amount of particles containing saponin and lipid can be, for example, the saponin content of the formulation used to prepare the particles. During step (1.3), the particles can be loaded onto the reversed-phase solid-phase extraction sorbent at a saponin content of, for example, 0.1 mg to 2 mg, 0.2 mg to 1 mg, 0.3 mg to 0.7 mg, 0.4 mg to 0.6 mg, or about 0.5 mg per 500 mg bed mass of the reversed-phase solid-phase extraction sorbent. According to the techniques discussed above for calculating the desired sample volume to load onto a reversed-phase solid-phase extraction sorbent, a sample of a liquid dispersion of particles having a saponin content in these ranges of 0.1 mg to 2 mg, 0.2 mg to 1 mg, 0.3 mg to 0.7 mg, 0.4 mg to 0.6 mg, or about 0.5 mg, and having a saponin content concentration of approximately 5 mg / mL, will have a desired sample loading volume of approximately 0.02 to 0.4 mL, 0.04 to 0.2 mL, 0.06 to 0.14 mL, 0.08 to 0.12 mL, or about 0.1 mL.

[0080] Reversed-phase solid-phase extraction sorbents can include, among other sorbents, for example, octadecyl sorbent active groups, which have been determined to be preferred over hydrophilic-lipophilic balanced reversed-phase sorbents (also called HLB) and octyl sorbents (also called C8) for iscom matrix particles in terms of repeatability and lipid adsorption, although the suitability of these and other reversed-phase solid-phase extraction sorbents for other particles containing saponin and lipid may vary depending, for example, on the lipids present in the saponin and lipid-containing particles.

[0081] In some embodiments, step (1) further comprises (1.6) adding water to the mixture comprising the isolated saponin and the polar organic solvent to a final water content of 5% to 20%, 7% to 15%, 9% to 12%, or about 10% by volume, which may be useful in preventing methanolysis of the isolated saponin.

[0082] Considering step (1) in more detail, loading particles containing saponin and lipids onto a reversed-phase solid-phase extraction sorbent at a level well below the conventional range is crucial for obtaining isolated saponins. This can be illustrated in the context of solid-phase extraction of saponins from iscom matrix particles using a solid-phase extraction cartridge as follows: The mass of iscom matrix particles in a sample can be defined as the saponin concentration in mg. The iscom matrix particles also contain phospholipids and cholesterol. The loading can correspond, for example, to approximately 0.5 mg of saponin (approximately 0.1% of the bed mass) per 500 mg of sorbent bed mass. In contrast, typical recommendations from manufacturers of solid-phase extraction cartridges are 25–100 mg of sample (approximately 5% of the bed mass) per 500 mg of bed mass. The reason for using a relatively low loading is due to solubility considerations and the need to disrupt the iscom matrix particles, as well as the overall loading capacity of the solid-phase extraction sorbent. Considering solubility reasons first, saponin, phosphatidylcholine, and / or cholesterol may have limited solubility in methanol and methanol:water (5:95 (V:V)) eluents. This is true even though these eluents are determined herein to provide maximum saponin recovery and repeatability, and to fully retain phosphatidylcholine and cholesterol in the sorbent. Turning to the need to disrupt the iscom matrix particles, they must be disrupted early in the stationary phase prior to separation of the saponin from the lipid. Methanol is effective for this purpose. Considering the overall loading capacity of the solid-phase extraction sorbent, this depends on the combined loading from all sample components, including saponin (0.5 mg), lipid (approximately 0.5 mg), and PBS buffer (approximately 1 mg salt). Advantageously, a sample size of 0.5 mg was determined herein to be sufficient for accurate quantitative proton NMR measurements.

[0083] The method also includes the step of (2) lyophilizing the isolated saponin, thereby obtaining a lyophilized saponin.

[0084] The method also includes (3) preparing a solution of the lyophilized saponin and a predetermined amount of an internal standard compound containing one or more protons in deuterated methanol. The internal standard compound can include, for example, maleic acid, and the one or more protons of the internal standard compound can include, for example, the two magnetically equivalent olefinic protons of maleic acid. Alternatively, other suitable internal standard compounds containing one or more protons can also be used. The solution can be prepared, for example, by pre-dissolving the internal standard compound in deuterium oxide, and then combining the pre-dissolved internal standard compound, the lyophilized saponin, and deuterated methanol to obtain the solution. Alternatively, other suitable techniques can be used to prepare the solution.

[0085] The method also includes (4) generating a quantitative proton NMR spectrum of the lyophilized saponin and internal standard compound solution, the spectrum including signals for the three protons of the C26 methyl group of the triterpene core of the saponin and signals for one or more protons of the internal standard compound.

[0086] In some embodiments, quantitative proton NMR spectra are generated according to the following parameters: (a) a pulse flip angle of 30°; (b) a relaxation delay of at least 10 seconds; and (c) an acquisition time of at least 2.7 seconds. Based on the structures of typical saponins and the results provided below, these parameters are expected to be appropriate.

[0087] In some of these embodiments, the quantitative proton NMR spectrum is further generated according to one or more of the following parameters: (d) a temperature of 12 to 30°C; (e) a magnetic field strength of at least 400 MHz or at least 600 MHz; (f) a probe comprising a proton channel having a probe diameter of 1 mm to 10 mm, e.g., 5 mm; (g) at least 64 scans or at least 128 scans or at least 256 scans; (h) a sweep width of at least 12 ppm or at least 16 ppm; or (i) a line broadening function of at least 0.3 Hz or 1 Hz. Based on the structures of exemplary saponins and the results provided below, these parameters are also expected to be suitable.

[0088] The method also includes (5) comparing the signals of the three protons of the C26 methyl group of the triterpene core of the saponin with the signals of one or more protons of the internal standard compound. The comparison of NMR signals may be, for example, a comparison of peak integrals. This is a standard technique and is good in terms of accuracy. The comparison of NMR signals may also be, for example, a comparison of peak intensities, e.g., peak heights, but this is generally less accurate, especially for signals containing multiple peaks. For example, the comparison of NMR signals can be performed as discussed in the experimental section below.

[0089] In some embodiments, step (5) comprises manual phase correction, automatic polynomial baseline correction, calibration of the chemical shift scale, manual polynomial baseline correction, and integration of the quantitative proton NMR spectrum signal.

[0090] Considering saponins in more detail, in some of these embodiments, the one or more triterpene cores of the saponin further comprise a C23 aldehyde group, and in some of these embodiments, the calibration of the chemical shift scale includes placing the main peak of the C23 aldehyde group at 9.45 ppm.

[0091] In some embodiments, (i) manual polynomial baseline correction comprises correcting the signals of the three protons of the C26 methyl group of the triterpene core of the saponin in a chemical shift range of 0.60 to 0.85 ppm; and / or (ii) signal integration comprises applying integration limits to the signals of the three protons of the C26 methyl group of the triterpene core of the saponin based on a chemical shift range of 0.73 to 0.85 ppm.

[0092] In some embodiments, step (5) comprises performing area normalization of the signals of the three protons of the C26 methyl group of the triterpene core of the saponin and the signals of one or more protons of the internal standard compound to reveal differences in the number of magnetically equivalent protons between them.

[0093] Considering saponins again in more detail, in some embodiments, the triterpene core of the saponin further comprises the C3 and C28 positions, and the saponin further comprises a disaccharide or trisaccharide group attached to the C3 position of the triterpene core and an oligosaccharide group attached to the C28 position of the triterpene core.

[0094] In some of these embodiments, the triterpene core comprises one or more of a quilacic acid triterpene core, a quilacic acid 22β-OH triterpene core, a phytolaccagenic acid triterpene core, a phytolaccagenic acid 23-OAc triterpene core, a gypsogenin triterpene core, or an echinocystic acid triterpene core. As described above, these are saponin triterpene cores comprising a C26 methyl group, a C3 position, and a C28 position.

[0095] Also in some of these embodiments, the oligosaccharide group comprises a fucosyl group comprising the O-3 and O-4 positions. Also in some of these embodiments, one or more of the saponins further comprise a fatty acyl group and / or an acyl II group attached to the O-3 or O-4 position of the fucosyl group. In some of these embodiments, the proton NMR spectrum of the lyophilized saponin and internal standard solution further comprises signals for one or more protons of the fatty acyl group attached to the O-3 or O-4 position of the fucosyl group, and the method further comprises step (6) of comparing the signals for one or more protons of the fatty acyl group attached to the O-3 or O-4 position of the fucosyl group with one or more signals for the three protons of the C26 methyl group of the triterpene core of the saponin or with one or more proton signals of the internal standard compound.

[0096] According to these embodiments, the degree of fatty acylation of the saponin can be determined as the percentage of fatty acyl and / or acyl II groups linked to the O-4 or O-3 of the fucosyl residue per triterpene residue. Alternatively or additionally, the degree of fatty acylation of the saponin can be determined as the percentage of fatty acyl and / or acyl II groups linked to the O-4 or O-3 of the fucosyl residue per internal standard compound. Again, the comparison of NMR signals can be, for example, a comparison of peak integrals or a comparison of peak intensities. Also, the comparison of NMR signals can be performed, for example, as discussed in the experimental section below. [Example]

[0097] 1. Purpose A procedure is described to determine the saponin content in iscom matrix particles and to characterize saponin from iscom matrix particles by proton NMR spectroscopy (H-NMR).

[0098] 2. Scope The scope includes (1) the steps of preparing iscom matrix particle samples containing various saponins from Quillaja Saponaria Molina and / or modified saponins from Quillaja Saponaria Molina, (2) solid-phase extraction (SPE) for sample pretreatment, and (3) proton NMR spectroscopy as the analytical methodology.

[0099] 3. Introduction Applying qNMR to the disintegrated iscom matrix particles allows for the determination of saponin content as well as the measurement of the relative proportions of important structural features in the material, such as triterpene fatty acyl groups.

[0100] 4. Experimental section The quantitative NMR analysis procedure includes a sample workup step for the iscom matrix particle sample to avoid signal overlap between the phospholipids and cholesterol of the sample and the saponin of the sample, thereby enabling quantification. Supelco Discovery DSC-18 SPE tubes with a bed mass of 500 mg / 3 mL tube are used according to the following steps: 1. Conditioning by adding 1000 μL of methanol. 2. Equilibration by adding 1000 μL of 5:95 (V:V) methanol:water solution. 3. Sample loading to add approximately 0.5 mg total saponin content, assuming 100% conversion of saponin to iscom matrix particles upon iscom matrix particle formation. 4. Wash with 1000 μL of 5:95 (V:V) methanol:water solution, then elute the column to dryness. 5. Elute the analytes by adding 1000 μL of methanol, then eluting the column to dryness. 6. Add 100 μl of water to the sample before evaporation; the final water content should be approximately 10% (V:V). 7. Evaporate the sample by centrifugal evaporation at 40°C and 2000 RPM. 1.5 mL will evaporate in approximately 6 hours.

[0101] Maleic acid of certified purity is used as an internal standard. Prepare a stock solution by adding 4-6 mg of maleic acid to 6 g of deuterated water (≥99.9% isotopic purity). Weigh out 50.0 μL of the stock solution into each lyophilized sample.

[0102] The sample containing the saponin and internal standard is then reconstituted in 0.57 mL of deuterated methanol.

[0103] H-NMR spectra were recorded at 12 °C on a Bruker 600 MHz spectrometer using a 5 mm broadband probe. For quantitative H-NMR measurements, a standard proton run (zg30), a pulse flip angle of 30°, 128 or 256 scans, a relaxation delay (D1) of 30 s, a sweep width of 20 ppm, and an acquisition time (AQ) of 2.7 s were applied. This setting allowed a total of 32.7 s of spin relaxation between scans to avoid spin saturation. Before Fourier transformation, a window function was applied using a 1 Hz line broadening function. The chemical shift scale was calibrated by setting the main peak of the aldehyde signal at 9.45 ppm. Manual phasing and polynomial baseline correction (A+Bx+Cx) were performed. 2 ), an integration of the signal can be applied.

[0104] The settings of each of the parameters defined in this section can all have some effect on the NMR spectrum in terms of peak position, peak splitting and / or peak amplitude. Reasonably acceptable settings and / or ranges include: temperature 12-30°C, magnetic field strength 600 MHz or greater (lower magnetic field strengths increase peak overlap, which is undesirable), any 5 mm probe with a proton channel and equal or greater sensitivity, any single pulse test for proton detection (e.g., zg30), a pulse flip angle of 30° (optimized sensitivity and response for this setup), 128 or 256 scans (or fewer scans if using a more sensitive instrument / probe), a relaxation delay of 30 seconds or greater (tested and optimized for the saponin / maleic acid (standard) combination; reasonably, 10 seconds or greater is reasonable depending on the work in progress), a sweep width of at least 16 ppm, AQ = 2.7 seconds or greater (sufficient digital resolution is required for reliable peak integration), and a 1 Hz line broadening function (optimized for 1 Hz in our system for optimal resolution and sensitivity).

[0105] 4.1. Characterization by measuring the ratios of important structural features The application of quantitative NMR to collapsed iscom matrix particles allows the measurement of ratios of important structural features in the material, such as aldehyde and fatty acyl groups.

[0106] The degree of aliphatic acylation can be determined by comparing the signals Fa-2 and Fa-2' with Sap26 (shown in Figures 1 and 2). The signals Fa-2 and Fa-2' originate from two geminal proton pairs present in the aliphatic acyl and acyl II groups. The acyl I group results in a signal that overlaps with Fa-2'. The degree of aliphatic acylation is intended to be evaluated for samples that primarily contain aliphatic acyl and acyl II, i.e., saponin type II or saponin type III samples. The methyl signal Sap26 is common to all reported triterpenes from the tree Quillaja saponaria Molina, including quillaric acid, quillaric acid 22β-OH, phytolaccagenic acid, phytolaccagenic acid 23-OAc, gypsogenin, and echinocystic acid (Fleck et al., Molecules 2019, 24(1), 171; doi.org / 10.3390 / molecules24010171 and references therein). The Sap26 peak is composed of three magnetically equivalent protons. Because it is common to most triterpenes related to Quillaja saponaria, and because different substitution patterns (e.g., sugars or acyl groups) do not change its position (chemical shift) in the spectrum, signal Sap26 can be used as a reference. The degree of fatty acylation, given by Equation 1, reports the percentage of fatty acyl-substituted saponins in the saponin material or iscom matrix particles. The theoretical maximum is 100%. The sum of the signal integrals is divided by the total number of protons in the combined signal (n=4). formula 1

[0107]

number

[0108] The degree of fatty acylation may be used, for example, to characterize the composition of a naturally occurring or modified saponin sample, or to study the degradation of a saponin sample over time. Equation 1 is strictly applicable to saponin type II and / or III samples containing fatty acyl and / or acyl II. Samples containing primarily acyl I may be evaluated by Equation 1, however, the total number of protons may be adjusted to (n=1) to obtain a more realistic degree of acylation.

[0109] 4.2. Determination of Saponin Content Quantitative H-NMR spectroscopy is suitable for determining the saponin content in disintegrated iscom matrix particles. Manual phase correction followed by polynomial baseline correction (A + Bx + Cx) as described above is used. 2 ), further manual baseline correction is performed according to TABLE 2 and FIGS. 3A-C, and signal integration is performed manually according to TABLE 3 and FIG.

[0110] [Table 2]

[0111] [Table 3]

[0112] Area normalization is necessary because the Sap26 peak consists of three magnetically equivalent protons and the maleic acid peak consists of two magnetically equivalent protons. From the normalized areas (integrals) of the saponin and maleic acid peaks, the molar ratio between saponin and maleic acid can be directly determined by the formula given in Equation 2. formula 2

[0113]

number

[0114] This means that the relationship between the normalized peak integrals is directly proportional to the molar ratio. Furthermore, the following formula can be used to obtain the saponin content:

[0115] Calculate the molar concentration of the maleic acid stock solution, C(ma), according to Equation 3. formula 3

[0116]

number

[0117] C(ma) is expressed in mmol / L. Weight(ma) and Weight(D2O) are both in mg and are recorded during preparation of the maleic acid stock solution. Purity(ma) is the certified purity (mass / mass) of maleic acid. MW(ma) is the molecular weight of maleic acid (116.1 g / mol). Density(D2O) is the density of deuterium oxide at room temperature (1.156 g / mL). Conversion factor: 10 6 is introduced, resulting in the molar concentration in mmol / L.

[0118] Calculate the molar amount of maleic acid in the NMR sample, Mol(ma), by the formula in Equation 4. formula 4 Mol(ma)=C(ma, stock solution)×Volume(ma) Volume(ma) is the volume of maleic acid stock solution added to the NMR sample (50.0 microliters).

[0119] The molar amount of saponin in the NMR sample, Mol(sap), can be calculated by the formula given in Equation 5. formula 5

[0120]

number

[0121] The molar concentration of saponin in an NMR sample, C(sap), can be calculated by the formula given in Equation 6, which is a combination of Equations 4 and 5. formula 6

[0122]

number

[0123] Finally, the saponin content (mmol / L) of the matrix can be calculated by the formula given in Equation 7. formula 7

[0124]

number

[0125] In Equation 7, C(ma) (mmol / L) is obtained from Equation 3, and the remaining variables are obtained from the sample preparation procedure and NMR spectra. Volume(sap) is the volume of the iscom matrix particle dispersion loaded onto the SPE column in the sample workup.

[0126] 5. Results 5.1. SPE Sample Preparation - Levels of Purification and Recovery Prior to determination of saponin content by quantitative NMR spectroscopy, SPE (solid phase extraction) was applied to remove non-saponin lipid components and salts from the samples.

[0127] The SPE process was evaluated on a sample of iscom matrix particles designated matrix type III. Fractions collected after the SPE process were analyzed by high-performance liquid chromatography (HPLC) for saponins and lipids (phosphatidylcholine and cholesterol). The recovery of each component was calculated and the results are summarized in Table 4.

[0128] [Table 4]

[0129] The results were satisfactory, showing both an adequate level of purification and recovery of saponins. The relative standard deviation (RSD) from six replicates was 1% for both saponin types, indicating excellent repeatability.

[0130] 5.2. Saponin content in the matrix The saponin content was determined in two samples of iscom matrix particles, the first designated matrix type I and the second designated matrix type II. Three replicates were prepared from each sample. The saponin content was determined and the results are shown in TABLE 5.

[0131] [Table 5]

[0132] The precision of the method was observed to be approximately 2%. The corresponding NMR spectra are shown in Figures 5-10.

[0133] 5.3. Saponin content in modified matrices (deacylated) The saponin content of samples of deacylated iscom matrix particles was determined. Two replicates were prepared for most samples. The saponin content of a reference sample was also determined. The results are shown in Table 6.

[0134] [Table 6]

[0135] Additional results are shown in TABLE 7 for saponin content.

[0136] [Table 7A]

[0137] [Table 7B]

[0138] 5.4. Degree of fatty acylation in matrix and digested samples The degree of fatty acylation was determined for various samples of disassembled iscom matrix particles and a reference iscom matrix particle sample, all of which were disintegrated before determining the fatty acylation as described above. The results are shown in TABLE 8.

[0139] [Table 8]

[0140] In TABLE 8, unstressed matrix refers to the conventional disintegration reference sample.

[0141] The degree of fatty acylation was also determined for a sample of deacylated iscom matrix particles and for a reference disintegrated iscom matrix sample. The results are shown in TABLE 9.

[0142] [Table 9]

Claims

1. 1. A method for quantifying saponin present in a particle comprising saponin and lipid, wherein the saponin of the particle comprises a triterpene core comprising a C26 methyl group containing three protons, said method comprising: (1) isolating the saponin from lipids of particles comprising the saponin and lipids by reversed-phase solid-phase extraction of the saponin from a predetermined amount of the particles comprising the saponin and lipids, thereby obtaining an isolated saponin; (2) freeze-drying the isolated saponin, thereby obtaining a freeze-dried saponin; (3) preparing a solution of the lyophilized saponin and a predetermined amount of an internal standard compound containing one or more protons in deuterated methanol; (4) generating a quantitative proton NMR spectrum of the solution of the lyophilized saponin and the internal standard compound, the spectrum including signals for the three protons of the C26 methyl group of the triterpene core of the saponin and signals for one or more protons of the internal standard compound; and (5) A step of comparing the signals of the three protons of the C26 methyl group of the triterpene core of the saponin with the signals of one or more protons of the internal standard compound. A method comprising:

2. Step (1) is (1.1) conditioning a reversed-phase solid-phase extraction sorbent with a polar organic solvent; (1.2) equilibrating the reversed-phase solid-phase extraction sorbent with a mixture comprising the polar organic solvent and water; (1.3) loading the particles comprising saponin and lipid onto the reversed-phase solid-phase extraction sorbent; (1.4) washing the reversed-phase solid-phase extraction sorbent with a mixture comprising the polar organic solvent and water; and (1.5) eluting the saponin from the reversed-phase solid-phase extraction sorbent with the polar organic solvent, thereby obtaining the isolated saponin in a mixture comprising the polar organic solvent and the saponin.

2. The method of claim 1, comprising:

3. 3. The method of claim 2, wherein the polar organic solvent comprises methanol.

4. 4. The method of claim 3, wherein the mixture comprising a polar organic solvent and water in one or more of steps (1.2) or (1.4) comprises methanol and water in a ratio of 1:99 to 20:80 (V:V), 2:98 to 10:90 (V:V), 3:97 to 7:93 (V:V), or about 5:95 (V:V).

5. 5. The method of any one of claims 2 to 4, wherein during step (1.3), the particles comprising saponin and lipid are loaded onto the reversed-phase solid-phase extraction sorbent at a saponin content of 0.1 mg to 2 mg, a saponin content of 0.2 mg to 1 mg, a saponin content of 0.3 mg to 0.7 mg, a saponin content of 0.4 mg to 0.6 mg, or about 0.5 mg per 500 mg bed mass of the reversed-phase solid-phase extraction sorbent.

6. 6. The method of any one of claims 2 to 5, wherein the reversed-phase solid-phase extraction sorbent comprises an octadecyl sorbent active group.

7. 7. The method of any one of claims 2 to 6, wherein step (1) further comprises step (1.6) of adding water to a mixture comprising the isolated saponin and the polar organic solvent to a final water content of between 5% and 20% by volume, between 7% and 15% by volume, between 9% and 12% by volume, or about 10% by volume.

8. 8. The method of claim 1, wherein the predetermined amount of particles comprising saponin and lipid is the saponin content of the formulation used to make the particles comprising saponin and lipid.

9. 9. The method of claim 1, wherein the internal standard compound comprises maleic acid, and the one or more protons of the internal standard compound comprise two magnetically equivalent olefinic protons of maleic acid.

10. The quantitative proton NMR spectrum has the following parameters: (a) Pulse flip angle of 30°; (b) a relaxation delay of at least 10 seconds; and (c) a collection time of at least 2.7 seconds The method of any one of claims 1 to 9, wherein the compound is produced according to

11. The quantitative proton NMR spectrum has the following parameters: (d) temperature between 12 and 30°C; (e) a magnetic field strength of at least 400 MHz or at least 600 MHz; (f) a probe comprising a proton channel with a probe diameter of 1 mm to 10 mm, e.g., 5 mm; (g) at least 64 scans, or at least 128 scans, or at least 256 scans; (h) a sweep width of at least 12 ppm or at least 16 ppm; or (i) a line broadening function of at least 0.3 Hz or 1 Hz 11. The method of claim 10, further produced according to one or more of:

12. 12. The method of claim 1, wherein step (5) comprises manual phase correction, automatic polynomial baseline correction, calibration of the chemical shift scale, manual polynomial baseline correction, and integration of quantitative proton NMR spectrum signals.

13. 13. The method of claim 12, wherein the triterpene core of one or more of the saponins further comprises a C23 aldehyde group.

14. 14. The method of claim 13, wherein the calibration of the chemical shift scale comprises setting the main peak of the C23 aldehyde group to 9.45 ppm.

15. (i) the manual polynomial baseline correction comprises correcting the signals of the three protons of the C26 methyl group of the triterpene core of saponin in the chemical shift range of 0.60 to 0.85 ppm; and / or (ii) the integration of signals comprises applying integration limits to the signals of the three protons of the C26 methyl group of the triterpene core of the saponin based on a chemical shift range of 0.73 to 0.85 ppm.

16. 16. The method of claim 1, wherein step (5) comprises performing area normalization of the signals of the three protons of the C26 methyl group of the triterpene core of the saponin and the signals of one or more protons of an internal standard compound to reveal the difference in the number of magnetically equivalent protons between them.

17. 17. The method of any one of claims 1 to 16, wherein the triterpene core of the saponin further comprises the C3 and C28 positions, and the saponin further comprises a disaccharide or trisaccharide group attached to the C3 position of the triterpene core and an oligosaccharide group attached to the C28 position of the triterpene core.

18. 18. The method of claim 17, wherein the triterpene core comprises one or more of a quilacic acid triterpene core, a quilacic acid 22β-OH triterpene core, a phytolaccagenic acid triterpene core, a phytolaccagenic acid 23-OAc triterpene core, a gypsogenin triterpene core, or an echinocystic acid triterpene core.

19. 19. The method of claim 17 or claim 18, wherein the oligosaccharide group comprises a fucosyl group comprising the O-3 and O-4 positions.

20. 20. The method of claim 19, wherein one or more of the saponins further comprises a fatty acyl group and / or an acyl II group attached to the O-3 or O-4 position of the fucosyl group.

21. 21. The method of claim 20, wherein the proton NMR spectrum of the lyophilized saponin and internal standard solution further comprises signals for one or more protons of a fatty acyl group attached to the O-3 or O-4 position of the fucosyl group, and the method further comprises step (6) of comparing the signals for one or more protons of the fatty acyl group attached to the O-3 or O-4 position of the fucosyl group with one or more of the signals for the three protons of the C26 methyl group of the triterpene core of the saponin or the signals for one or more protons of the internal standard compound.

22. 22. The method of any one of claims 1 to 21, wherein the saponin comprises saponin extracted from the bark of the South American soap tree Quillaja Saponaria Molina.

23. 23. The method of any one of claims 1 to 22, wherein the particles comprising saponin and lipid comprise or consist of one or more of iscom matrix particles, iscom antigen-presenting particles, liposomal adjuvant system 01 particles, or Army liposomal formulation Q particles.

24. 24. The method of any one of claims 1 to 23, wherein the lipids comprise one or more phospholipids and cholesterol.

25. 25. The method of claim 24, wherein the one or more phospholipids comprise one or more phosphatidylcholines.

26. 26. The method of any one of claims 23 to 25, wherein the particle comprising saponin and lipid is an iscom matrix particle consisting essentially of the saponin, the one or more phospholipids, and the cholesterol.

27. 26. The method of any one of claims 23 to 25, wherein the particle comprising saponin and lipid is an iscom antigen-presenting particle consisting essentially of the saponin, the one or more phospholipids, the cholesterol, and one or more antigens.

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