Method for detecting content of cane sugar in solution

Abstract

The invention relates to a method for quantitatively detecting the content of sucrose in an mRNA (messenger ribonucleic acid) composition, in particular to a UPLC-CAD (Ultra Performance Liquid Chromatography-Computer Aided Design) sucrose residue detection method operated in an HILIC (Hydrophilic Interaction Chromatography) mode. According to the method, liquid chromatography (HILIC) suitable for hydrophilic interaction is adopted, a polyvinyl alcohol silica gel column and an electrospray detector (CAD) are selected, and acetonitrile-water is used as a mobile phase. The method is accurate, reliable, good in chromatographic separation degree and high in sensitivity, and the content of sucrose in the mRNA solution can be accurately measured in the mRNA composition.

Description

Technical Field

[0001] The present invention relates to a method for quantitatively detecting the sucrose content in an mRNA solution, and more particularly to a method capable of detecting the sucrose content in an mRNA vaccine solution. Specifically, the method described in the present application involves using UPLC-CAD operated in a hydrophilic interaction chromatography (HILIC) mode to detect sucrose. Background Art

[0002] mRNA vaccine technology has become the most promising vaccine against new viruses due to its rapid and flexible advantages. For mRNA, it requires a specific carrier to deliver it into animal or human cells. With the development and maturity of nano-lipid carrier technology, the advantages of mRNA vaccines in the development of various virus vaccines have become increasingly prominent.

[0003] In the production process of mRNA lipid nanoparticle injection, a high concentration of sucrose buffer is mixed with LNP according to the actual detected content of mRNA in LNP to adjust the final content of mRNA in the injection so that the content of mRNA reaches a specific concentration. Therefore, it is necessary to develop an analytical method that can accurately determine the sucrose content in the mRNA composition to examine whether the sucrose content in the mRNA lipid nanoparticle injection prepared by a specific process meets the acceptance criteria.

[0004] Sucrose is a natural non-reducing disaccharide with strong polarity. It cannot be effectively retained on a reverse chromatographic column, and it is difficult to effectively separate oligosaccharides with similar chemical structures. The current mainstream methods for sucrose analysis all use silica amino columns or strong cation exchange chromatography columns, which have the disadvantages of severe column bleed and poor durability. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for detecting sucrose content by UPLC-CAD operated in a hydrophilic interaction chromatography (HILIC) mode. The method obtains the best conditions for detecting sucrose content using UPLC-CAD by optimizing the mobile phase, pH value, and chromatographic parameters. The electrospray ionization detector (CAD) selected in the method is a new type of liquid phase universal detector, which can accurately detect the sucrose content in the mRNA solution in combination with the HILIC chromatography analysis mode.

[0006] The invention discloses a method for detecting sucrose content in an mRNA composition by using UPLC-CAD. Specifically, the method comprises the following steps: respectively analyzing a test solution and a reference solution by using hydrophilic interaction liquid chromatography (HILIC), and recording the results by using a high performance liquid chromatograph connected in series with a charged aerosol detector, using a polyvinyl alcohol silica gel column as a chromatographic column, using water as a mobile phase A, and using acetonitrile as a mobile phase B for elution.

[0007] In some embodiments, when diluting the mRNA composition, alcohol is used as a diluent; preferably, the alcohol is methanol; preferably, the alcohol is isopropanol; preferably, the methanol is 50% methanol.

[0008] In some embodiments, when a high performance liquid chromatography column is used to detect a sample, a YMC-PackPVA-Sil polyvinyl alcohol silica gel column is used.

[0009] In some embodiments, when a sample is detected using a high performance liquid chromatography column, the water in the mobile phase A used contains 0.2% triethylamine; preferably, the mobile phase A is 0.2% triethylamine water, and the pH value is 7.8.

[0010] In some embodiments, when a high performance liquid chromatography column is used to detect a sample, the mobile phase B used is 0.2% triethylamine acetonitrile and the pH value is 7.6.

[0011] In some embodiments, the ratio of mobile phase A to mobile phase B is 10-15%:90-85%; preferably, the ratio of mobile phase A to mobile phase B is 15:85. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Chromatographic results using different diluents;

[0013] Figure 2 :Chromatographic results of mobile phases with different components;

[0014] Figure 3 :Chromatographic results of different proportions of mobile phase;

[0015] Figure 4 : Sucrose standard curve results Example

[0016] Example 1 Selection of diluent

[0017] The matrix in the LNP-mRNA solution is complex, containing mRNA with a large molecular weight and a variety of liposomes. Therefore, the choice of diluent is more important. A suitable diluent can reduce interference in sucrose detection. In order to find the best diluent for detecting sucrose in LNP-mRNA solution, we selected 50% methanol solution, methanol and isopropanol to examine the response and interference of LNP-mRNA solution in these diluents.

[0018] Instruments and reagents:

[0019] Thermo Vanquish Flex high performance liquid chromatograph coupled with charged aerosol detector (Thermo Fisher Scientific, USA)

[0020] Chromatographic column: YMC-Pack PVA-Sil-NP, 12nm, S-5μm, 100x 3.0mm (LC-PSN-002)

[0021] Sucrose is a standard substance of China National Institute for Food and Drug Control (Batch No.: 111507-202105)

[0022] Acetonitrile was chromatographic grade (Merck, batch number: JB1218130)

[0023] Methanol was chromatographic grade (Merck, batch number: L1231407231)

[0024] Isopropanol was chromatographic grade (Merck, batch number: I1231407231)

[0025] Water: Ultrapure water (Merck Mili-Q, homemade).

[0026] In order to optimize the conditions for detecting sucrose, the best diluent is selected using the test sample (the solution containing mRNA in actual production) as the sample.

[0027] Preparation of test solution: Take 10uL of the test sample into a 2mL centrifuge tube, add 990uL of the corresponding diluent, and mix well.

[0028] The chromatographic conditions were as follows:

[0029]

[0030] For detailed results, see Figure 1 and Table 1 below:

[0031] Table 1

[0032]

[0033] Figure 1The chromatograms of sucrose in the test sample (the solution containing mRNA in actual production) obtained by the chromatographic method disclosed in the present invention using methanol, 50% methanol and isopropanol as diluents are shown. Figure 1 The results show that when using the chromatographic method disclosed in the present invention, sucrose in the test sample can be detected by using methanol, 50% methanol and isopropanol as diluents. After the test sample is diluted by the same multiple, when the diluent is isopropanol, the sucrose peak height is the highest and the symmetry is the best.

[0034] Example 2: Selection of mobile phase

[0035] Triethylamine was GC grade (Sigma-Aldrich, lot number: STBK1748)

[0036] Acetic acid was of analytical grade (Nanjing Chemical Reagent Co., Ltd., batch number: 20220824)

[0037] Using the chromatographic conditions described in Example 1, using isopropanol as the diluent, three groups of mobile phases with different components were designed, and the specific components are as follows:

[0038]

[0039] The three groups of mobile phases mentioned above were mixed in a ratio of mobile phase A: mobile phase B = 15:85, and the test sample was analyzed by a chromatographic column.

[0040] Figure 2 The chromatographic results are obtained using three different mobile phase combinations.

[0041] Table 2 is an analysis of the results of the three mobile phase combinations:

[0042] Table 2

[0043]

[0044] According to Table 2 and Figure 2 As can be seen from the content, combinations 1, 2, and 3 can all detect the test sample sucrose. Under the condition of combination 3, the peak area RSD of the parallel samples of the test sample is 0.03%, and the peak height RSD% is 0.24%. The parallelism of the test sample results is good. Combination 1 is simple and convenient to prepare, and has little effect on the life of the chromatographic column. Under this condition, the sucrose response is good and meets the requirements of the content method. Therefore, in summary, combination 1 and combination 3 can both be used for the detection of sucrose content.

[0045] Example 3: Optimization of mobile phase ratio

[0046] Under the same conditions of diluent and mobile phase components, the ratio of mobile phase was optimized. The specific experimental design is as follows:

[0047]

[0048]

[0049] Figure 3 This is the chromatographic result diagram using different mobile phase ratios;

[0050] Table 3 is an analysis of the results of using different proportions of mobile phase:

[0051] Table 3

[0052]

[0053] according to Figure 3 As shown in the results in Table 3, both mobile phase ratios can complete the detection of sucrose in the mRNA composition, and the peak heights of the two combinations are basically the same. The higher the aqueous phase ratio, the faster the peak emission and the smaller the peak width.

[0054] Example 4: Detection of sucrose content in actual samples

[0055] In order to test whether the method disclosed in this application can be used to detect the content of sucrose in an actual mRNA vaccine composition, the following experiment is designed:

[0056] The chromatographic conditions described in Example 1 were used, with isopropanol as the diluent, water as the mobile phase A, acetonitrile as the mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B being 15%:85%.

[0057] The standard samples were prepared into a standard curve with a certain concentration, and the relationship between the peak area and the concentration was fitted using a log-log linear method to calculate the sucrose content in the test sample. The recovery rate was calculated compared with the theoretical sucrose content to verify the accuracy of this analytical method in detecting sucrose content.

[0058] Preparation of standard curve: Accurately weigh 40 mg of sucrose standard into a 20 mL volumetric flask, add about 14 mL of isopropanol, sonicate the solution, cool to room temperature after complete dissolution, dilute to the scale with isopropanol, and shake well to obtain the standard curve mother solution.

[0059] According to the table below, transfer the corresponding volume of mRNA vaccine composition stock solution into the HPL injection vial, add a certain volume of isopropanol and mix well. The batch numbers are marked as SAC-1 to 6.

[0060]

[0061]

[0062] Figure 4 The corresponding positions of the batch numbers SAC-1 to SAC-6 in the standard curve are shown. Figure 4The results show that under this analytical method, when the sucrose concentration in the mRNA composition is within the linear range of 0.2002-2.0040 mg / ml, the results obtained by the test have a good linear relationship, with an R square of 0.9989. The linear relationship is used to quantify the sucrose content in the test sample, and the recovery rate of the sucrose content measured in the test sample and the theoretical content can reach 103%, and the test results are accurate and good.

Claims

1. A method for detecting the sucrose content in an mRNA composition by UPLC-CAD, specifically, the method comprises the following steps: analyzing a test solution and a reference solution by hydrophilic interaction liquid chromatography (HILIC), The results were recorded using a high performance liquid chromatograph connected in series with a charged aerosol detector, using a polyvinyl alcohol silica gel column as the chromatographic column, water or triethylamine aqueous solution as the mobile phase A, and acetonitrile or triethylamine acetonitrile as the mobile phase B for elution.

2. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: Alcohols were used as diluents for mRNA compositions.

3. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: Methanol was used as diluent for the mRNA compositions.

4. The method for detecting sucrose content in an mRNA composition as claimed in claim 3, wherein: The methanol is 50% methanol.

5. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: Isopropanol was used as a diluent for the mRNA compositions.

6. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: When the samples were tested using a high performance liquid chromatography column, a YMC-Pack PVA-Sil polyvinyl alcohol silica gel column was used.

7. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: The water in the mobile phase A contains 0.2% triethylamine; preferably, the mobile phase A is 0.2% triethylamine water, and the pH value is 7.

8.

8. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: Mobile phase B used 0.2% triethylamine in acetonitrile and had a pH of 7.

6.

9. The method for detecting sucrose content in an mRNA composition according to claim 1, wherein: The volume ratio of mobile phase A to mobile phase B is 10-15%:90-85%.

10. The method for detecting sucrose content in an mRNA composition according to claim 8, wherein: The volume ratio of mobile phase A to mobile phase B was 15%:85%.

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