Method for detecting MPL in sample and related application thereof

Abstract

The invention discloses a method for detecting MPL in a sample and related application of the method, and relates to the field of biological detection. The adsorbent comprises aluminum phosphate or a product obtained by mixing aluminum hydroxide and a phosphate solution, and can effectively adsorb free MPL in a sample, so that effective separation of the free MPL in the sample and MPL combined with lipidosome is realized, and the proportion of the free MPL in the sample and the MPL combined with the lipidosome can be determined by detecting the content difference of the MPL in the sample before and after adsorption. The method has the advantages of being easy to implement, low in cost and high in efficiency, expensive reagents and instruments are not needed, the encapsulation efficiency of the MPL on the lipidosome can be rapidly and accurately calculated, and a new way is provided for quality control of adjuvants or vaccines containing the MPL.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular, to a method for detecting MPL in a sample and related applications thereof. Background Art

[0002] MPLA, the same as MPL, with the Chinese name of lipid A phosphate, is mainly extracted from the innermost lipid A part of lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria. It is an amphiphilic structure and is also the key structure of Gram-negative bacteria with toxicity and immunogenicity, which can cause an immune response in the body.

[0003] MPL acts on Toll-like receptor 4 (TLR4) and has the advantages of strong immunogenicity, clear mechanism, and low toxicity. It can activate macrophages, especially dendritic cells (DCs), promote the differentiation of antigen-specific CD4+ T cells and the production of IFN-γ, and cause a Th1-type immune response. MPL is widely used as an adjuvant for vaccines, allergy drugs, and immunotherapy to enhance the immune response. It has been used in multiple vaccine products, such as the cervical cancer vaccine Cervarix® and the hepatitis B vaccine Fendrix®.

[0004] Due to the relatively large toxic and side effects of MPL when used alone, MPL is often combined with other components to form an adjuvant system, such as AS01 and AS02. In the adjuvant system, MPL is usually tightly bound to lipids, thereby reducing its toxic and side effects while retaining its adjuvant effect.

[0005] Currently, the detection method of MPL can only detect the total amount of MPL in a sample and cannot distinguish between free MPL and MPL bound to liposomes. To ensure the batch-to-batch consistency of MPL bound to liposomes in products and improve the safety of vaccines, there is an urgent need for a new detection method that can distinguish between free MPL and MPL bound to liposomes.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting MPL in a sample and related applications thereof.

[0008] The present invention is implemented as follows: In a first aspect, an embodiment of the present invention provides a method for detecting MPL in a sample, which includes the following steps: mixing the sample with an adsorbent, taking the portion not bound to the adsorbent, and detecting the MPL content, which is used as the MPL content bound to liposomes; wherein, the adsorbent includes adsorbent A and / or adsorbent B; the adsorbent A includes aluminum phosphate; the preparation method of the adsorbent B includes: mixing aluminum hydroxide and a phosphate solution.

[0009] In a second aspect, an embodiment of the present invention provides the use of the method as described in the foregoing embodiments or a reagent for implementing the method as described in the foregoing embodiments in the quality control of differentiating free MPL and MPL bound to liposomes or products containing free MPL and / or MPL bound to liposomes.

[0010] The present invention has the following beneficial effects: By using aluminum phosphate or a product obtained by mixing aluminum hydroxide and a phosphate solution as a new adsorbent, the present invention can effectively adsorb free MPL in the sample, thereby realizing the effective separation of free MPL and MPL bound to liposomes in the sample. By detecting the difference in MPL content in the sample before and after adsorption, the proportions of free MPL and MPL bound to liposomes in the sample can be determined. This method has the advantages of easy implementation, low cost, and high efficiency. It does not require expensive reagents and instruments and can quickly and accurately calculate the encapsulation rate of MPL on liposomes, providing a new way for the quality control of products containing MPL (such as adjuvants or vaccines). Detailed Embodiments

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0012] On the one hand, an embodiment of the present invention provides a method for detecting MPL in a sample, which includes the following steps: Mixing the sample with an adsorbent, taking the portion not bound to the adsorbent, and detecting the MPL content, which is used as the MPL content bound to liposomes; Wherein, the adsorbent includes adsorbent A and / or adsorbent B; the adsorbent A includes aluminum phosphate; the preparation method of the adsorbent B includes: mixing aluminum hydroxide and a phosphate solution.

[0013] In some embodiments, the phosphate solution contains phosphate ions, and the phosphate ions include (dihydrogen phosphate ion), (hydrogen phosphate ion) and Any one or more of (phosphate ions).

[0014] In some embodiments, in the adsorbent B, the molar ratio of the aluminum ions to the phosphate ions is 37:22 to 50.

[0015] The inventors found that when using an adsorbent for adsorption treatment, if the concentration of phosphate ions is too low, both MPL bound to liposomes and free MPL will be adsorbed simultaneously. If the concentration of phosphate ions is too high, no significant adsorption effect will be produced on either MPL bound to liposomes or free MPL. By regulating the molar ratio of aluminum ions to phosphate ions, the adsorption effect of the adsorbent on free MPL can be significantly improved while adsorbing MPL bound to liposomes.

[0016] The specific value of 37:22 to 50 can be any one or the range between any two of 37:22, 37:24, 37:26, 37:28, 37:30, 37:32, 37:34, 37:36, 37:38, 37:40, 37:42, 37:44, 37:46, 37:48, and 37:50.

[0017] In some embodiments, in the adsorbent B, the concentration of the aluminum ions is in the range of any one or between any two of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 mg / mL.

[0018] In some embodiments, in the adsorbent B, the concentration of the phosphate ions is in the range of any one or between any two of 2.2, 5, 10, 15, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500 mM.

[0019] In some embodiments, the phosphate solution includes any one or more of PB, PBS, PBST, KPBS, and TPBS.

[0020] Specifically, the main components of PBS (Phosphate-Buffered Saline): The main components of PB (Phosphate Buffer): PBST (PBS + Tween-20), with Tween-20 (a non-ionic surfactant) added to the basis of PBS. KPBS (Potassium PBS), using to replace such as KCl, ). TPBS (Tris-PBS, Tris-buffered PBS), with Tris-HCl added to the basis of PBS to enhance the buffering capacity.

[0021] In some embodiments, the pH of the phosphate solution is 5 to 8, and specifically can be any one or the range between any two of 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, and 8.

[0022] In some embodiments, sodium chloride is contained in the mixed system of aluminum hydroxide and the phosphate solution, and the mass-volume fraction of the sodium chloride in the mixed system is 0.8% to 5% (w / v). The situation that sodium chloride is contained in the mixed system of aluminum hydroxide and the phosphate solution includes two cases: the phosphate solution used is a phosphate solution containing sodium chloride, such as PBS; or the phosphate solution used is a phosphate solution without sodium chloride, then sodium chloride needs to be added to the phosphate solution or system without sodium chloride (such as PB + NaCl) to make the action concentration of sodium chloride in the system reach the above-set range. The action concentration of the sodium chloride in the system can specifically be any one or the range between any two of 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, and 5%.

[0023] In some embodiments, the volume ratio of the sample to the adsorbent is (1~3):(1~3), and specifically can be any one or the range between any two of 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2.

[0024] In some embodiments, the mass - volume fraction of sodium chloride in the mixed system of the adsorbent and the sample is 0.4% - 2.5% (w / v), specifically, it can be any one of 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4% and 2.5%, or the range between any two of them.

[0025] In some embodiments, the method further includes: detecting the MPL content of the sample not treated with the adsorbent as the total MPL amount in the sample; the proportion of free MPL in the sample = (total MPL amount - MPL content bound to liposomes) / total MPL amount × 100%.

[0026] In some embodiments, the method for detecting the MPL content includes any one or more of the following: high - performance liquid chromatography (HPLC) detection, liquid chromatography - mass spectrometry (LC - MS / MS), and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (MALDI - TOF MS).

[0027] In some embodiments, the sample includes a solution containing MPL; the solution containing MPL includes an adjuvant or a vaccine.

[0028] In some embodiments, the MPL includes any one or more of MPL in its natural form, 2D - MPL (dideacylated MPL), 3D - MPL (3 - O - deacylated MPL), and 6D - MPL (hexa - deacylated MPL).

[0029] The natural form of MPL is a natural hydrolysis product of lipopolysaccharide (LPS) from Gram - negative bacteria (such as Salmonella), retaining a single phosphate group and an intact lipid chain. 2D - MPL: The natural MPL is partially deacylated to remove 2 acyl chains (usually branched chains). 3D - MPL: A derivative of natural MPL by 3 - position deacylation (selective removal of the acyl chain at the 3 - O position). 6D - MPL: The natural MPL is completely deacylated to remove all 6 acyl chains.

[0030] In some embodiments, the liposomes include any one or more of neutral liposomes and positive liposomes.

[0031] Specifically, neutral liposomes are usually composed of neutral (uncharged) lipids (such as phosphatidylcholine (PC), cholesterol), with a net surface charge of zero. Positive liposomes are usually composed of cationic lipids (such as DOTAP, DDAB) and / or cholesterol, with a positive surface charge.

[0032] In addition, the embodiments of the present invention provide the application of the method described in the foregoing embodiments or the reagent for implementing the method described in the foregoing embodiments in the quality control of distinguishing free MPL from MPL bound to liposomes or products containing free MPL and / or MPL bound to liposomes.

[0033] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0034] Example 1 This example provides a method for detecting the MPL content (applicable to different MPL forms, such as natural form and 3D-MPL) as follows: (1) Experimental instruments and reagents HPLC: Agilent 1260 InfinityⅡ, equipment number: ME-D-070(J); Chromatographic column: shim-pack GIS 5μm C18 4.6×150mm (HSS), number: C18-A-23-018; Mobile phase A: 95% methanol - triethylamine - glacial acetic acid (volume ratio 1000:1:1); Mobile phase B: isopropanol - triethylamine - glacial acetic acid (volume ratio 1000:1:1); MPL (purity: 93.17%); Solvent: isopropanol - tetrahydrofuran (volume ratio 1:2).

[0035] (2) Chromatographic conditions Mobile phase A: 95% methanol - triethylamine - glacial acetic acid (volume ratio 1000:1:1); Mobile phase B: isopropanol - triethylamine - glacial acetic acid (volume ratio 1000:1:1); Flow rate: 1 ml / min; detector: ELSD (drift tube temperature: 45°C, carrier gas: 1.5 L / min); column temperature: 45°C; injection volume: 100 μl; gradient elution; time: 35 min; Mobile phase A and mobile phase B are used for gradient elution according to the elution gradient shown in Table 1 below.

[0036] Table 1 Elution gradient

[0037] (3) Preparation of reference solution Reference stock solution: Weigh 5.367 mg of MPL solid powder (purity: 93.17%) into a 25 ml volumetric flask, dissolve it with the solvent, and make up to the mark. The concentration of the reference stock solution is 200 μg / ml.

[0038] Standard curve: Take 50 μl, 100 μl, 150 μl, 200 μl, and 250 μl of the stock solution respectively and place them in 5 1.5-ml EP tubes. Add solvent to each tube to make the volume up to 1 ml. In this way, standard curves of 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, and 50 μg / ml are prepared.

[0039] Table 2 Standard curve

[0040] Table 3 Injection sequence

[0041] (4)Preparation of test solution Take 200 μl of the sample solution at room temperature + 800 μl of solvent (the solvent should be taken with a pipette). Dilute the sample 5 times and use it as the test solution for injection. The sample should be diluted at least 4 times to be completely dissolved.

[0042] Example 2 This example provides a method for detecting MPL in a sample, which includes the following steps.

[0043] 1. Before adsorption Before adsorption with the adsorbent, take 2.5 mL of the sample not treated with the adsorbent and add 2.5 mL of ultrapure water. Detect the MPL content (the detection method is shown in Example 1), and use the obtained result as the total MPL amount in the test sample.

[0044] 2. Adsorption Take 2.5 mL of the test sample and mix it evenly with 2.5 mL of the adsorbent. Adsorb at room temperature for 15 min, centrifuge at 6000 rpm for 5 min, and then take the supernatant to detect the MPL content (the detection method is shown in Example 1). Use the obtained result as the MPL content bound to liposomes.

[0045] Among them, the preparation method of the adsorbent is as follows: Take an aluminum hydroxide solution, add PB (sodium dihydrogen phosphate and disodium hydrogen phosphate, pH = 6.0) to the aluminum hydroxide solution according to the molar ratio of aluminum ions to phosphate ions of 1.48:1, and add an NaCl solution. Mix for more than 30 min, and then dilute with water to make the final concentration of Al ions 2 mg / ml, the working concentration of PB 50 mM, and the working concentration of NaCl 1.8% (w / v). It should be noted that the final concentration of Al ions in the mixture of the adsorbent and the sample is 1 mg / ml, the working concentration of PB in the mixture is 25 mM, and the working concentration of NaCl in the mixture is 0.9% (w / v).

[0046] 3. Calculation Calculate the proportion of free MPL in the sample based on the following formula: The proportion of free MPL in the sample = (total amount of MPL - amount of MPL bound to liposomes) / total amount of MPL × 100%.

[0047] Example 3 The method of Example 2 was used to detect MDL in its natural form (Samples 1 - 2) and 3D-MDL (Samples 3 - 4), and the results are as follows.

[0048] Table 4 Results of detecting the content and encapsulation efficiency of MPL

[0049] Note: Encapsulation efficiency = MPL concentration after adsorption by adsorbent / MPL concentration after mixing with ultrapure water * 100%.

[0050] Example 4 Verify the adsorption effect of adsorbents containing different molar ratios of aluminum ions and phosphate ions on free MPL and MPL bound to liposomes.

[0051] In this example, multiple experimental groups and control groups were set up. The method for detecting the MPL concentration referred to Example 1 and is as follows: Control 1: Directly detect the MPL concentration of a 0.1 mg / ml free MPL solution; Samples 1-1 to 1-11: Based on the method of Example 2, set adsorbents with different molar ratios of aluminum ions and phosphate ions, and respectively detect the 0.1 mg / ml free MPL solution; Sample 1-12: Replace the adsorbent in Example 2 with aluminum phosphate and detect the MPL of the 0.1 mg / ml free MPL solution; Control 2: Directly detect the MPL concentration of a 0.1 mg / ml solution of MPL (neutral-MPL) bound to neutral liposomes (composed of cholesterol and DOPC); Samples 2-1 to 2-11: Based on the method of Example 2, set adsorbents with different molar ratios of aluminum ions and phosphate ions, and respectively detect the 0.1 mg / ml neutral-MPL solution; Sample 2-12: Replace the adsorbent in Example 2 with aluminum phosphate and detect the MPL of the 0.1 mg / ml neutral-MPL solution.

[0052] The results are as follows.

[0053] Table 5 Comparison of adsorbents with different molar ratios of aluminum ions and phosphate ions

[0054] Note: The concentrations of Al, Al in aluminum phosphate, PB, and NaCl recorded in the "Formulation" column are their effective concentrations in the mixture of the sample and the adsorbent; the concentrations of MPL and neutral-MPL recorded in the table are the concentrations of MPL and neutral-MPL in the sample; the pH is the pH of the mixture of the sample and the adsorbent.

[0055] It can be seen from Samples 1-1 to 1-11 that when the concentration of aluminum ions in aluminum hydroxide is 1 mg / ml and the concentration of phosphate ions is 60 mM, the adsorbent cannot completely adsorb free MPL. When the concentration of phosphate ions ≤ 50 mM, the adsorbent can completely adsorb free MPL. It can be seen from Samples 2-1 to 2-11 that when the concentration of aluminum ions in aluminum hydroxide is 1 mg / ml and the concentration of phosphate ions is 5-15 mM, the adsorbent will adsorb MPL bound to liposomes. When the concentration of phosphate ions ≥ 22 mM, the adsorbent no longer adsorbs MPL bound to liposomes. Therefore, when the concentration of aluminum ions in aluminum hydroxide is 1 mg / ml and the concentration of phosphate ions is 22-50 mM, that is, when the molar ratio of aluminum ions to phosphate ions is 37:22 to 37:50, the adsorbent only adsorbs free MPL and does not adsorb MPL bound to liposomes, so free MPL and MPL bound to liposomes can be separated. It can be seen from Sample 1-12 and Sample 2-12 that aluminum phosphate can completely adsorb free MPL and does not adsorb MPL bound to liposomes.

[0056] Example 5 Verify the influence of different adsorbents on the detection of MPL.

[0057] Using adsorbents with different molar ratios of aluminum ions and phosphate ions (based on the adsorbent in Example 2, the molar ratio of aluminum:phosphate ions = 37:22 to 37:50) and aluminum phosphate as adsorbents, the detection of MPL was carried out based on the method in Example 2, and the verification of sample addition and recovery was carried out to judge the accuracy and applicability of the method, as follows.

[0058] Control 3: Directly detect the MPL concentration of a 100 μg / ml neutral-MPL solution; Samples 3-1 to 3-7: Based on the method in Example 2, for a 100 μg / ml neutral-MPL solution (or additionally supplemented with MPL solutions with final concentrations of 10 μg, 50 μg, and 100 μg), where the effective concentration of Al ions (Al in aluminum hydroxide) in the mixture is set to 1 mg / mL, and the effective concentration of phosphate ions in the mixture is set to 22 mM; Samples 3-8 to 3-14: Based on the method of Example 2, a solution of 100 μg / ml neutral-MPL (or additionally supplemented with MPL solutions with final concentrations of 10 μg, 50 μg, and 100 μg), wherein the effective concentration of Al ions (Al in aluminum hydroxide) in the mixture is set to 1 mg / mL, and the effective concentration of phosphate ions in the mixture is set to 50 mM; Samples 3-15 to 3-21: Based on the method of Example 2, a solution of 100 μg / ml neutral-MPL (or additionally supplemented with free MPL or neutral / MPL, with the additional concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL), wherein the adsorbent is replaced with aluminum phosphate, and the effective concentration of aluminum ions in aluminum phosphate in the mixture is 1 mg / mL.

[0059] Table 6 Spike recovery

[0060] Note: The concentrations of Al, Al in aluminum phosphate, PB, and NaCl recorded in the "Preparation" column are their effective concentrations in the mixture of the sample and the adsorbent, and the pH is the pH of the mixture of the sample and the adsorbent.

[0061] From the results, it can be seen that the spike recoveries of the samples with different concentrations of MPL added additionally (Samples 3-2 to 3-4, Samples 3-9 to 3-11, Samples 3-16 to 3-18) are all close to 0%, indicating that aluminum phosphate or the adsorbent obtained by mixing aluminum hydroxide and phosphate solution (molar ratio of aluminum:phosphate ions = 37:22 to 37:50) can effectively adsorb free MPL; The spike recoveries of the samples with different concentrations of "neutral-MPL" added additionally (Samples 3-5 to 3-7, Samples 3-12 to 3-14, Samples 3-19 to 3-21) are close to 100%, indicating that aluminum phosphate or the adsorbent obtained by mixing aluminum hydroxide and phosphate solution (molar ratio of aluminum:phosphate ions = 37:22 to 37:50) does not adsorb "neutral-MPL", and at the same time proves that the quality control measures of this analysis method are effective and the analysis data is reliable.

[0062] Example 6 Effect of different sodium chloride concentrations on detection Verify the adsorption effect of adsorbents containing different sodium chloride concentrations on free MPL and MPL bound to liposomes.

[0063] In this example, multiple experimental groups and control groups are set up, and the MPL concentration detection method refers to Example 1, specifically as follows: Control 4: Directly detect the MPL concentration in the 0.15 mg / ml free MPL solution; Samples 4-1 to 4-5: Based on the method of Example 2, adsorbents with different sodium chloride concentrations were set to detect the 0.15 mg / ml free MPL solution respectively; Control 5: Directly detect the MPL concentration in the solution of 0.15 mg / ml MPL combined with neutral liposomes (neutral-MPL); Samples 5-1 to 5-5: Based on the method of Example 2, adsorbents with different sodium chloride concentrations were set to detect the 0.15 mg / ml neutral-MPL solution respectively.

[0064] Table 7 Detection Results

[0065] Note: The concentrations of Al, PB, and NaCl recorded in the "Preparation" column are their effective concentrations in the mixture of the sample and the adsorbent, and the pH is the pH of the mixture of the sample and the adsorbent.

[0066] It can be seen from Samples 4-1 to 4-5 that when the concentration of aluminum ions in aluminum hydroxide is maintained at 1 mg / ml and the concentration of phosphate ions is 25 mM unchanged, when the sodium chloride concentration is 0, the adsorbent cannot completely adsorb free MPL, and when the sodium chloride concentration ≥ 0.4%, the adsorbent can completely adsorb free MPL. It can be seen from Samples 5-1 to 5-5 that when the concentration of aluminum ions in aluminum hydroxide is maintained at 1 mg / ml and the concentration of phosphate ions is 25 mM unchanged, when the sodium chloride concentration is 0% - 2.5%, the adsorbent does not adsorb MPL bound to liposomes. Therefore, when the sodium chloride concentration is 0.4% - 2.5%, the adsorbent can effectively adsorb free MPL and does not adsorb MPL bound to liposomes.

[0067] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting MPL in a sample, characterized in that, It includes the following steps: The sample is mixed with an adsorbent, and the portion not bound to the adsorbent is taken for testing the MPL content, which is used as the MPL content bound to the liposome; Wherein, the adsorbent includes adsorbent A and / or adsorbent B; the adsorbent A includes aluminum phosphate; and the preparation method of the adsorbent B includes: mixing aluminum hydroxide and phosphate solution.

2. The method according to claim 1, wherein The phosphate solution contains phosphate ions, and the phosphate ions include , and any one or more thereof; in the adsorbent B, the molar ratio of aluminum ions to phosphate ions is 37:22 to 50.

3. The method according to claim 2, wherein In the adsorbent B, the concentration of the aluminum ions is 0.1-10 mg / mL, and the concentration of the phosphate ions is 2.2-500 mM.

4. The method according to claim 2, wherein The phosphate solution includes: any one or more of PB, PBS, PBST, KPBS and TPBS; The pH of the phosphate solution is 5-8.

5. The method according to claim 1, wherein The mixed system of aluminum hydroxide and phosphate solution contains sodium chloride, and the mass volume fraction of the sodium chloride in the mixed system is 0.8% to 5%.

6. The method according to any one of claims 1 to 5, characterized in that, The method further comprises: detecting the MPL content of the sample not treated with the adsorbent as the total amount of MPL in the sample; The proportion of free MPL in the sample = (total MPL amount - content of MPL bound to liposomes) / total MPL amount × 100%.

7. The method according to any one of claims 1 to 5, characterized in that The method for detecting the MPL content includes any one or more of the following: high performance liquid chromatography detection, liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

8. The method according to any one of claims 1 to 5, characterized in that, The sample includes a solution containing MPL; the solution containing MPL includes an adjuvant or a vaccine.

9. The method according to any one of claims 1 to 5, characterized in that The MPL includes any one or more of the natural form of MPL, 2D-MPL, 3D-MPL and 6D-MPL; The liposomes include any one or more of neutral liposomes and cationic liposomes.

10. Use of the method according to any one of claims 1 to 9 in distinguishing free MPL from liposome-bound MPL or in quality control of a product containing free MPL and / or liposome-bound MPL.

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