Method for determining purity of vaccine adjuvant MPLA

Abstract

The invention discloses a method for determining the purity of a vaccine adjuvant MPLA, which adopts a high performance liquid chromatography for detection, and the high performance liquid chromatography adopts alkyl bonded silica gel as a chromatographic column of a stationary phase, an organic solvent-ion pair reagent buffer solution as a mobile phase, an ultraviolet spectrophotometer as a detector, and adopts a gradient elution mode for detection. The invention provides a stable method for accurately completing the MPLA purity detection method by selecting proper stationary phase and mobile phase and proper determination conditions. The detection method disclosed by the invention is high in accuracy, convenient to operate and high in efficiency, and is a method suitable for being widely popularized and used.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a method for determining the purity of vaccine adjuvant MPLA. Background Art

[0002] Gram-negative bacterial lipopolysaccharides (LPS), also known as endotoxins, are one of the most effective stimulants of the host's innate immune system, and the recognition of LPS is crucial for the host body to eliminate the infection of invading bacterial pathogens. It is generally composed of three parts (such as Figure 1 Shown): O-antigen (O-antigen) or O-specific polysaccharide, core oligosaccharide (oligosaccharide) and lipid A (lipid A).

[0003] The detailed structure of Escherichia coli lipid A is considered to be the prototype of all lipid A structures (e.g. Figure 2 ). It consists of a diphosphorylated glucosamine disaccharide backbone (light brown). Due to factors such as bacterial adaptation, selection pressure, incomplete biosynthesis, changes in environmental conditions and the presence of external stimuli, the lipid A portion can show subtle chemical structural differences, mainly in the number, nature and distribution of acyl chains, such as acyl chains can be identified in four to seven acylated forms; fatty acids can present unusual structural features such as methyl branches, different functional groups and hydroxyl groups, chain lengths up to 30 carbon atoms and odd carbon chains. The acylation pattern of lipid A (small structural differences) determines the endotoxin activity of these compounds.

[0004] Most intestinal bacteria produce hexaacylated bisphospholipid A, which has the strongest immunostimulatory capacity in mammals, i.e., the highest pro-inflammatory cytokine induction activity. Tetraacylated lipid A and some pentaacylated lipid A lack activity on human immune cells. Structures similar to E. coli lipid A, with two phosphates but seven or five fatty acids, are about 100 times less active.

[0005] MPLA currently approved for vaccine adjuvants is a product of GSK After S.minnesota R59 cells are grown, harvested and dried under culture conditions, LPS is obtained by solvent extraction of the cell mass, followed by acid hydrolysis, neutralization, purification and salt formation. Bulk product for use in vaccine formulations.

[0006] Synthetic MPLA and bacterial origin The reason for the differences lies in the degree of variability in the acyl chain structure; synthetic MPLA contains a fixed composition of six acyl chains attached to two sugar moieties; Similar natural lipid A products have 3 to 6 acyl chains. Relevant experiments have shown that the synthetic phospholipid series products have similar activity and safety as bacterial-derived MPLA.

[0007] The new adjuvant MPLA product is hexaacylated 3-O-deacylated-4'-monophosphoryl lipid A triethylamine salt. From the structural point of view, it is of bacterial origin. One of the main ingredients of the product; in terms of content, it is The most abundant ingredient in the product line; from a functional point of view, it is The most important stimulator of immune activity, therefore, hexaacylated 3-O-deacylated-4'-monophosphoryl lipid A is The core component of Figure 3 shown.

[0008] The new adjuvant MPLA belongs to the MPL class of adjuvants. The combination of MPL and QS-21 enhances the cell-mediated immune response and promotes antigen-specific CD8 + T cell responses and CD4 + T cell responses, inducing the production of IFN-γ, IgG1 and IgG3 antibodies. The combination of immunostimulants in different adjuvant formulations maximizes vaccine efficacy while minimizing toxicity. Adjuvants containing MPL have been used in prostate cancer containing the MUC1 antigen. or with For melanoma vaccines TLR4 receptor agonist MPL shows strong development potential.

[0009] Since MPLA is a new type of MPL adjuvant, the CAS number of this adjuvant has not been found so far, and its detection method has not been reported. Summary of the invention

[0010] The purpose of the present invention is to provide a method for determining the purity of vaccine adjuvant MPLA, so as to achieve rapid and convenient detection of the purity of vaccine adjuvant MPLA and ensure the quality control of MPLA adjuvant, which has practical significance.

[0011] To this end, the present invention provides the following technical solutions.

[0012] One aspect of the present invention provides a method for determining the purity of the vaccine adjuvant MPLA, which is detected by high performance liquid chromatography. The high performance liquid chromatography uses an alkyl bonded silica gel as a chromatographic column with a stationary phase, an organic solvent-ion pair reagent buffer as a mobile phase, an ultraviolet spectrophotometer as a detector, and gradient elution for detection.

[0013] In a preferred embodiment of the present invention, the alkyl bonded silica gel is selected from any one of butyl bonded silica gel, octyl bonded silica gel and octadecyl bonded silica gel.

[0014] In a preferred embodiment of the present invention, the alkyl bonded silica gel is octyl bonded silica gel.

[0015] In a preferred embodiment of the present invention, the detection temperature of the chromatographic column is 20-45°C.

[0016] In a preferred embodiment of the present invention, the chromatographic column detection temperature is 40°C.

[0017] In a preferred embodiment of the present invention, the organic solvent is an acid-containing organic solvent or an alkali-containing organic solvent.

[0018] In a preferred embodiment of the present invention, the organic solvent is an acid-containing organic solvent.

[0019] In a preferred embodiment of the present invention, the acid is selected from any one of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid.

[0020] In a preferred embodiment of the present invention, the acid is trifluoroacetic acid.

[0021] In a preferred embodiment of the present invention, the organic solvent is selected from any one of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, or a mixture of two or more thereof.

[0022] In a preferred embodiment of the present invention, the organic solvent is methanol-tetrahydrofuran-trifluoroacetic acid; wherein the volume ratio of the methanol to tetrahydrofuran is 100-400:700, and the trifluoroacetic acid accounts for 0.02-0.2% of the total volume of methanol-tetrahydrofuran.

[0023] In a preferred embodiment of the present invention, the volume ratio of methanol-tetrahydrofuran is 300:700, and the trifluoroacetic acid accounts for 0.1% of the total volume of methanol-tetrahydrofuran.

[0024] In a preferred embodiment of the present invention, the ion pair reagent buffer is selected from any one of water-soluble phosphates, ammonium salts, alkyl sulfonate sodium salts, alkyl sulfate sodium salts, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, or a mixture of two or more thereof.

[0025] In a preferred embodiment of the present invention, the ion pair reagent buffer is an ammonium formate-trifluoroacetic acid buffer solution; wherein the concentration of the ammonium formate is 0.01 mol / L to 0.03 mol / L, and the volume of the trifluoroacetic acid accounts for 0.005 to 0.2% of the volume of the buffer solution.

[0026] In a preferred embodiment of the present invention, the concentration of the ammonium formate is 0.01 mol / L; the volume of the trifluoroacetic acid accounts for 0.1% of the volume of the buffer solution.

[0027] In a preferred embodiment of the present invention, the volume ratio of the organic solvent to the ion pair reagent buffer varies with the elution time and has a gradient as shown in the following table:

[0028] Time / min Buffer ratio (%) Organic solvent ratio (%) 0 98％～2％ 2％～98％ 3 98％～2％ 2％～98％ 15 98％～2％ 2％～98％ 33 98％～2％ 2％～98％ 36 98％～2％ 2％～98％ 40 98％～2％ 2％～98％

[0029] In a preferred embodiment of the present invention, the volume ratio of the organic solvent to the ion pair reagent buffer varies with the elution time and has a gradient as shown in the following table:

[0030] Time / min Buffer ratio (%) Organic solvent ratio (%) 0 70％～20％ 30％～80％ 3 70％～20％ 30％～80％ 15 15％～0％ 85％～100％ 33 15％～0％ 85％～100％ 36 70％～20％ 30％～80％ 40 70％～20％ 30％～80％

[0031] In a preferred embodiment of the present invention, the flow rate of the mobile phase is 0.5-1.5 ml / min.

[0032] In a preferred embodiment of the present invention, the flow rate of the mobile phase is 0.7 ml / min.

[0033] In a preferred embodiment of the present invention, the detection wavelength of the detector violet is 190nm to 250nm.

[0034] In a preferred embodiment of the present invention, the detection wavelength of the detector violet is 210 nm.

[0035] In a preferred embodiment of the present invention, the detection method comprises the following steps:

[0036] Take an appropriate amount of the sample to be tested and dissolve it in tetrahydrofuran-water-trifluoroacetic acid in a volume ratio of 90:10:0.01 to prepare a sample solution;

[0037] Accurately measure 50ul of the sample solution, inject it into the high performance liquid chromatograph, and detect it under the conditions of a flow rate of 0.7ml / min, a detection wavelength of 210nm, and a column temperature of 40°C, thereby completing the purity determination of the vaccine adjuvant MPLA.

[0038] By means of the above technical solution, the present invention has at least the following advantages:

[0039] The present invention provides a method for detecting the purity of vaccine adjuvant MPLA by high performance liquid chromatography, wherein a chromatographic column with alkyl bonded silica gel as the stationary phase, an organic solvent-ion pair reagent buffer as the mobile phase, an ultraviolet spectrophotometer as the detector, and gradient elution are used for detection, which can achieve efficient, accurate and convenient determination of the purity of vaccine adjuvant MPLA. The present invention provides a quick method for accurately completing the MPLA purity detection method by selecting a suitable stationary phase and mobile phase, as well as appropriate determination conditions. The detection method of the present invention has high accuracy, is easy to operate, and is highly efficient, and is a method suitable for widespread promotion and use.

[0040] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the structural composition of lipopolysaccharide of Gram-negative bacteria;

[0042] Figure 2 is the structural formula of Escherichia coli lipid A;

[0043] Figure 3 is the structural formula of hexaacylated 3-O-deacyl-4'-monophosphoryl lipid A;

[0044] Figure 4 is the HPLC chromatogram of blank reagent;

[0045] Figure 5 is the HPLC chromatogram of the novel adjuvant MPLA to be tested in Example 1;

[0046] Figure 6 is the HPLC chromatogram of the novel adjuvant MPLA to be tested in Example 2;

[0047] Figure 7 is the HPLC chromatogram of the novel adjuvant MPLA to be tested in Example 3;

[0048] Figure 8 This is the HPLC chromatogram of the new adjuvant MPLA to be tested in Comparative Example 1. DETAILED DESCRIPTION

[0049] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0051] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0052] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0053] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] Example 1: Testing instruments and testing conditions

[0056] 1.1 Testing instruments

[0057] High performance liquid chromatograph: U3000 Thermo Fisher Scientific high performance liquid chromatograph and Thermo Fisher Scientific chromatography workstation;

[0058] Chromatographic column: Agilent ZORBAX SB-C8 3.0mm*150mm.

[0059] 1.2 Detection Reagents

[0060] Organic solvent: methanol-tetrahydrofuran-trifluoroacetic acid (300:700:1);

[0061] Ion pair reagent buffer solution: ammonium salt buffer solution. The specific preparation method is as follows: measure 900 ml of the filtered 10 mmol / L ammonium formate solution, add 0.9 ml of trifluoroacetic acid, mix well, and obtain the solution.

[0062] 1.3 Test conditions

[0063] Flow rate: 0.7ml / min;

[0064] Column temperature: 40°C;

[0065] Wavelength: 210nm;

[0066] Injection volume: 50ul;

[0067] The elution gradient is shown in Table 1 below:

[0068] Table 1 Elution gradient

[0069] Time / min Buffer ratio (%) Organic solvent ratio (%) 0.0 40 60 3 40 60 15 5 95 33 5 95 36 40 60 40 40 60

[0070] Note: The buffer in the table refers to the ion pair reagent buffer; the buffer ratio refers to the ratio of the ion pair reagent buffer to the total volume of the organic solvent-ion pair reagent buffer.

[0071] 1.4 Detection of blank solvent

[0072] Take 50ul of tetrahydrofuran-water-trifluoroacetic acid (volume ratio of 90:10:0.01), inject it into the high performance liquid chromatograph, and detect it according to the detection conditions described in Example 1, and record the chromatogram at the same time. The results are as follows: Figure 4 shown.

[0073] like Figure 4 shown.

[0074] 1.5 Testing of samples to be tested

[0075] Take an appropriate amount of the MPLA sample to be tested, weigh it accurately, dissolve it with tetrahydrofuran-water-trifluoroacetic acid (volume ratio of 90:10:0.01) to prepare a sample solution with a concentration of 2.2 mg / ml.

[0076] Take 50ul of the sample solution and inject it into a high performance liquid chromatograph. Perform the test according to the test conditions described in Example 1 and record the chromatogram. The results are as follows: Figure 5 shown.

[0077] like Figure 5 As shown, the purity of the process synthesized product MPLA was determined to be 97.18%.

[0078] Example 2: Detection Instrument and Detection Conditions

[0079] 2.1 Testing instruments

[0080] High performance liquid chromatograph: U3000 Thermo Fisher Scientific high performance liquid chromatograph and Thermo Fisher Scientific chromatography workstation;

[0081] Chromatographic column: YMC-Triact C8 250mm*4.6mm.

[0082] 2.2 Detection Reagents

[0083] Organic solvent: methanol-tetrahydrofuran-trifluoroacetic acid (300:700:1);

[0084] Ion pair reagent buffer solution: ammonium salt buffer solution. The specific preparation method is as follows: measure 900 ml of the filtered 10 mmol / L ammonium formate solution, add 0.9 ml of trifluoroacetic acid, mix well, and obtain the solution.

[0085] 2.3 Test conditions

[0086] Flow rate: 1.0ml / min;

[0087] Column temperature: 40°C;

[0088] Wavelength: 210nm;

[0089] Injection volume: 50ul;

[0090] The elution gradient is shown in Table 2 below:

[0091] Table 2 Elution gradient

[0092]

[0093]

[0094] Note: The buffer in the table refers to the ion pair reagent buffer; the buffer ratio refers to the ratio of the ion pair reagent buffer to the total volume of the organic solvent-ion pair reagent buffer.

[0095] 2.4 Testing of samples to be tested

[0096] Take an appropriate amount of the MPLA sample to be tested, weigh it accurately, dissolve it with tetrahydrofuran-water-trifluoroacetic acid (volume ratio of 90:10:0.01) to prepare a sample solution with a concentration of 2.2 mg / ml.

[0097] Take 50ul of the sample solution and inject it into a high performance liquid chromatograph. Perform the test according to the test conditions described in Example 2 and record the chromatogram. The results are as follows: Figure 6 shown.

[0098] like Figure 6 As shown, the purity of the process synthesized product MPLA was determined to be 98.57%.

[0099] Example 3: Testing instruments and testing conditions

[0100] 3.1 Testing instruments

[0101] High performance liquid chromatograph: U3000 Thermo Fisher Scientific high performance liquid chromatograph and Thermo Fisher Scientific chromatography workstation;

[0102] Chromatographic column: YMC-Triact C8 250mm*4.6mm.

[0103] 3.2 Detection Reagents

[0104] Organic solvent: methanol-tetrahydrofuran-trifluoroacetic acid (300:700:1);

[0105] Ion pair reagent buffer solution: ammonium salt buffer solution. The specific preparation method is as follows: measure 900 ml of the filtered 10 mmol / L ammonium formate solution, add 0.9 ml of trifluoroacetic acid, mix well, and obtain the solution.

[0106] 3.3 Test conditions

[0107] Flow rate: 0.7ml / min;

[0108] Column temperature: 40°C;

[0109] Wavelength: 210nm;

[0110] Injection volume: 50ul;

[0111] The elution gradient is shown in Table 3 below:

[0112] Table 3 Elution gradient

[0113] Time / min Buffer ratio (%) Organic solvent ratio (%) 0.0 40 60 3 40 60 15 5 95 33 5 95 36 40 60 40 40 60

[0114] Note: The buffer in the table refers to the ion pair reagent buffer; the buffer ratio refers to the ratio of the ion pair reagent buffer to the total volume of the organic solvent-ion pair reagent buffer.

[0115] 3.4 Testing of samples to be tested

[0116] Take an appropriate amount of the MPLA sample to be tested, weigh it accurately, dissolve it with tetrahydrofuran-water-trifluoroacetic acid (volume ratio of 90:10:0.01) to prepare a sample solution with a concentration of 2.2 mg / ml.

[0117] Take 50ul of the sample solution and inject it into a high performance liquid chromatograph. Perform the test according to the test conditions described in Example 3 and record the chromatogram. The results are as follows: Figure 7 shown.

[0118] like Figure 7 As shown, the purity of the process synthesized product MPLA was determined to be 98.14%.

[0119] Comparative Example 1: Testing Instruments and Testing Conditions

[0120] 1.1 Testing instruments

[0121] High performance liquid chromatograph: U3000 Thermo Fisher Scientific high performance liquid chromatograph and Thermo Fisher Scientific chromatography workstation;

[0122] Chromatographic column: Agilent ZORBAX SB-C8 3.0mm*150mm.

[0123] 1.2 Detection Reagents

[0124] Organic solvent: methanol-tetrahydrofuran-trifluoroacetic acid (900:100:1);

[0125] Ion pair reagent buffer solution: ammonium salt buffer solution. The specific preparation method is: measure 1000 ml of filtered water, add 1.0 ml of trifluoroacetic acid, mix well, and obtain the solution.

[0126] 1.3 Test conditions

[0127] Flow rate: 0.7ml / min;

[0128] Column temperature: 40°C;

[0129] Wavelength: 210nm;

[0130] Injection volume: 50ul;

[0131] The elution gradient is shown in Table 4 below:

[0132] Table 4 Elution gradient

[0133] Time / min Buffer ratio (%) Organic solvent ratio (%) 0.0 40 60 3 40 60 15 5 95 33 5 95 36 40 60 40 40 60

[0134] Note: The buffer in the table refers to the ion pair reagent buffer; the buffer ratio refers to the ratio of the ion pair reagent buffer to the total volume of the organic solvent-ion pair reagent buffer.

[0135] 1.4 Testing of samples to be tested

[0136] Take an appropriate amount of the MPLA sample to be tested, weigh it accurately, dissolve it with tetrahydrofuran-water-trifluoroacetic acid (volume ratio of 90:10:0.01) to prepare a sample solution with a concentration of 2.2 mg / ml.

[0137] Take 50ul of the sample solution and inject it into a high performance liquid chromatograph. Perform the test according to the test conditions described in Example 1 and record the chromatogram. The results are as follows: Figure 8 shown.

[0138] like Figure 8 As shown, the purity of the process synthetic product MPLA is 95.41%, the main peak front and the peak shape are supplementary, which are not suitable for purity detection.

[0139] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for determining the purity of vaccine adjuvant MPLA, characterized in that: The detection is performed by high performance liquid chromatography, wherein the high performance liquid chromatography uses an alkyl bonded silica gel as a chromatographic column with a stationary phase, an organic solvent-ion pair reagent buffer as a mobile phase, an ultraviolet spectrophotometer as a detector, and a gradient elution method for detection.

2. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 1, characterized in that: The alkyl bonded silica gel is selected from any one of butyl bonded silica gel, octyl bonded silica gel and octadecyl bonded silica gel.

3. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 1, characterized in that: The detection temperature of the chromatographic column is 20-45°C.

4. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 1, characterized in that: The organic solvent is an acid-containing organic solvent or an alkali-containing organic solvent.

5. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 4, characterized in that: The acid is selected from any one of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid.

6. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 4, characterized in that: The organic solvent is selected from any one of methanol, ethanol, isopropanol, acetonitrile and tetrahydrofuran, or a mixture of two or more thereof.

7. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 4, characterized in that: The organic solvent is methanol-tetrahydrofuran-trifluoroacetic acid; wherein the volume ratio of the methanol to tetrahydrofuran is 100-400:700, and the trifluoroacetic acid accounts for 0.02-0.2% of the total volume of the methanol-tetrahydrofuran.

8. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 1, characterized in that: The ion pair reagent buffer is selected from any one of water-soluble phosphates, ammonium salts, alkyl sulfonic acid sodium salts, alkyl sulfate sodium salts, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, or a mixture of two or more thereof.

9. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 8, characterized in that: The ion pair reagent buffer is an ammonium formate-trifluoroacetic acid buffer solution; wherein the concentration of the ammonium formate is 0.01 mol / L to 0.03 mol / L; and the volume of the trifluoroacetic acid accounts for 0.005 to 0.2% of the total volume of the buffer solution.

10. The method for determining the purity of the seed vaccine adjuvant MPLA according to claim 1, characterized in that: The flow rate of the mobile phase is 0.5 to 1.5 ml / min; The detection wavelength of the detector is 190nm to 250nm.

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