Method of quantifying saponin present in particles comprising saponin and lipid

Abstract

A method of quantifying a saponin present in a particle comprising a saponin and a lipid is disclosed. The method comprises the steps of: (1) separating saponins from lipids of particles by reverse phase solid phase extraction of the saponins from a predetermined amount of particles; (2) freeze-drying the separated saponin; (3) preparing a solution of freeze-dried saponin and a predetermined amount of internal standard compound in deuterated methanol; (4) generating a quantitative proton NMR spectrum of the solution comprising a signal of three protons of the C26 methyl group of the triterpenoid core of the saponin and a signal of one or more protons of an internal standard compound; and (5) comparing the signal of the three protons of the C26 methyl group of the triterpene core to the signal of the one or more protons of the internal standard compound.

Description

Technical Field

[0001] The present invention generally relates to methods for quantifying saponins present in particles comprising saponins and lipids, and more particularly to methods for quantifying saponins present in particles comprising saponins and lipids, wherein the saponins of the particles comprise a triterpene core that comprises a C26 methyl group containing three protons. Background Art

[0002] Saponins are a large family of glycoconjugates that share a triterpene structure with multiple glycosidic side chains and can have potent immunostimulatory properties. Saponins extracted from the bark of the South American soapbark 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. Saponins from Quillaja saponaria can have a high degree of glyceryl O-acylation, a low degree of glyceryl O-acylation, or no glyceryl O-acylation in their native form. Saponins can also be chemically modified, for example, by partial or complete deacylation or degradation.

[0003] Saponins from Quillaja saponaria can particularly have potent adjuvant activity, but can also be chemically unstable, exhibit hemolytic activity, and be associated with immediate pain at the injection site. For example, Cox et al., PCT / AU1995 / 000670 (WO96011711) describe saponin formulations based on a defined composition of purified saponin fractions from Quillaja saponaria.

[0004] Incorporating saponins from Quillaja saponaria into particles comprising saponins and lipids can attenuate the chemical instability, hemolytic activity, and immediate pain upon injection associated with saponins. Examples of particles comprising saponins and lipids include immunostimulating complex (iscom) matrix particles, immunostimulating complex 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] Immunostimulating complex matrix particles, also known as immunostimulating complex matrix or Matrix, are unique and stable nanostructures made of saponins, phospholipids (such as phosphatidylcholine), and cholesterol. Immunostimulating complex matrix particles are described, for example, in Morein et al., PCT / SE1989 / 000528 (WO9003184) and Morein et al., PCT / SE2003 / 001180 (WO2004004762). Immunostimulating complex matrix particles exhibit potent adjuvant activity similar to saponins, but with reduced chemical instability, hemolytic activity, and immediate pain upon injection associated with saponins.

[0006] Immunostimulating complex antigen presenting particles, also known as immunostimulating complex particles or ISCOMs, are stable nanostructures prepared from a co-formulation of an antigen with saponins, phospholipids, and cholesterol. Immunostimulating complex antigen presenting particles are described, for example, in Morein et al., PCT / SE1986 / 000480 (WO87002250) and Morein et al., PCT / SE2003 / 001180 (WO2004004762). Immunostimulating complex antigen presenting particles contain multiple copies of an antigen physically incorporated into a matrix of saponins, phospholipids, and cholesterol. Like immunostimulating complex matrix particles, immunostimulating complex antigen presenting particles also exhibit potent adjuvant activity, but with reduced chemical instability, hemolytic activity, and immediate pain upon injection associated with saponins.

[0007] Liposome-based adjuvant system 01 particles are liposome-based vaccine adjuvants that contain the saponin Quillaja saponaria 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), incorporation of the QS-21 saponin into liposome-based adjuvant system 01 particles eliminates the hemolytic activity of the saponin.

[0008] Army liposome formulation Q particles are liposome-based vaccine adjuvants that contain the saponin Quillaja saponaria QS-21, saturated phospholipids, cholesterol, and monophosphoryl lipid A, as described, for example, by Alving et al., Expert Review of Vaccines, 2020, 19(3):279-292. According to Alving et al. (2020), incorporation of the QS-21 saponin into army liposome formulation Q particles inhibits the hemolytic activity of the saponin on red blood cells.

[0009] As described above, particles comprising saponins and lipids (such as immunostimulatory complex matrix particles, immunostimulatory complex antigen presenting particles, liposome-based adjuvant system 01 particles, and Army liposomal formulation Q particles) are all composed of saponins and other components. The precise saponin concentration in the particles typically needs to be determined during product development. For most analytical techniques, quantification of the saponin concentration will involve the use of an appropriate saponin reference standard. Unfortunately, in most cases, such a standard is not available, limiting the availability of analytical techniques for quantifying saponins in particles comprising saponins and lipids.

[0010] Nuclear magnetic resonance (NMR) spectroscopy techniques, particularly quantitative proton NMR (also known as qNMR, proton NMR, or 1H-NMR) techniques, can allow determination of the saponin concentration in a liquid composition relative to a common reference standard, which is 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 the sample conditions and experimental settings have been adjusted to ensure a complete quantitative signal response. Important parameters include, for example, signal selection, relaxation delay, and digital resolution. The spectral processing routine should be robust. The signal response is preferably measured as peak area, but peak height can be used in special cases. The resulting signal response can be readily converted to molar ratios and / or concentrations.

[0012] Thus, quantitative proton NMR spectroscopy can be used as an analytical method for quantifying compounds containing protons. The quantification in proton NMR is based on signal comparison, since the signal is directly proportional to the number of protons with the same resonance frequency. The ratio of the signal areas observed in the NMR spectrum is proportional to the ratio of the number of nuclei at the respective molecular sites. The ratio of the observed signal intensities can also be used as an alternative, although this is less preferred. In absolute quantification, one signal of the analyte is compared with a signal from an internal reference standard. Additionally, a normalization procedure 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 techniques to determine the saponin concentration in a liquid composition containing particles comprising saponins and lipids. Adding particles comprising saponins and lipids 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. Additionally, the dispersed particles are not suitable for solution NMR spectroscopy due to the slow molecular tumbling and rapid NMR relaxation caused by their relatively large size. Furthermore, the presence of components other than saponins in the particles and the rest of the composition complicates the direct measurement of the sample.

[0014] Accordingly, there is a need for analytical techniques for quantifying saponins present in particles containing saponins and lipids, and in particular for quantitative proton NMR spectroscopy techniques for this purpose. SUMMARY OF THE INVENTION

[0015] A method for quantifying saponins present in particles containing saponins and lipids is disclosed. The saponins of the particles comprise a triterpene core that includes a C26 methyl group containing three protons. The method includes the steps of:

[0016] (1) Separating saponins from the lipids of particles containing saponins and lipids by reverse-phase solid-phase extraction of saponins from a predetermined amount of the particles containing saponins and lipids, thereby obtaining separated saponins;

[0017] (2) Freeze-drying the separated saponins, thereby obtaining freeze-dried saponins;

[0018] (3) Preparing a solution of the freeze-dried saponins and a predetermined amount of an internal standard compound containing one or more protons in deuterated methanol;

[0019] (4) Generating a quantitative proton NMR spectrum of the solution of the freeze-dried saponins and the internal standard compound, which includes signals of three protons of the C26 methyl group of the triterpene core of the saponins and signals of one or more protons of the internal standard compound; and

[0020] (5) Comparing the signals of the three protons of the C26 methyl group of the triterpene core of the saponins with the signals of one or more protons of the internal standard compound.

[0021] In some embodiments, step (1) includes the steps of:

[0022] (1.1) Conditioning a reverse-phase solid-phase extraction adsorbent with a polar organic solvent;

[0023] (1.2) Equilibrating the reverse-phase solid-phase extraction adsorbent with a mixture containing a polar organic solvent and water;

[0024] (1.3) Loading the particles containing saponins and lipids onto the reverse-phase solid-phase extraction adsorbent;

[0025] (1.4) Washing the reverse-phase solid-phase extraction adsorbent with a mixture containing a polar organic solvent and water; and

[0026] (1.5) Eluting the saponins from the reverse-phase solid-phase extraction adsorbent with a polar organic solvent, thereby obtaining separated saponins in a mixture containing the separated saponins and the polar organic solvent.

[0027] In some of these embodiments, the polar organic solvent includes methanol.

[0028] In addition, in some of these embodiments, 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).

[0029] In addition, in some of these embodiments, during step (1.3), the particles comprising saponin and lipid are loaded onto a reversed-phase solid-phase extraction adsorbent at a saponin content 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 of saponin per 500 mg of bed weight of the reversed-phase solid-phase extraction adsorbent.

[0030] In addition, in some of these embodiments, the reversed-phase solid-phase extraction adsorbent comprises an octadecyl adsorbent active group.

[0031] In addition, in some of these embodiments, step (1) further comprises step (1.6): adding water to the mixture comprising the separated saponin and the polar organic solvent to a final water content of 5 vol% to 20 vol%, 7 vol% to 15 vol%, 9 vol% to 12 vol%, or about 10 vol%.

[0032] In some embodiments, the predetermined amount of the particles comprising saponin and lipid is the saponin content of the preparation for preparing the particles comprising saponin and lipid.

[0033] In some embodiments, the internal standard compound comprises maleic acid, and one or more protons of the internal standard compound comprise two magnetically equivalent vinylic protons of maleic acid.

[0034] In some embodiments, the quantitative proton NMR spectrum is generated according to the following parameters:

[0035] (a) The pulse flip angle is 30°;

[0036] (b) The relaxation delay is at least 10 seconds; and

[0037] (c) The acquisition time is at least 2.7 seconds.

[0038] In some of these embodiments, the quantitative proton NMR spectrum is further generated according to one or more of the following parameters:

[0039] (d) The temperature is 12 - 30 °C;

[0040] (e) The field strength is at least 400 MHz or at least 600 MHz;

[0041] (f) A probe with a proton channel, having a probe diameter of 1 mm to 10 mm, such as 5 mm;

[0042] (g) At least 64 scans, or at least 128 scans, or at least 256 scans;

[0043] (h) A sweep frequency width of at least 12 ppm or at least 16 ppm; or

[0044] (i) A line broadening function of at least 0.3 Hz or 1 Hz.

[0045] In some embodiments, step (5) includes manual phase correction, automatic polynomial baseline correction, calibration of the chemical shift scale, manual polynomial baseline correction, and integration of the quantitative proton NMR spectral signal.

[0046] In some of these embodiments, the triterpene core of one or more saponins further comprises a C23 aldehyde group.

[0047] 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.

[0048] Additionally, in some of these embodiments, (i) the manual polynomial baseline correction includes correcting the signal of three protons of the C26 methyl group of the triterpene core of the saponin in the chemical shift range of 0.60 to 0.85 ppm; and / or (ii) the signal integration includes applying an integration limit to the signal of three protons of the C26 methyl group of the triterpene core of the saponin based on the chemical shift range of 0.73 to 0.85 ppm.

[0049] In some embodiments, step (5) includes area normalization of the signal of three protons of the C26 methyl group of the triterpene core of the saponin and the signal of one or more protons of the internal standard compound to account for the difference in the number of magnetically equivalent protons.

[0050] In some embodiments, the triterpene core of the saponin further comprises C3 and C28 positions, and the saponin further comprises a disaccharide group or a trisaccharide group linked to the C3 position of the triterpene core, and an oligosaccharide group linked to the C28 position of the triterpene core.

[0051] In some of these embodiments, the triterpene core includes one or more of a quillaic acid triterpene core, a quillaic acid 22β-OH triterpene core, a phytolaccagenic acid triterpene core, a phytolaccagenic acid 23-OAc triterpene core, a gypsogenin triterpene core, or an echinoic acid triterpene core.

[0052] In addition, in some of these embodiments, the oligosaccharide group includes fucosyl groups containing O-3 and O-4 positions.

[0053] In addition, in some of these embodiments, one or more saponins further comprise an acyl group and / or an acyl II group linked to the O-3 or O-4 position of the fucosyl group.

[0054] In addition, in some of these embodiments, the proton NMR spectrum of the solution of the lyophilized saponin and the internal standard further comprises signals of one or more protons of the acyl group linked to the O-3 or O-4 position of the fucosyl group, and the method further comprises step (6): comparing the signals of one or more protons of the acyl group linked to the O-3 or O-4 position of the fucosyl group with one or more signals of three protons of the C26 methyl group of the triterpene core of the saponin or one or more signals of one or more protons of the internal standard compound.

[0055] In some embodiments, the saponin includes saponins extracted from the bark of Quillaja saponaria Molina.

[0056] In some embodiments, the particles comprising saponin and lipid include one or more of or consist of one or more of the following: immunostimulating complex matrix particles, immunostimulating complex antigen-presenting particles, liposome-based adjuvant system 01 particles, or Army liposome formulation Q particles.

[0057] In some embodiments, the lipid includes one or more phospholipids and cholesterol.

[0058] In some of these embodiments, one or more phospholipids include one or more phosphatidylcholines.

[0059] In some embodiments, the particles comprising saponin and lipid are immunostimulating complex matrix particles consisting essentially of saponin, one or more phospholipids, and cholesterol.

[0060] In some embodiments, the particles comprising saponin and lipid are immunostimulating complex antigen-presenting particles consisting essentially of saponin, one or more phospholipids, cholesterol, and one or more antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Shows the common structure of saponins from Quillaja saponaria Molina. The asterisk indicates the sites of the Fa-2 and Fa-2' protons

[0062] Figure 2 Shows the proton NMR spectrum of the chromatographic fraction of saponins from Quillaja saponaria Molina, designated as saponin type II. The doublet peaks of Fa-2 and Fa-2' and the methyl Sap26 are shown.

[0063] Figures 3A - 3C Showing baseline correction of the proton NMR spectrum of saponins from an immunostimulatory complex matrix particle sample. First, as Figure 3A shown, the spectrum is corrected in the range of 0 - 10 ppm. Second, as Figure 3B shown, the signal at 0.8 ppm (Sap26) is corrected. Finally, as Figure 3C shown, the signal at 6.35 ppm (maleic acid) is corrected.

[0064] Figure 4 Showing the integrals of quantitative NMR corresponding to (A) the integral of the Sap26 signal (0.0022), and (B) the integral of the maleic acid signal (0.0045).

[0065] Figure 5 Showing the proton NMR spectrum of the first matrix type I sample.

[0066] Figure 6 Showing the proton NMR spectrum of the second matrix type I sample.

[0067] Figure 7 Showing the proton NMR spectrum of the third matrix type I sample.

[0068] Figure 8 Showing the proton NMR spectrum of the first matrix type II sample.

[0069] Figure 9 Showing the proton NMR spectrum of the second matrix type II sample.

[0070] Figure 10 Showing the proton NMR spectrum of the third matrix type II sample. DETAILED DESCRIPTION OF THE INVENTION

[0072] Surprisingly, we have determined that saponins present in particles containing saponins and lipids can be quantified by a method comprising the steps of: (1) separating the saponins from the lipids of the particles by reverse phase solid phase extraction of the saponins from a pre - quantified amount of the particles; (2) lyophilizing the separated saponins; (3) preparing a solution of the lyophilized saponins and a pre - quantified internal standard compound in deuterated methanol; (4) generating a quantitative proton NMR spectrum of the solution of the lyophilized saponins and the internal standard compound, which includes the signal of three protons of the C26 methyl group of the triterpene core of the saponins and the signal of one or more protons of the internal standard compound; and (5) comparing the signal of the three protons of the C26 methyl group of the triterpene core and the signal of one or more protons of the internal standard compound.

[0073] Focusing specifically on immunostimulatory complex matrix particles, we determined that the pretreatment step involves subjecting a predetermined amount of immunostimulatory complex matrix particles in a dispersion sample of immunostimulatory complex matrix particles in a saline solution of known volume to reversed-phase solid-phase extraction, which results in the disintegration of the immunostimulatory complex matrix particles during reversed-phase solid-phase extraction and allows for the adequate separation of saponins from other components present in the sample, thereby enabling the quantification of the separated saponins by qNMR. We also determined that by performing reversed-phase solid-phase extraction on a predetermined amount of immunostimulatory complex matrix particles in a known volume of sample (the predetermined amount being measured based on the saponin content of the formulation initially used to prepare the immunostimulatory complex matrix particles), the qNMR results of the separated saponins can be used to quantify the saponins present in the immunostimulatory complex matrix particles of the sample.

[0074] Specifically, we tested several solvent systems and reversed-phase solid-phase extraction sorbents, resulting in a method that includes a methanol-water system and an octadecyl sorbent active group (also known as C18). We observed reasonable saponin recoveries and good reproducibility, while other components, especially lipids and salts, were either retained on the sorbent (in the case of lipids) or washed away (in the case of salts). We quantitatively collected the eluted saponins, added water to prevent degradation, and lyophilized the resulting solution.

[0075] We then redissolved the dried extract together with an internal standard previously dissolved in fully deuterated water in fully deuterated methanol. We determined suitable conditions for quantitative proton NMR spectroscopy regarding peak selection, digital resolution, relaxation (repetition) delay, and signal-to-noise ratio. Additionally, we determined suitable data processing conditions, including phase and baseline correction and integration limits. After correcting for the number of protons contained in each peak, we calculated the molar ratio from the peak integrals of the selected saponin and internal standard peaks in the resulting NMR spectra.

[0076] Based on our method, since the content of the internal standard is known, we determined that the molar amount of saponins in the NMR sample can be calculated. Additionally, by performing reversed-phase solid-phase extraction on a predetermined amount of immunostimulatory complex matrix particles in a known volume of sample, we were able to relate the molar amount of saponins in the NMR sample to the molar content of saponins in the immunostimulatory complex matrix sample.

[0077] Our method is also applicable to other particles containing saponins and lipids (such as immunostimulatory complex antigen-presenting particles, liposome-based adjuvant system 01 particles, or Army liposome formulation Q particles), which also rely on the use of reversed-phase solid-phase extraction to disintegrate other particles and separate the saponins of the disintegrated particles.

[0078] Particular consideration is given to immunostimulating complex antigen - presenting particles, which are generally made from a protein antigen solution containing a non - ionic surfactant such as polysorbate 80 (PS - 80), Triton, or NP - 40 to stabilize the antigen and form the immunostimulating complex antigen - presenting particles. Like the lipids of the immunostimulating complex matrix particles, the lipids and non - ionic surfactants (such as polysorbate 80) of the immunostimulating complex antigen - presenting particles will remain on the adsorbent. Small amounts of impurities and degradation products of non - ionic surfactants (such as polysorbate 80) may co - elute with the saponin, but are not expected to interfere with the integration of signals in NMR.

[0079] Accordingly, a method for quantifying saponin present in particles containing saponin and lipids is disclosed.

[0080] The particles contain saponin and lipids. The particles can comprise or consist of one or more of the following: for example, immunostimulating complex matrix particles, immunostimulating complex antigen - presenting particles, liposome - based adjuvant system 01 particles, or Army liposome formulation Q particles, as well as other particles containing saponin and lipids.

[0081] Immunostimulating complex matrix particles can be prepared by the methods described, for example, in Morein et al., PCT / SE1989 / 000528 and Morein et al., PCT / SE2003 / 001180. As taught by Morein et al., PCT / SE2003 / 001180, immunostimulating complex matrix particles consist of quillaja saponin, cholesterol, and phospholipids. These particles can be present in a mixture with an antigen, but do not associate with the antigen.

[0082] Immunostimulating complex antigen - presenting particles can be prepared by the methods described, for example, in Morein et al., PCT / SE1989 / 000528 and Morein et al., PCT / SE2003 / 001180. As taught by Morein et al., PCT / SE2003 / 001180, immunostimulating complex antigen - presenting particles are nanoparticle complexes containing quillaja saponin, cholesterol, and phospholipids, into which a vaccine antigen is incorporated.

[0083] Liposome - based adjuvant system 01 particles are described, for example, in Didierlaurent et al., Expert Rev Vaccines 2017, 16(1):55 - 63.

[0084] Army liposome formulation Q particles are described, for example, by Alving et al., Expert Rev Vaccines 2020, 19(3):279 - 292.

[0085] The lipid of the particle can include, for example, one or more phospholipids and cholesterol. One or more phospholipids can include, for example, one or more phosphatidylcholines. These are the lipids of the immunostimulatory complex matrix particle and the immunostimulatory complex antigen presenting particle.

[0086] Thus, in some embodiments, the particle is an immunostimulatory complex matrix particle consisting essentially of saponin, one or more phospholipids and cholesterol. These are the components that provide the structure and function of the immunostimulatory complex matrix particle.

[0087] In addition, in some embodiments, the particle is an immunostimulatory complex antigen presenting particle consisting essentially of saponin, one or more phospholipids, cholesterol and one or more antigens. These are the components that provide the structure and function of the immunostimulatory complex antigen presenting particle.

[0088] The saponin contains a triterpene core that contains a C26 methyl group with three protons. The saponin can include, for example, saponins extracted from the bark of Quillaja saponaria Molina.

[0089] Quillaja saponins generally contain three main parts: (1) a triterpene core, usually the triterpene core of quillaic acid, but also to some extent the triterpene core of quillaic acid 22β-OH, esculentic acid, esculentic acid 23-OAc, gypsogenin or echinocystic acid, the triterpene core containing a C26 methyl group (also known as the Sap26 methyl group), a C3 position and a C28 position; (2) a disaccharide / trisaccharide linked to the C3 position of the triterpene core; and (3) an oligosaccharide linked to the C28 position of the triterpene core. The triterpene core may optionally further contain a C23 aldehyde group (also known as the Sap23 aldehyde group). The triterpene cores of quillaic acid, gypsogenin and echinocystic acid all contain a C23 aldehyde group. The oligosaccharide may optionally contain an acyl group.

[0090] For the common structure of Quillaja saponins, see Figure 1 and Table 1.

[0091] Table 1. Figure 1 The supplementary information shows different structural possibilities of the main components of the sample containing Quillaja saponins.

[0092]

[0093] Regarding the acyl groups in Table 1, the saponin materials can be divided into different categories. Type I saponins can contain a small amount of acyl I, but do not contain fatty acyls or acyl II, while type II saponins can contain a large amount of fatty acyls and / or acyl II, but only contain trace amounts of acyl I. The third type, type III saponins, can contain all types of acyl groups.

[0094] The degree of acylation is the percentage of fatty acyl groups and / or acyl II groups per triterpene residue linked to O-4 or O3 of the fucosyl residue.

[0095] For the desired signal, see Figure 2 .

[0096] The method comprises the steps of: (1) separating saponins from the lipids of particles containing saponins and lipids by reversed-phase solid-phase extraction of the saponins from a predetermined amount of the particles, thereby obtaining separated saponins.

[0097] The predetermined amount of particles containing saponins and lipids can be, for example, the saponin content of the formulation used to prepare the particles. According to the method, the predetermined amount of the particles is pre-determined based on the assumption that 100% of the saponins in the formulation are incorporated into the particles, from the amount of saponins contained in the formulation used to prepare the particles. Thus, for example, when applied to immunostimulatory complex matrix particles according to this method, the predetermined amount of immunostimulatory complex matrix particles can be the saponin content of the formulation used to prepare the immunostimulatory complex matrix particles (assuming 100% of the saponins are incorporated into the immunostimulatory complex matrix particles). Similarly, when applied to immunostimulatory complex antigen-presenting particles according to the method, the predetermined amount of immunostimulatory complex antigen-presenting particles can be the saponin content of the formulation used to prepare the immunostimulatory complex antigen-presenting particles (assuming 100% of the saponins are incorporated into the immunostimulatory complex antigen-presenting particles). The method can also be applied to liposome-based adjuvant system 01 particles and Army liposome formulation Q particles, as well as other particles containing saponins and lipids.

[0098] As described above, by performing reversed-phase solid-phase extraction on a predetermined amount of immunostimulatory complex matrix particles in a known volume of sample (the predetermined amount being measured according to the saponin content of the formulation used to prepare the immunostimulatory complex matrix particles), the qNMR results of the separated saponins can be used to quantify the saponins present in the immunostimulatory complex matrix particles of the sample.

[0099] According to the method, in practice, the required sample volume (mL) loaded onto the reversed-phase solid-phase extraction adsorbent in step (1) can be calculated, for example, as follows. The amount of particles containing saponins and lipids in the sample is defined as the mass (mg) of the saponin content of the formulation used to prepare the particles in the sample, assuming 100% of the saponins are incorporated into the particles, and this mass is known. The volume (mL) of the sample is also known. Thus, also assuming 100% of the saponins are incorporated into the particles, the saponin content concentration (mg / mL) can be calculated by dividing the saponin content mass by the sample volume. Then, the sample volume required for loading is calculated by dividing the desired saponin content for loading (e.g., about 0.5 mg saponin content) by the concentration of the saponin content of the sample (e.g., 5 mg / mL), and in this example, the loading volume is 0.1 mL.

[0100] As will be appreciated, the methods disclosed herein can be used in particular to determine the actual percentage of saponins incorporated into particles comprising saponins and lipids during particle formulation.

[0101] In some embodiments, step (1) comprises the steps of:

[0102] (1.1) conditioning a reversed-phase solid-phase extraction sorbent with a polar organic solvent;

[0103] (1.2) equilibrating the reversed-phase solid-phase extraction sorbent with a mixture comprising a polar organic solvent and water;

[0104] (1.3) loading particles comprising saponins and lipids onto the reversed-phase solid-phase extraction sorbent;

[0105] (1.4) washing the reversed-phase solid-phase extraction sorbent with a mixture comprising a polar organic solvent and water; and

[0106] (1.5) eluting saponins from the reversed-phase solid-phase extraction sorbent with a polar organic solvent to obtain separated saponins in a mixture comprising separated saponins and a polar organic solvent.

[0107] In some of these embodiments, the polar organic solvent comprises methanol. Methanol can be used to condition the reversed-phase solid-phase extraction sorbent to prepare for separating saponins from particles comprising saponins and lipids. A mixture of methanol and water can be used to equilibrate the reversed-phase solid-phase extraction sorbent. A mixture of methanol and water can also be used to wash the reversed-phase solid-phase extraction sorbent to remove salts, particularly when removing salts from a sample of a dispersion of particles comprising saponins and lipids in an aqueous salt solution when loaded onto the reversed-phase solid-phase extraction sorbent. After loading particles comprising saponins and lipids, methanol can also be used to complete the disintegration of particles comprising saponins and lipids in an initial portion of the stationary phase of the solid-phase extraction. Methanol can also be used to elute saponins from the reversed-phase solid-phase extraction sorbent without eluting lipids, such as phospholipids and cholesterol, thereby providing separated saponins in a mixture comprising separated saponins and methanol while the lipids remain on the reversed-phase solid-phase extraction sorbent.

[0108] The mixture comprising a 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), etc.

[0109] It should be understood that polar organic solvents other than methanol can also be used for one or more of conditioning, completing the disintegration of particles comprising saponins and lipids, and eluting saponins without eluting lipids. Such polar organic solvents can also be combined with methanol.

[0110] In addition, mixtures of other polar organic solvents, with or without methanol, and water can be used for equilibration and washing, and can contain other polar organic solvents and water in proportions such as 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), etc.

[0111] In step (1.3), the particles containing saponin and lipid can be loaded onto a reversed-phase solid-phase extraction adsorbent as a liquid dispersion of the particles in a saline solution. This is a common form, for example, for formulating and testing immunostimulatory complex matrix particles and immunostimulatory complex antigen-presenting particles. Conveniently, this form also works well for loading onto a reversed-phase solid-phase extraction adsorbent.

[0112] As described above, a predetermined amount of the particles containing saponin and lipid can be, for example, the saponin content of the formulation for preparing the particles. During step (1.3), the particles can be loaded onto a reversed-phase solid-phase extraction adsorbent, for example, with a saponin content 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 per 500 mg bed weight of the reversed-phase solid-phase extraction adsorbent. According to the method for calculating the required sample volume to be loaded onto the reversed-phase solid-phase extraction adsorbent discussed above, a sample of a liquid dispersion of particles with 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 a saponin content concentration of about 5 mg / mL will have a desired sample loading volume of about 0.02 mL to 0.4 mL, 0.04 mL to 0.2 mL, 0.06 mL to 0.14 mL, 0.08 mL to 0.12 mL, or about 0.1 mL.

[0113] The reversed-phase solid-phase extraction adsorbent can contain, for example, octadecyl adsorbent active groups, etc. For immunostimulatory complex matrix particles, octadecyl adsorbent active groups have been determined to be superior to hydrophilic-lipophilic balance reversed-phase adsorbents (also known as HLB) and octyl adsorbents (also known as C8) in terms of reproducibility and lipid adsorption. However, the suitability of these and other reversed-phase solid-phase extraction adsorbents for other particles containing saponin and lipid can vary depending on, for example, the lipid present in the particles containing saponin and lipid.

[0114] In some embodiments, step (1) further comprises step (1.6): adding water to the mixture comprising the isolated saponin and the polar organic solvent to bring the final water content to 5 vol% to 20 vol%, 7 vol% to 15 vol%, 9 vol% to 12 vol% or about 10 vol%. This can help prevent methanolysis of the isolated saponin.

[0115] Considering step (1) in more detail, loading the particles comprising saponin and lipid onto the reversed-phase solid-phase extraction sorbent in amounts far below the conventional range is crucial for obtaining the isolated saponin. This can be illustrated in the context of solid-phase extraction of saponin from immunostimulatory complex matrix particles using a solid-phase extraction column as shown below. The mass of the immunostimulatory complex matrix particles in the sample can be defined as the saponin concentration (in mg). The immunostimulatory complex matrix particles also contain phospholipids and cholesterol. For example, the loading amount can be equivalent to, for example, about 0.5 mg saponin per 500 mg bed weight of the sorbent (about 0.1% of the bed weight). In contrast, the general recommendation from solid-phase extraction column manufacturers is 25 - 100 mg sample per 500 mg bed weight (about 5% of the bed weight). The reason for using a relatively low loading amount, in addition to the total loading capacity of the solid-phase extraction sorbent, is due to solubility considerations and the need to disintegrate the immunostimulatory complex matrix particles. Considering the solubility reason first, the solubility of saponin, phosphatidylcholine, and / or cholesterol in methanol and methanol:water (5:95 (V:V)) eluents may be limited. This is the case even though these eluents have been determined herein to result in the maximum saponin recovery and reproducibility and complete retention of phosphatidylcholine and cholesterol on the sorbent. Turning to the need to disintegrate the immunostimulatory complex matrix particles, the immunostimulatory complex matrix particles must be disintegrated in the initial part of the stationary phase before separating the saponin from the lipid. Methanol is effective for this. Considering the total loading capacity of the solid-phase extraction sorbent, this depends on the total loading amount of all sample components including saponin (0.5 mg), lipid (about 0.5 mg), and PBS buffer (about 1 mg salt). Advantageously, a sample amount of 0.5 mg has been determined herein to be sufficient for accurate quantitative proton NMR measurements.

[0116] The method further comprises step (2): lyophilizing the isolated saponin to obtain lyophilized saponin.

[0117] The method further includes step (3): preparing a solution of 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 one or more protons of the internal standard compound can include, for example, two magnetically equivalent vinylic protons of maleic acid. Alternatively, other suitable internal standard compounds that also contain one or more protons can be used. The solution can be prepared, for example, by pre-dissolving the internal standard compound in deuterium oxide and then mixing the pre-dissolved internal standard compound, lyophilized saponin, and deuterated methanol to obtain the solution. Alternatively, other suitable methods for preparing the solution can be used.

[0118] The method further includes step (4): generating a quantitative proton NMR spectrum of the solution of lyophilized saponin and the internal standard compound, which includes signals of three protons of the C26 methyl group of the saponin triterpene core and signals of one or more protons of the internal standard compound.

[0119] In some embodiments, the quantitative proton NMR spectrum is 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 structure of typical saponins and the results provided below, these parameters are expected to be suitable.

[0120] 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 - 30 °C; (e) a field strength of at least 400 MHz or at least 600 MHz; (f) a probe diameter of the probe with a proton channel of 1 mm to 10 mm, such as 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 structure of typical saponins and the results provided below, these parameters are also expected to be suitable.

[0121] The method further includes step (5): comparing the signals of three protons of the C26 methyl group of the saponin triterpene core with the signals of one or more protons of the internal standard compound. The comparison of NMR signals can be, for example, a comparison of peak integrals. This is a standard method and has high accuracy. The comparison of NMR signals can also be, for example, a comparison of peak intensities, such as peak height comparison, but this is usually less accurate, especially for signals containing multiple peaks. The comparison of NMR signals can be carried out, for example, in the manner discussed in the experimental section below.

[0122] In some embodiments, step (5) includes manual phase correction, automatic polynomial baseline correction, calibration of chemical shift scale, manual polynomial baseline correction, and integration of the signals of the quantitative proton NMR spectrum.

[0123] Considering saponins in more detail, in some of these embodiments, the triterpene core of one or more saponins further comprises a C23 aldehyde group. Additionally, in some of these embodiments, calibration of the chemical shift scale includes setting the main peak of the C23 aldehyde group to 9.45 ppm.

[0124] In some embodiments, (i) manual polynomial baseline correction includes correcting the signal of three protons of the C26 methyl group of the triterpene core of saponins in the chemical shift range of 0.60 to 0.85 ppm; and / or (ii) signal integration includes applying an integration limit to the signal of three protons of the C26 methyl group of the triterpene core of saponins based on the chemical shift range of 0.73 to 0.85 ppm.

[0125] In some embodiments, step (5) includes area normalization of the signal of three protons of the C26 methyl group of the triterpene core of saponins and the signal of one or more protons of an internal standard compound to account for the difference in the number of magnetically equivalent protons.

[0126] Considering saponins again in more detail, in some embodiments, the triterpene core of saponins further comprises positions C3 and C28, and the saponin further comprises a disaccharide group or a trisaccharide group linked to position C3 of the triterpene core, and an oligosaccharide group linked to position C28 of the triterpene core.

[0127] In some of these embodiments, the triterpene core includes one or more of the following: the triterpene core of quillaic acid, the 22β-OH triterpene core of quillaic acid, the triterpene core of esculentic acid, the 23-OAc triterpene core of esculentic acid, the triterpene core of gypsogenin, or the triterpene core of echinoic acid. As described above, these are saponin triterpene cores containing a C26 methyl group, positions C3 and C28.

[0128] Additionally, in some of these embodiments, the oligosaccharide group contains a fucosyl group containing O-3 and O-4 positions. Additionally, in some of these embodiments, one or more saponins further comprise a fatty acyl group and / or an acyl II group linked to the O-3 or O-4 position of the fucosyl group. In some of these embodiments, the proton NMR spectrum of the freeze-dried solution of saponins and the internal standard further includes the signal of one or more protons of a fatty acyl group linked to the O-3 or O-4 position of the fucosyl group, and the method further includes step (6): comparing the signal of one or more protons of a fatty acyl group linked to the O-3 or O-4 position of the fucosyl group with one or more of the signal of three protons of the C26 methyl group of the triterpene core of saponins or the signal of one or more protons of the internal standard compound.

[0129] 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 attached to 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 attached to O-4 or O-3 of the fucosyl residue per internal standard compound. Similarly, the comparison of NMR signals can be, for example, a comparison of peak integrals or a comparison of peak intensities. In addition, the comparison of NMR signals can be carried out, for example, in the manner discussed in the experimental section below. Example

[0130] 1. Purpose

[0131] A procedure for determining the saponin content in immunostimulatory complex matrix particles and for characterizing the saponins from immunostimulatory complex matrix particles by proton NMR spectroscopy (1H-NMR) is described.

[0132] 2. Scope

[0133] The scope includes (1) providing a sample of immunostimulatory complex matrix particles containing various saponins from Quillaja saponaria and / or modified saponins from Quillaja saponaria, (2) solid-phase extraction (SPE) for sample pretreatment, and (3) proton NMR spectroscopy as the analytical method.

[0134] 3. Introduction

[0135] Applying qNMR to disrupted immunostimulatory complex matrix particles enables determination of the saponin content and measurement of the relative proportions of key structural features (such as triterpene fatty acyl groups) in the material.

[0136] 4. Experiments

[0137] The procedure for quantitative NMR analysis involves sample processing steps for the immunostimulatory complex matrix particle sample to avoid signal overlap between the phospholipids and cholesterol of the sample and the saponins of the sample, thereby enabling quantification. The following steps are used with a Supelco Discovery DSC-18 SPE tube with a bed weight of 500 mg / 3 mL tube:

[0138] 1. Condition by adding 1000 μL of methanol.

[0139] 2. Equilibrate by adding 1000 μL of 5:95 (V:V) methanol:aqueous solution.

[0140] 3. Sample loading, add approximately 0.5 mg of total saponin content, assuming 100% conversion of saponin to immunostimulatory complex matrix particles during the formation of immunostimulatory complex matrix particles.

[0141] 4. Rinse with 1000 µL of 5:95 (V:V) methanol: aqueous solution, then elute the column to dryness.

[0142] 5. Elute the analyte by adding 1000 µL of methanol, then elute the column to dryness.

[0143] 6. Add 100 µl of water to the sample before evaporation, and the final water content should be approximately 10% (V:V).

[0144] 7. Evaporate the sample by centrifugal evaporation at 40 °C and 2000 RPM. 1.5 mL is evaporated in approximately 6 hours.

[0145] Use certified purity maleic acid as the internal standard. Prepare the stock solution by adding 4 - 6 mg of maleic acid to 6 g of deuterated water (isotope purity 99.9% or higher). Weigh 50.0 µL of the stock solution into each lyophilized sample.

[0146] Then reconstitute the sample containing saponin and internal standard in 0.57 mL of deuterated methanol.

[0147] Record the 1H-NMR spectrum at 12 °C on a Bruker 600 MHz spectrometer using a 5 mm broadband probe. For quantitative 1H-NMR measurements, apply the standard proton experiment (zg30), 30° pulse flip angle, 128 or 256 scans, 30 s relaxation delay (D1), 20 ppm sweep width, and 2.7 s acquisition time (AQ). This setting allows a total of 32.7 s of spin relaxation between scans to avoid spin saturation. Apply a window function with a line broadening function of 1 Hz before Fourier transform. Calibrate the chemical shift scale by setting the main peak of the aldehyde signal to 9.45 ppm. After manual phasing and polynomial baseline correction (A + Bx + Cx 2 )), signal integration can be applied.

[0148] The settings of each parameter defined in this section may slightly affect the NMR spectrum in terms of peak position, peak splitting, and / or peak amplitude. Reasonably acceptable settings and / or ranges are as follows: temperature 12 - 30 °C, field strength 600 MHz or higher (lower field strengths increase peak overlap; not desirable), any 5 mm probe with a proton channel and the same or better sensitivity, any single - pulse experiment for proton detection (e.g., zg30), pulse flip angle of 30° (sensitivity and response are optimized at this setting), 128 or 256 scans (fewer scans if a more sensitive instrument / probe is used), relaxation delay of 30 s or longer (tested and optimized for the saponin / maleic acid (standard) combination, and reasonably 10 s or longer is justified depending on the work being done), sweep width of at least 16 ppm, AQ = 2.7 s or longer (reliable peak integration requires sufficient digital resolution), line - broadening function of 1 Hz (optimized to 1 Hz for the best resolution and sensitivity of our system).

[0149] 4.1 Characterization by Measuring Ratios of Key Structural Features

[0150] Applying quantitative NMR to disintegrated immunostimulatory complex matrix particles enables the measurement of the proportion of key structural features (such as aldehyde and fatty acyl groups) in the material.

[0151] The degree of acylation can be determined by comparing the signals Fa - 2 and Fa - 2' with Sap26 (as Figure 1 and Figure 2 shown). The signals Fa - 2 and Fa - 2' originate from two geminal proton pairs present in the fatty acyl and acyl II groups. The acyl I group will produce a signal that overlaps with Fa - 2'. The degree of acylation is intended to evaluate samples mainly containing fatty acyl and acyl II, i.e., saponin type II or saponin type III samples. The methyl signal Sap26 is common to all triterpenes, such as quillaic acid, quillaic acid 22β - OH, phytolaccagenic acid, phytolaccagenic acid 23 - OAc, gypsogenin, and echinocystic acid, which are triterpenes reported from soapbark tree (Fleck et al., Molecules 2019, 24(1), 171; doi.org / 10.3390 / molecules24010171, and references therein). The Sap26 peak consists of three magnetically equivalent protons. The signal Sap26 can be used as a reference because it is common to most soapbark - related triterpenes and the fact that different substitution patterns (such as sugar or acyl groups) do not change its position (chemical shift) in the spectrum. The degree of acylation (given in Equation 1) reports the percentage of saponins substituted with fatty acyl groups in the saponin material or immunostimulatory complex matrix particles. The maximum theoretical value is 100%. The sum of the signal integrals is divided by the total number of protons in the combined signals (n = 4).

[0152] Equation 1.

[0153]

[0154] The degree of acylation can be used, for example, to characterize the composition of natural or modified saponin samples, or to study the degradation of saponin samples over time. Equation 1 strictly applies to type II and / or type III saponin samples containing fatty acyl groups and / or acyl II. Samples mainly containing acyl I can be evaluated by Equation 1, but in order to obtain a more realistic degree of acylation, the total number of protons can be adjusted to (n = 1).

[0155] 4.2 Determination of Saponin Content

[0156] Quantitative 1H-NMR spectroscopy is suitable for determining the saponin content in disintegrated immunostimulatory complex matrix particles. After performing manual phase correction as described above, followed by polynomial baseline correction (A + Bx + Cx 2 ), further manual baseline correction is carried out according to Table 2 and Figure 3A -C, and signal integration is performed manually according to Table 3 and Figure 4

[0157] Table 2. Baseline correction ranges for determining the saponin content in samples of disintegrated immunostimulatory complex matrix particles.

[0158]

[0159] Table 3. Integration limits for determining the saponin content.

[0160]

[0161] Since the Sap26 peak consists of three magnetically equivalent protons and the maleic acid peak consists of two magnetically equivalent protons, area normalization is required. The molar ratio between the saponin and maleic acid can be directly determined from the normalized areas (integrations) of the saponin and maleic acid peaks using the formula given in Equation 2.

[0162] Equation 2.

[0163]

[0164] This means that the relationship between the normalized peak integrations is directly proportional to the molar ratio. Further equations can be used to obtain the saponin content, as described below.

[0165] The molar concentration C(ma) of the stock maleic acid solution is calculated using the formula in Equation 3.

[0166] Equation 3.

[0167] ​

[0168] C(ma) is expressed in mmol / L. The weight (ma) and the weight of D2O (both in mg) are recorded during the preparation of the maleic acid stock solution. Purity (ma) is the certified purity of maleic acid (weight / weight). 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). A conversion factor of 10 6 is introduced to obtain the resulting molar concentration in mmol / L.

[0169] The molar amount of maleic acid, Mol(ma), in the NMR sample is calculated by the formula in Equation 4.

[0170] Equation 4.

[0171]

[0172] Volume (ma) is the volume of the maleic acid stock solution added to the NMR sample (50.0 microliters).

[0173] The molar amount of saponin, Mol(sap), in the NMR sample can be calculated by the formula in Equation 5.

[0174] Equation 5.

[0175]

[0176] The molar concentration of saponin, C(sap), in the NMR sample can be calculated by the formula given in Equation 6, which is a combination of Equation 4 and Equation 5.

[0177] Equation 6.

[0178]

[0179] Finally, the saponin content in the matrix in mmol / L can be calculated by the formula given in Equation 7.

[0180] Equation 7.

[0181]

[0182] In Equation 7, C(ma) (in mmol / L) is obtained from Equation 3, and the remaining variables are obtained from the sample preparation procedure and the NMR spectrum. Volume (sap) refers to the volume of the dispersion of immunostimulatory complex matrix particles loaded on the SPE column during sample treatment.

[0183] 5. Results

[0184] 5.1 Sample Preparation by SPE - Purification Level and Recovery Rate

[0185] SPE (Solid Phase Extraction) was applied to remove non-saponin lipid components and salts from the samples, and then the saponin content was determined by quantitative NMR spectroscopy.

[0186] The SPE step was evaluated on immunostimulatory complex matrix particle samples designated as Matrix Type III. The fractions collected after the SPE step were analyzed for saponins and lipids (phosphatidylcholine and cholesterol) by high performance liquid chromatography (HPLC). The recovery rates of each component were calculated and the results are summarized in Table 4.

[0187] Table 4. Recovery rates of matrix components.

[0188]

[0189] The results were found to be satisfactory and indicated that both the purification level and recovery rate of saponins were appropriate. The relative standard deviation (RSD) for six replicate samples of both saponin types was 1%, indicating excellent reproducibility.

[0190] 5.2 Saponin Content in Matrix

[0191] The saponin content was determined in two samples of immunostimulatory complex matrix particles, with the first sample designated as Matrix Type I and the second sample designated as Matrix Type II. Three replicates were prepared for each sample. The saponin content was measured and the results are shown in Table 5.

[0192] Table 5. Saponin content of matrix samples.

[0193]

[0194] The precision of the method was observed to be approximately 2%. The corresponding NMR spectra are as Figures 5 - 10 shown.

[0195] 5.3 Saponin Content in Modified Matrix (Deacylated)

[0196] For samples of deacylated immunostimulatory complex matrix particles, the saponin content was determined. Two replicates were prepared for most samples. The saponin content of the reference samples was also determined. The results are shown in Table 6.

[0197] Table 6. Saponin content (mmol / L) determined by NMR for modified matrix (deacylated) and reference matrix samples.

[0198]

[0199] Additional results of the saponin content are provided in Table 7.

[0200] Table 7. Saponin content (mmol / L) determined by NMR for additional samples.

[0201]

[0202] 5.4 Degree of Fatty Acylation in Matrix and Degraded Samples

[0203] The degree of acylation of various samples of degraded immunostimulatory complex matrix particles and reference immunostimulatory complex matrix particle samples was determined. All samples disintegrated prior to determination of acylation as described above. The results are shown in Table 8.

[0204] Table 8. Degree of acylation of degraded and reference disintegrated immunostimulatory complex matrix particle samples.

[0205]

[0206] In Table 8, the stress-free matrix refers to the reference sample that disintegrated conventionally.

[0207] The degree of acylation of samples of deacylated immunostimulatory complex matrix particles and reference disintegrated immunostimulatory complex matrix samples was also determined. The results are shown in Table 9.

[0208] Table 9. Degree of acylation of deacylated and reference disintegrated immunostimulatory complex matrix samples.

[0209]

Claims

1. A method for quantifying saponins present in particles comprising saponins and lipids, wherein the saponins of the particles comprise a triterpene core containing a C26 methyl group having three protons, the method comprising the steps of: (1) Separating the saponins from the lipids of the particles comprising saponins and lipids by reversed-phase solid-phase extraction of the saponins from a predetermined amount of the particles comprising saponins and lipids, thereby obtaining separated saponins; (2) Freeze-drying the separated saponins, thereby obtaining freeze-dried saponins; (3) Preparing a solution of the freeze-dried saponins and a predetermined amount of an internal standard compound having one or more protons in deuterated methanol; (4) Generating a quantitative proton NMR spectrum of the solution of the freeze-dried saponins and the internal standard compound, which comprises signals of three protons of the C26 methyl group of the triterpene core of the saponins and signals of one or more protons of the internal standard compound; and (5) Comparing the signals of the three protons of the C26 methyl group of the triterpene core of the saponins with the signals of one or more protons of the internal standard compound.

2. The method according to claim 1, wherein step (1) comprises the steps of: (1.1) Conditioning a reversed-phase solid-phase extraction adsorbent with a polar organic solvent; (1.2) Equilibrating the reversed-phase solid-phase extraction adsorbent with a mixture comprising the polar organic solvent and water; (1.3) Loading the particles comprising saponins and lipids onto the reversed-phase solid-phase extraction adsorbent; (1.4) Washing the reversed-phase solid-phase extraction adsorbent with a mixture comprising the polar organic solvent and water; and (1.5) Eluting the saponins from the reversed-phase solid-phase extraction adsorbent with the polar organic solvent, thereby obtaining the separated saponins in a mixture comprising the separated saponins and the polar organic solvent.

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

4. The method according to claim 3, wherein in one or more of steps (1.2) or (1.4), the mixture comprising the polar organic solvent and water 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. The method according to any one of claims 2-4, wherein during step (1.3), the particles comprising saponins and lipids are loaded onto the reversed-phase solid-phase extraction adsorbent at a saponin content 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 of saponins per 500 mg of bed weight of the reversed-phase solid-phase extraction adsorbent.

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

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

8. The method according to any one of claims 1 - 7, wherein the predetermined amount of the particles comprising saponin and lipid is the saponin content of the formulation for preparing the particles comprising saponin and lipid.

9. The method according to any one of claims 1 - 8, wherein the internal standard compound comprises maleic acid, and one or more protons of the internal standard compound comprise two magnetically equivalent vinylic protons of the maleic acid.

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

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

12. The method according to any one of claims 1 - 11, wherein step (5) comprises manual phase correction, automatic polynomial baseline correction, calibration of the chemical shift scale, manual polynomial baseline correction, and signal integration of the quantitative proton NMR spectrum.

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

14. The method according to 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. The method according to any one of claims 12 - 14, wherein: (i) The manual polynomial baseline correction comprises correcting the signal of three protons of the C26 methyl group of the triterpene core of the saponin in the chemical shift range of 0.60 to 0.85 ppm; and / or (ii) The signal integration comprises applying an integration limit to the signal of three protons of the C26 methyl group of the triterpene core of the saponin based on the chemical shift range of 0.73 to 0.85 ppm.

16. The method according to any one of claims 1 - 15, wherein step (5) comprises area normalization of the signal of three protons of the C26 methyl group of the triterpene core of the saponin and the signal of one or more protons of the internal standard compound to account for the difference in the number of magnetically equivalent protons.

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

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

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

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

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

22. The method according to any one of claims 1-21, wherein the saponin comprises saponins extracted from the bark of Quillaja saponaria Molina.

23. The method according to any one of claims 1-22, wherein the particles comprising saponins and lipids comprise one or more of the following or consist of one or more of the following: Immunostimulating complex matrix particles, immunostimulating complex antigen-presenting particles, liposome-based adjuvant system 01 particles or army liposome formulation Q particles.

24. The method according to any one of claims 1-23, wherein the lipid comprises one or more phospholipids and cholesterol.

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

26. The method according to any one of claims 23-25, wherein the particles comprising saponin and lipid are immunostimulating complex matrix particles consisting essentially of the saponin, one or more phospholipids and cholesterol.

27. The method according to any one of claims 23-25, wherein the particles comprising saponin and lipid are immunostimulating complex antigen-presenting particles consisting essentially of the saponin, one or more phospholipids, cholesterol and one or more antigens.

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