Method for detecting purity of CpG1018 adjuvant

Abstract

The invention discloses a purity detection method of a CpG1018 adjuvant, and belongs to the technical field of chemical analysis. The detection method comprises the following steps: (1) preparing a solution; (2) setting chromatographic conditions; (3) carrying out a system applicability test; (4) judging whether the system applicability test is passed or not according to the acceptance standard of the system applicability; and (5) analyzing and calculating the sample purity of the CpG1018 adjuvant after the system applicability test is passed. The CpG1018 adjuvant is a full-thio-modified oligonucleotide, and the conventional detection method is difficult to separate phosphorus-oxygen (P = O) impurities generated in the thio-modification process from the CpG1018; according to the detection method disclosed by the invention, phosphorus-oxygen (P = O) impurities can be successfully separated from CpG1018 in the detection process, and the separation degree can reach 0.8, so that the purity detection result of the CpG1018 adjuvant is relatively accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a purity detection method for a CpG1018 adjuvant. Background Art

[0002] CpG1018 adjuvant is a fully thiolated oligonucleotide with a length of 22 nucleotides. These oligodeoxynucleotide units are linked together by phosphodiester bonds to form a linear polymer structure. In the CpG adjuvant structure, the order and number of oligodeoxynucleotide units can be adjusted as needed to achieve specific regulatory effects on the immune system. Therefore, CpG1018 adjuvant is widely used in various immunotherapy programs, such as vaccine development, tumor treatment, and infectious disease prevention and control.

[0003] The DNA backbone of the CpG1018 adjuvant is thio-modified, that is, the oxygen in the phosphodiester bond is replaced by sulfur to form a thiophosphate backbone. This thio modification enhances the stability of the adjuvant. During the synthesis of the CpG1018 adjuvant, the non-bridging oxygen atoms on the phosphate backbone in its structure are replaced by sulfur atoms. This thiophosphate backbone enhances the resistance of CpG1018 to nucleases, thereby extending its half-life in the body. Compared with natural phosphodiester nucleotides, fully thio-CpG1018 has greater stability and longer in vivo retention time, which makes them more effective in immune stimulation.

[0004] During the thiolation process, the newly added nucleotide is connected to the oligonucleotide chain on the CpG through a phosphite bond (trivalent phosphorus). This phosphite bond needs to be oxidized to a stable pentavalent phosphorus to form a thiophosphate bond. Incomplete thiolation will result in the inability of the phosphodiester bond to form a thiophosphate bond, thereby generating phosphorus oxygen (P=O) impurities. The properties of phosphorus oxygen (P=O) impurities are similar to those of CpG1018, and conventional detection methods are difficult to separate them from CpG1018. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting the purity of CpG1018 adjuvant, so as to solve the problem that it is difficult to separate phosphorus oxygen (P=O) impurities and CpG1018 when detecting CpG1018 adjuvant.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for detecting the purity of a CpG1018 adjuvant, the detection steps comprising: (1) preparing a solution: the solution comprises a mobile phase A, a mobile phase B, a diluent, a blank solution, a reference solution, a sensitivity solution and a test solution; (2) setting chromatographic conditions; (3) performing a system suitability test; (4) judging whether the system suitability test has passed according to the acceptance criteria of the system suitability; (5) analyzing and calculating the sample purity of the CpG1018 adjuvant after the system suitability test has passed;

[0008] The sequence of the tested CpG1018 adjuvant is: TsGsAs CsTsGs TsGsAs AsCsGs TsTsCs GsAsGsAsTsGs As.

[0009] The detection instrument is Thermo Vanquish Flex liquid chromatograph or equivalent instrument.

[0010] Further, the mobile phase A is an aqueous solution containing 3% acetonitrile, 0.2% triethylamine and 1.0% hexafluoroisopropanol prepared with chromatographic grade or higher grade acetonitrile, hexafluoroisopropanol and triethylamine, recorded as 3% ACN + 0.2% TEA + 1.0% HFIP aqueous solution;

[0011] The mobile phase B is an aqueous solution containing 12% acetonitrile, 0.2% triethylamine and 1.0% hexafluoroisopropanol prepared with chromatographic grade or higher grade acetonitrile, hexafluoroisopropanol and triethylamine, recorded as 12% ACN+0.2% TEA+1.0% HFIP aqueous solution.

[0012] Furthermore, acetonitrile, hexafluoroisopropanol and triethylamine of chromatography grade or higher are used.

[0013] Furthermore, the diluent is ultrapure water;

[0014] The blank solution is a diluent solution, i.e., an ultrapure aqueous solution.

[0015] Furthermore, the reference solution is a 1 mg / mL CpG1018 adjuvant standard solution prepared from a CpG1018 adjuvant standard;

[0016] The sensitivity solution is a 0.002 mg / mL CpG1018 adjuvant standard solution prepared from a CpG1018 adjuvant standard.

[0017] Furthermore, the test solution is a 1 mg / mL solution prepared from a sample of CpG1018 adjuvant; two portions are prepared in parallel and marked as test sample 1 and test sample 2, respectively.

[0018] Furthermore, the chromatographic conditions are set as follows:

[0019] Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C181.7μm, 2.1×150mm, P / N: 186009486;

[0020] Wavelength: 260nm;

[0021] Column temperature: 65-75°C;

[0022] Flow rate: 0.30-0.40mL / min;

[0023] Sample plate temperature: 10°C;

[0024] Injection volume: 5-10 μL;

[0025] Mobile phase A: 3% ACN + 0.2% TEA + 1.0% HFIP aqueous solution;

[0026] Mobile phase B: 12% ACN + 0.2% TEA + 1.0% HFIP aqueous solution;

[0027] Diluent: ultrapure water;

[0028] The gradient elution program was:

[0029] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 30 70 27 0 100 30 0 100 30.1 90 10 35 90 10

[0030] Data collection time: 35min;

[0031] Running time: 35min;

[0032] Needle washing solvent: 10% methanol.

[0033] Furthermore, the injection sequence for the system suitability test is as follows:

[0034] 1) Blank solution: ≥ 1 injection;

[0035] 2) Sensitivity solution: 1 needle;

[0036] 3) Reference solution: 6 injections;

[0037] 4) 1:1 injection of test solution;

[0038] 5) 2:1 injection of test solution;

[0039] 6) Reference solution: 1 injection.

[0040] Furthermore, the acceptance criteria for system suitability are as follows:

[0041] Acceptance criteria for blank solution spectrum: Ensure that the system is clean and stable, and the spectrum of the blank solution has no obvious interfering peaks at the retention time of the target peak; if an interfering peak appears, the peak area of ​​the interfering peak should be less than half of the peak area of ​​the sensitivity test solution;

[0042] Separation acceptance criteria: In the reference solution, the separation between the main peak and the phosphorus and oxygen impurity peaks is ≥ 0.5;

[0043] Sensitivity acceptance criteria: The signal-to-noise ratio (S / N) of the main peak in the sensitivity solution should be ≥10;

[0044] Precision acceptance criteria:

[0045] 1) The RSD of the main peak area in the reference solution for 6 consecutive measurements is ≤2.0%;

[0046] 2) The RSD of the retention time of the main peak in the reference solution for 6 consecutive measurements is ≤1.0%;

[0047] 3) The RSD of the main peak area in all reference solutions is ≤2.0%.

[0048] Furthermore, the purity of the sample is automatically given by the liquid chromatography software Chromeleon7 or calculated according to the area normalization method.

[0049] Beneficial effects of the present invention: CpG1018 adjuvant is a fully thio-modified oligonucleotide, and phosphorus oxygen (P=O) impurities will be generated during the thio-modification process. The phosphorus oxygen (P=O) impurities have properties similar to those of CpG1018, and conventional detection methods are difficult to separate them from CpG1018. The detection method of the present invention can successfully separate the phosphorus oxygen (P=O) impurities from CpG1018 during the detection process, and the separation degree can reach 0.8, thereby making the purity detection result of the CpG1018 adjuvant more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further described below in conjunction with the accompanying drawings.

[0051] Figure 1 is the spectrum of the blank solution measured in Example 1 of the present invention;

[0052] Figure 2 is the spectrum of the sensitive solution measured in Example 1 of the present invention;

[0053] Figure 3 It is the spectrum of the CpG1018 adjuvant sample test solution 1 measured in Example 1 of the present invention;

[0054] Figure 4 It is the spectrum of the CpG1018 adjuvant sample test solution 2 measured in Example 1 of the present invention;

[0055] Figure 5 It is the spectrum of the CpG1018 adjuvant sample measured in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0056] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0057] Example 1

[0058] This embodiment provides a method for detecting the purity of CpG1018 adjuvant, and the detection instrument adopts a Thermo VanquishFlex liquid chromatograph;

[0059] The detection steps include:

[0060] (1) Solution preparation:

[0061] 1) Prepare mobile phase A and mobile phase B:

[0062] Mobile phase A: accurately measure 30 mL of acetonitrile, place in a 1000 mL volumetric flask, add 1 / 3 volume of ultrapure water, then add 10 mL of hexafluoroisopropanol and 2 mL of triethylamine in sequence, dilute to scale with ultrapure water, shake well, filter with a 0.22 μm filter membrane, and ultrasonically degas to obtain an aqueous solution containing 3% acetonitrile, 0.2% triethylamine and 1.0% hexafluoroisopropanol, recorded as 3% ACN + 0.2% TEA + 1.0% HFIP aqueous solution;

[0063] Mobile phase B: accurately measure 120 mL of acetonitrile, place it in a 1000 mL volumetric flask, add 1 / 3 volume of ultrapure water, then add 10 mL of hexafluoroisopropanol and 2 mL of triethylamine in sequence, dilute to the mark with ultrapure water, shake well, filter with a 0.22 μm filter membrane, and ultrasonically degas to obtain an aqueous solution containing 12% acetonitrile, 0.2% triethylamine and 1.0% hexafluoroisopropanol, recorded as 12% ACN+0.2% TEA+1.0% HFIP aqueous solution; the purity of the acetonitrile, hexafluoroisopropanol and triethylamine used is chromatographic grade;

[0064] 2) The diluent is ultrapure water;

[0065] 3) Prepare blank solution:

[0066] Pipette about 1.0 mL of diluent, i.e., 1.0 mL of ultrapure water, into the injection vial and seal it with a cap to obtain a blank solution;

[0067] 4) Prepare reference solution and sensitivity solution:

[0068] Reference solution: accurately weigh 10 mg of the standard of CpG1018 adjuvant (the purity of the standard is 95.8%), place it in a 10 mL volumetric flask, dissolve it with a diluent, then dilute to the mark, shake well, and obtain a reference solution;

[0069] Sensitivity solution: accurately measure 1 mL of the reference solution, place it in a 100 mL volumetric flask, dilute to the mark with a diluent, and shake well; accurately measure 2 mL of the above solution, place it in a 10 mL volumetric flask, dilute to the mark with a diluent, and shake well to obtain the sensitivity solution;

[0070] 5) Prepare the test solution:

[0071] Accurately weigh 10 mg of CpG1018 adjuvant sample into a 10 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain the test solution; prepare 2 portions in parallel and mark them as test sample 1 and test sample 2 respectively.

[0072] (2) Set the chromatographic conditions:

[0073] Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C181.7μm, 2.1×150mm, P / N: 186009486;

[0074] Wavelength: 260nm;

[0075] Column temperature: 70°C;

[0076] Flow rate: 0.35 mL / min;

[0077] Sample plate temperature: 10°C;

[0078] Injection volume: 5 μL;

[0079] Mobile phase A: 3% ACN + 0.2% TEA + 1.0% HFIP aqueous solution;

[0080] Mobile phase B: 12% ACN + 0.2% TEA + 1.0% HFIP aqueous solution;

[0081] Diluent: ultrapure water;

[0082] The gradient elution program was:

[0083] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 30 70 27 0 100 30 0 100 30.1 90 10 35 90 10

[0084] Data collection time: 35min;

[0085] Running time: 35min;

[0086] Needle washing solvent: 10% methanol.

[0087] (3) Perform a system suitability test. The injection sequence of the system suitability test is as follows:

[0088] 1) Blank solution: 1 injection;

[0089] 2) Sensitivity solution: 1 needle;

[0090] 3) Reference solution: 6 injections;

[0091] 4) 1:1 injection of test solution;

[0092] 5) 2:1 injection of test solution;

[0093] 6) Reference solution: 1 injection.

[0094] The system suitability test results are as follows:

[0095] Blank solution spectrum: The spectrum of the blank solution has no obvious interfering peaks at the retention time of the target peak;

[0096] Separation: In the reference solution, the separation between the main peak and the phosphorus and oxygen impurity peaks is 0.8;

[0097] Sensitivity: The signal-to-noise ratio (S / N) of the main peak in the sensitivity solution is 11;

[0098] Precision:

[0099] 1) The RSD of the main peak area in the reference solution for 6 consecutive measurements is ≤2.0%;

[0100] 2) The RSD of the retention time of the main peak in the reference solution for 6 consecutive measurements is ≤1.0%;

[0101] 3) The RSD of the main peak area in all reference solutions is ≤2.0%;

[0102] (4) Judge whether the system suitability test has passed according to the acceptance criteria of the system suitability;

[0103] (5) Calculate sample purity according to the area normalization method:

[0104] The spectra of the samples of CpG1018 adjuvant, i.e., test solution 1 and test solution 2, are as follows: Figure 3 and Figure 4 As shown, Figure 3 The peak time of CpG1018 in the sample was about 15.3 min, and the sample purity of CpG1018 adjuvant was calculated to be 95.7%; Figure 4The peak time of CpG1018 was about 15.4 min, and the sample purity of CpG1018 adjuvant was calculated to be 95.8%.

[0105] Comparative Example 1

[0106] Compared with Example 1, this comparative example has the following differences:

[0107] (1) Changing the mobile phase system: mobile phase A was changed to 0.1% TEA + 0.5% HFIP aqueous solution; mobile phase B was changed to 60% MeOH + 0.1% TEA + 0.5% HFIP aqueous solution;

[0108] (2) Change the gradient elution procedure in the chromatographic conditions to:

[0109] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 23 10 90 28 10 90 28.1 70 30 33 70 30

[0110] The remaining raw materials and preparation process remain the same as in Example 1.

[0111] The spectrum of the sample containing CpG1018 adjuvant was measured as follows: Figure 5 As shown, it can be seen from the spectrum that CpG1018 and phosphorus oxygen (P=O) impurities are difficult to separate, so it is difficult to obtain the sample purity of CpG1018 adjuvant.

[0112] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0113] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the purity of a CpG1018 adjuvant, characterized in that: The detection steps include: (1) solution preparation: the solution includes mobile phase A, mobile phase B, diluent, blank solution, reference solution, sensitivity solution and test solution; (2) setting chromatographic conditions; (3) performing a system suitability test; (4) judging whether the system suitability test is passed according to the acceptance criteria of the system suitability; (5) analyzing and calculating the sample purity of the CpG1018 adjuvant after the system suitability test is passed; The procedure for gradient elution in the chromatographic conditions is: The sequence of the tested CpG1018 adjuvant is: TsGsAs CsTsGs TsGsAs AsCsGs TsTsCs GsAsGs AsTsGsAs.

2. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: The mobile phase A is an aqueous solution containing 3% acetonitrile, 0.2% triethylamine and 1.0% hexafluoroisopropanol, recorded as 3% ACN+0.2% TEA+1.0% HFIP aqueous solution; The mobile phase B is an aqueous solution containing 12% acetonitrile, 0.2% triethylamine and 1.0% hexafluoroisopropanol, recorded as 12% ACN+0.2% TEA+1.0% HFIP aqueous solution.

3. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: The diluent is ultrapure water; The blank solution is a diluent solution, i.e., an ultrapure aqueous solution.

4. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: The reference solution is a 1 mg / mL CpG1018 adjuvant standard solution prepared from a standard of CpG1018 adjuvant; The sensitivity solution is a 0.002 mg / mL CpG1018 adjuvant standard solution prepared from a CpG1018 adjuvant standard.

5. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: The test solution is a 1 mg / mL solution prepared from a sample of CpG1018 adjuvant.

6. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: Set the chromatographic conditions as follows: Chromatographic column: Waters ACQUITY TM Premier Oligonucleotide BEH C181.7μm, 2.1×150mm, P / N: 186009486; Wavelength: 260nm; Column temperature: 65-75°C; Flow rate: 0.30-0.40mL / min; Sample plate temperature: 10°C; Injection volume: 5-10 μL; Mobile phase A: 3% ACN + 0.2% TEA + 1.0% HFIP aqueous solution; Mobile phase B: 12% ACN + 0.2% TEA + 1.0% HFIP aqueous solution; Diluent: ultrapure water; Data collection time: 35min; Running time: 35min; Needle washing solvent: 10% methanol.

7. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: The injection sequence for the system suitability test is as follows: 1) Blank solution: ≥ 1 injection; 2) Sensitivity solution: 1 needle; 3) Reference solution: 6 injections; 4) 1:1 injection of test solution; 5) 2:1 injection of test solution; 6) Reference solution: 1 injection.

8. The method for detecting the purity of a CpG1018 adjuvant according to claim 1, characterized in that: The acceptance criteria for system suitability are as follows: Acceptance criteria for blank solution spectrum: Ensure that the system is clean and stable, and the spectrum of the blank solution has no obvious interfering peaks at the retention time of the target peak; if an interfering peak appears, the peak area of ​​the interfering peak should be less than half of the peak area of ​​the sensitivity test solution; Separation acceptance criteria: In the reference solution, the separation between the main peak and the phosphorus and oxygen impurity peaks is ≥ 0.5; Sensitivity acceptance criteria: The signal-to-noise ratio (S / N) of the main peak in the sensitivity solution should be ≥10; Precision acceptance criteria: 1) The RSD of the main peak area in the reference solution for 6 consecutive measurements is ≤2.0%; 2) The RSD of the retention time of the main peak in the reference solution for 6 consecutive measurements is ≤1.0%; 3) The RSD of the main peak area in all reference solutions is ≤2.0%.

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