High performance liquid detection method for measuring recombinant small RNA (Ribonucleic Acid) and application of high performance liquid detection method

Abstract

The invention discloses a high performance liquid detection method for determining recombinant small RNA (Ribonucleic Acid) and application. The method comprises the following steps: preparing a recombinant small RNA test solution, providing a blank sample solution, preparing a mobile phase, carrying out gradient elution on the recombinant small RNA test solution and the blank sample solution by adopting a C18 chromatographic column, analyzing a chromatogram and the like. On the basis of a high performance liquid chromatography analysis technology, a set of detection method which is good in durability and high in accuracy and precision and is used for measuring the recombinant small RNA is established, the method is used for detecting the purity, the stability and the like of the recombinant small RNA, and then a quantitative analysis method is provided for enterprises to produce RNA raw materials and research and development of RNA drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of small RNA detection, and in particular to a high-performance liquid phase detection method for recombinant small RNA determination and application thereof. Background Art

[0002] In recent years, small RNA preparations are gradually becoming intervention strategies for various diseases and are one of the most innovative and promising treatments. However, the study of RNA function and the development of RNA-based therapies depend on obtaining a large amount of efficient, safe, homogeneous RNA raw materials and efficient and durable RNA quantitative analysis methods.

[0003] Patent application number CN201810595579.9 discloses a method for producing recombinant small RNA. This method uses tRNA as a scaffold, chimeric miRNA precursors, and expresses the target small RNA in Escherichia coli. Although this method is economical, practical, convenient, effective, easy to scale and has a short production cycle, it does not provide a method for detecting the target small RNA.

[0004] At present, there is a lack of rapid, efficient and accurate methods for detecting recombinant small RNA in the development of recombinant small RNA reagents and the corresponding RNA therapy research. Therefore, the development of detection methods for recombinant small RNA is particularly urgent for recombinant small RNA manufacturers, and it is also of great significance for the development of recombinant small RNA reagents and the corresponding therapy research. Summary of the invention

[0005] In view of the current lack of methods for quickly, efficiently and accurately detecting recombinant small RNA, the present invention, based on high performance liquid chromatography analysis technology, establishes a set of detection methods for measuring recombinant small RNA with good durability, high accuracy and precision, and is used for detecting the purity, stability and other aspects of recombinant small RNA, thereby providing a quantitative analysis method for enterprises to produce RNA raw materials and research and develop RNA drugs.

[0006] The technical solution provided by the present invention is as follows:

[0007] The MSA, recombinant miR-34a (180 nt), and recombinant MGA (Malachite green aptamer, MGA) mentioned in the following technical schemes or examples are all produced by biological fermentation methods.

[0008] A high performance liquid phase detection method for recombinant small RNA determination comprises the following steps:

[0009] S1. Prepare a recombinant small RNA test solution of a certain concentration and provide a blank sample solution;

[0010] S2. Prepare a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a triethylammonium acetate solution, and the mobile phase B is a mixed solution of the triethylammonium acetate solution and acetonitrile in a volume ratio of (70-80): (20-30);

[0011] S3, using a C18 chromatographic column to perform gradient elution on the recombinant small RNA test solution and the blank sample solution, setting the flow rate to 0.8-1.2 mL / min, the column temperature to 40-55° C., and recording the chromatogram;

[0012] S4. Select the recombinant small RNA test solution of different concentrations for sampling, record the chromatogram, and calculate the content, purity, separation, short-term storage stability, accuracy and precision of the recombinant small RNA test solution of different batches according to the chromatogram.

[0013] Further, in step S1, the blank sample solution is DEPC water;

[0014] The recombinant small RNA test sample comprises a tRNA scaffold, a miRNA precursor sequence and a target small RNA sequence, and the target small RNA sequence is miRNA, siRNA, shRNA or RNA aptamer.

[0015] Furthermore, the tRNA scaffold includes human serine tRNA, human glutamate tRNA, human cysteine ​​tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA and bacterial methionine tRNA.

[0016] Furthermore, the concentration of the recombinant small RNA test solution is 2.50-1200.00 μg / mL.

[0017] Furthermore, in step S2, the concentration of mobile phase A is 100 mM and the pH is 7.0.

[0018] Further, in step S3, the C18 chromatographic column has a column length of 150 mm, 200 mm or 300 mm, and an inner diameter of 5 μm;

[0019] In step S4, the gradient elution conditions are: the content of mobile phase B in the mobile phase is increased from 20% to 80% within 0-30 min, the content of mobile phase B in the mobile phase is reduced from 80% to 20% within 30-35 min, and the content of mobile phase B in the mobile phase is maintained at 20% within 35-45 min.

[0020] Furthermore, the recombinant small RNA sample to be tested is a small RNA produced by biological recombination;

[0021] The mobile phase B is triethylamine acetate solution and acetonitrile in a volume ratio of 80:20, and the concentration of the mobile phase A is 100 mM;

[0022] The C18 chromatographic column is nano-C18, and the flow rate during elution is 0.9 mL / min and the column temperature is 45°C.

[0023] The present invention also provides an application of the above-mentioned high-performance liquid phase detection method for determining recombinant small RNA in the quality detection and quantitative analysis of recombinant RNA products produced by microbial fermentation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention screens different chromatographic columns according to the structure and physicochemical properties of recombinant small RNA taking recombinant miR-34a as an example, and finds that the Nanomicro C18 chromatographic column is suitable for analyzing the recombinant small RNA, with good separation and excellent peak shape; establishes an HPLC analysis method for the recombinant small RNA, and performs methodological verification; according to the results of relevant embodiments of the present invention, it is shown that the liquid phase analysis method of the present invention has high accuracy, high precision and good stability, and all indicators meet the requirements, and the recombinant small RNA has a good linear relationship in the range of 10-1200 μg / mL, and can be used as a quantitative analysis method for the recombinant small RNA; the liquid chromatography analysis method of the present invention is used to determine the stability of the recombinant small RNA under different conditions, detect and analyze the purity of multiple recombinant small RNAs from different batches, and other properties of the recombinant small RNA, and provides a method specifically for analyzing the recombinant small RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a liquid chromatogram of a recombinant miR-34a sample tested using a Shimadzu Inertsil ODS-3 chromatographic column in an embodiment of the present invention, wherein A / B are liquid chromatograms of recombinant miR-34a samples at 10 μg / mL and 100 μg / mL, respectively;

[0027] Figure 2 In the present invention, Shimadzu Waters Atlantis TM Liquid chromatogram of the recombinant miR-34a sample tested by T3 column, where A / B are 10μL / 20μL liquid chromatograms of recombinant miR-34a respectively;

[0028] Figure 3 This is a liquid chromatogram of the recombinant miR-34a sample tested using the Yuexu Xtimate SEC-120 chromatographic column in the embodiment of the present invention;

[0029] Figure 4The liquid chromatograms of the analysis of the recombinant miR-34a rat plasma sample using the Yuexu Xtimate SEC-120 chromatographic column in the embodiment of the present invention, wherein (A) is the liquid chromatogram of the first blank plasma test solution, (B) is the liquid chromatogram of the first blank plasma test solution containing recombinant miR-34a, (C) is the liquid chromatogram of the second blank plasma test solution, and (D) is the liquid chromatogram of the second blank plasma test solution containing recombinant miR-34a;

[0030] Figure 5 This is a liquid chromatogram of a recombinant miR-34a sample analyzed using an Agilent PLRP-S 4000A chromatographic column in an embodiment of the present invention;

[0031] Figure 6 The liquid chromatogram of the recombinant miR-34a sample analyzed by Nano-C18 in the embodiment of the present invention, from top to bottom are the liquid chromatogram of the solvent DEPC water and the liquid chromatogram of the recombinant miR-34a sample;

[0032] Figure 7 This is a short-term stability test of recombinant miR-34a using liquid chromatography in the embodiment of the present invention, wherein A / B are short-term stability tests at 4°C / 25°C respectively;

[0033] Figure 8 The chromatograms of the analysis of different batches of recombinant miR-34a in the embodiment of the present invention, wherein A / B / C / D are the chromatograms of the analysis of 4 batches of recombinant miR-34a respectively;

[0034] Fig. 9 The figures are the liquid phase analysis chromatograms of recombinant small RNAs of different molecular weights in the embodiments of the present invention, wherein (A) is the liquid phase analysis chromatogram of recombinant MSA, (B) is the liquid phase analysis chromatogram of recombinant miR-34a, and (C) is the liquid phase analysis chromatogram of recombinant MGA aptamer. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the embodiments described below are part of the embodiments of the present application, rather than all of the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0036] The present invention provides a high-performance liquid phase detection method for recombinant small RNA determination, comprising the following steps:

[0037] S1. Prepare a recombinant small RNA test solution of a certain concentration and provide a blank sample solution;

[0038] S2. Prepare a mobile phase, wherein the mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is a triethylammonium acetate solution, and the mobile phase B is a mixed solution of the triethylammonium acetate solution and acetonitrile in a volume ratio of (70-80): (20-30);

[0039] S3. Use a C18 chromatographic column to perform gradient elution on the recombinant small RNA test solution and the blank sample solution, set the flow rate to 0.8-1.2 mL / min, the column temperature to 40-55°C, and record the chromatogram;

[0040] S4. Select recombinant small RNA test solutions of different concentrations for sampling, record the chromatogram, and calculate the content, purity, separation, short-term storage stability, accuracy and precision of different batches of recombinant small RNA test solutions based on the chromatogram.

[0041] In the present invention, the term "gradient elution" refers to an elution method in which the flow rate of the mobile phase remains constant, but the compositions of the mobile phase A and the mobile phase B show a gradient change during the analysis period of the sample components.

[0042] In the present invention, the term "external standard method" refers to a method of using a pure product of the component to be measured as a reference substance and comparing the response signals of the reference substance with those of the component to be measured in the sample for quantitative analysis.

[0043] In the present invention, the term "blank solution" refers to a solution that is measured under the same conditions as the sample in order to eliminate interference in the HPLC analysis method. The measured result is called the "blank value" and should be deducted from the sample measurement result to improve the accuracy of the measurement.

[0044] In the present invention, the term "test solution" refers to the solution of the sample to be tested in the high performance liquid chromatography analysis method.

[0045] In the present invention, the term "mobile phase" refers to a substance that carries the components to be measured forward during the liquid chromatography process.

[0046] Optionally, in step S1, the blank sample solution is DEPC water.

[0047] The recombinant small RNA sample to be tested includes a tRNA scaffold, a miRNA precursor sequence and a target small RNA sequence, and the target small RNA sequence is miRNA, siRNA, shRNA or RNA aptamer.

[0048] The tRNA scaffolds include human serine tRNA, human glutamate tRNA, human cysteine ​​tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA, and bacterial methionine tRNA.

[0049] Optionally, the concentration of the recombinant small RNA test solution is 2.50-1200.00 μg / mL.

[0050] Optionally, in step S2, the concentration of mobile phase A is 100 mM and the pH is 7.0.

[0051] Optionally, in step S3, the column length of the C18 chromatographic column is 150 mm, 200 mm or 300 mm, and the inner diameter of the column is 5 μm.

[0052] In step S4, the gradient elution conditions are: the content of mobile phase B in the mobile phase is increased from 20% to 80% within 0-30 min, the content of mobile phase B in the mobile phase is reduced from 80% to 20% within 30-35 min, and the content of mobile phase B in the mobile phase is maintained at 20% within 35-45 min.

[0053] Optionally, the recombinant small RNA sample to be tested is a small RNA produced by biological recombinant production, such as miR-34a.

[0054] Mobile phase B was a mixed solution of triethylamine acetate solution and acetonitrile in a volume ratio of 80:20, and the concentration of mobile phase A was 100 mM.

[0055] The C18 chromatographic column was Nano-C18, and the flow rate during elution was 0.9 mL / min and the column temperature was 45°C.

[0056] Example 1

[0057] In this embodiment, the preparation of the recombinant small RNA test solution takes recombinant miR-34a as an example.

[0058] Preparation of recombinant miR-34a standard stock solution: Take the biosynthesized recombinant miR-34a reagent, prepare a stock solution with a concentration of 1.5 mg / mL in sterile DEPC water, and store it in a -20°C refrigerator.

[0059] The miR-34a stock solution was diluted with DEPC water (ultrapure water treated with diethyl pyrocarbonate, DEPC and sterilized by high temperature and high pressure) to prepare a gradient solution of recombinant miR-34a with a series of concentrations as the test solution, preparing 2.50 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 25.00 μg / mL, 50.00 μg / mL, 100.00 μg / mL, 250.00 μg / mL, 500.00 μg / mL, 1000.00 μg / mL, and 1200.00 μg / mL, and the samples were stored in a -20°C refrigerator.

[0060] Example 2

[0061] Liquid chromatography column screening-1

[0062] (1) In this embodiment, the instruments and operating conditions are as follows:

[0063] Chromatographic column: Shimadzu Inertsil ODS-3 (4.6×250 mm, 5 μm);

[0064] Detection wavelength: 260nm;

[0065] Mobile phase A: DEPC water;

[0066] Mobile phase B: methanol;

[0067] Column temperature: 25°C;

[0068] Flow rate: 0.8 mL / min;

[0069] Elution conditions: Isocratic elution at 90% mobile phase A, 10% mobile phase B.

[0070] (2) Solution preparation:

[0071] DEPC water and methanol do not need to be prepared and can be used directly;

[0072] The test solution selected 10 μg / mL and 100 μg / mL recombinant miR-34a solutions.

[0073] (3) Testing:

[0074] Injection volume: 10 μL;

[0075] 10 μg / mL and 100 μg / mL of recombinant miR-34a solutions were injected and tested successively, and the chromatograms were recorded.

[0076] (4) Experimental results: Figure 1 As shown in the figure, when the Shimadzu InertsilODS-3 column was used to analyze the recombinant miR-34a solution, the target peak was extremely small and the peak shape was poor. This may be due to the interaction between the column filler and RNA, or the small pore size, which prevented the recombinant miR-34a sample from passing through due to its large molecular weight, resulting in column blockage. Therefore, the Shimadzu InertsilODS-3 column is not suitable for quantitative analysis of recombinant small RNA.

[0077] Example 3

[0078] Liquid chromatography column screening-2

[0079] (1) In this embodiment, the instruments and operating conditions are as follows:

[0080] Chromatographic column: Shimadzu Waters Atlantis TMT3 (3 × 150 mm, 3 μm);

[0081] Detection wavelength: 260nm;

[0082] Mobile phase A: DEPC water;

[0083] Mobile phase B: methanol;

[0084] Column temperature: 25°C;

[0085] Flow rate: 0.8 mL / min;

[0086] Elution conditions: Isocratic elution at 90% mobile phase A, 10% mobile phase B.

[0087] (2) Solution preparation:

[0088] DEPC water and methanol do not need to be prepared and can be used directly;

[0089] The test solution was a 100 μg / mL recombinant miR-34a solution.

[0090] (3) Testing:

[0091] Injection volume: 10μL, 20μL;

[0092] Different volumes of 100 μg / mL recombinant miR-34a solution were injected and tested successively, and the chromatograms were recorded.

[0093] (4) Experimental results: Figure 2 As shown, Shimadzu Waters Atlantis TM When the T3 column was used to analyze the recombinant miR-34a solution, the target peak appeared in about 1 minute, and the peak shape was poor, which was not suitable for quantitative analysis of the recombinant miR-34a solution.

[0094] Example 4

[0095] Liquid chromatography column screening-3

[0096] (1) In this embodiment, the instruments and operating conditions are as follows:

[0097] Chromatographic column: Yuexu Xtimate SEC-120 chromatographic column (7.8×300mm, 5μm);

[0098] Detection wavelength: 260nm;

[0099] Mobile phase: 50 mM potassium dihydrogen phosphate solution (pH = 7);

[0100] Column temperature: 35°C;

[0101] Flow rate: 0.5 mL / min;

[0102] Elution conditions: 50 mM potassium dihydrogen phosphate solution was used for direct elution.

[0103] (2) Solution preparation:

[0104] 50mM potassium dihydrogen phosphate solution: accurately weigh 8.709g of potassium dihydrogen phosphate, add 950ml of deionized water, adjust the pH to 7.0 with dilute hydrochloric acid, and finally make up to 1L;

[0105] The test solution was a 100 μg / mL recombinant miR-34a solution.

[0106] (3) Testing:

[0107] Injection volume: 50 μL;

[0108] Different volumes of 100 μg / mL recombinant miR-34a solution were injected and tested, and the chromatograms were recorded.

[0109] (4) Experimental results: Figure 3 As shown in Table 1, when the Yuexu Xtimate SEC-120 column was used to analyze the recombinant miR-34a solution, the peak shape was good and the retention time was acceptable, so it can be used for the quantitative analysis of the recombinant miR-34a aqueous solution.

[0110] Table 1 Corresponding parameters of the chromatogram of recombinant miR-34a analyzed by Yuexu Xtimate SEC-120 column

[0111]

[0112] To further test the analytical performance of the column, plasma samples of recombinant miR-34a were selected for analysis. Blank rat plasma was taken, and impurities such as plasma proteins were removed by liquid-liquid extraction to prepare a blank plasma test solution. Under the proposed chromatographic conditions, 50 μL was injected to analyze the blank plasma test solution and a mixture of equal volumes of blank plasma test solution and recombinant miR-34a, and each injection was repeated twice.

[0113] The results show that Figure 4 , Table 2), the analysis of recombinant miR-34a rat plasma samples took a long time, about 60 minutes. And after two consecutive injections, it was found that the sample residue in the chromatographic column was obvious (box), and the reproducibility was poor. Therefore, this chromatographic column is not suitable for quantitative analysis of recombinant miR-34a in biological samples.

[0114] Table 2 Analysis of recombinant miR-34a by Yuexu Xtimate SEC-120 column Figure 4 Chromatogram corresponding parameters

[0115]

[0116] Example 5

[0117] Liquid chromatography column screening-4

[0118] (1) In this embodiment, the instruments and operating conditions are as follows:

[0119] Chromatographic column: Agilent PLRP-S chromatographic column 4000A (4.6×250mm, 10μm);

[0120] Detection wavelength: 260nm;

[0121] Mobile phase A: 100 mM triethylamine acetate (PH = 7);

[0122] Mobile phase B: acetonitrile: mobile phase A = 20:80 (v / v);

[0123] Column temperature: 45°C;

[0124] Flow rate: 1.0 mL / min;

[0125] Elution conditions: gradient elution: 0-30 min (20% B-80% B), 30-31 min (80% B-20% B), 31-40 min (20% B).

[0126] (2) Solution preparation:

[0127] 100mM triethylamine acetate solution: accurately weigh 16.124g of triethylamine acetate, add 950ml of deionized water, adjust the pH to 7.0, and finally make up to 1L;

[0128] The test solution was a 100 μg / mL recombinant miR-34a solution.

[0129] (3) Testing:

[0130] Injection volume: 10 μL;

[0131] A 100 μg / mL recombinant miR-34a solution was injected and tested, and the chromatogram was recorded.

[0132] (4) Experimental results:

[0133] By liquid chromatography Figure 5As can be seen from the relevant parameters in Table 3, when the Agilent PLRP-S 4000A column was used to analyze the recombinant miR-34a solution, the peak shape and retention time were acceptable, but the baseline was unstable during the analysis, and a long time was required to flush and balance the instrument. Therefore, the Agilent PLRP-S 4000A column can be considered for the quantitative analysis of the recombinant miR-34a solution.

[0134] Table 3 Analysis of recombinant miR-34a using Agilent PLRP-S 4000A column Figure 5 Chromatogram corresponding parameters

[0135]

[0136] Example 6

[0137] Liquid chromatography column screening-5

[0138] (1) In this embodiment, the instruments and operating conditions are as follows:

[0139] Chromatographic column: Nano-C18 (4.6×200mm, 5μm);

[0140] Detection wavelength: 260nm;

[0141] Mobile phase A: 100 mM triethylamine acetate (pH = 7);

[0142] Acetonitrile in mobile phase B: mobile phase A = 20:80 (v / v);

[0143] Column temperature: 45°C;

[0144] Flow rate: 0.9 mL / min;

[0145] Elution conditions: 0-30min (20% B-80% B), 30-35min (80% B-20% B), 35-45min (20% B) gradient elution. That is, within 0-30min, the volume ratio of the mobile phase B in the whole elution solution increases from 20% to 80%, at 30-35min, the volume of the mobile phase B in the whole elution solution decreases from 80% to 20%, and at 35-45min, the volume of the mobile phase B remains unchanged at 20% of the whole eluent.

[0146] (2) Solution preparation:

[0147] 100mM triethylamine acetate solution: accurately weigh 16.124g of triethylamine acetate, add 950ml of deionized water, adjust the pH to 7.0, and finally make up to 1L;

[0148] The test solution was a 100 μg / mL recombinant miR-34a solution.

[0149] (3) Testing:

[0150] Injection volume: 10 μL;

[0151] DEPC water and 100 μg / mL recombinant miR-34a solution were injected and tested, and the chromatograms were recorded.

[0152] (4) Experimental results:

[0153] By liquid chromatography Figure 6 As can be seen from Table 4, when the Nano-C18 column was used to analyze the recombinant miR-34a solution, the theoretical plate number was 69931, indicating that the column had high column efficiency, good target peak shape, and short analysis time. Therefore, the Nano-C18 column can be used for quantitative analysis of recombinant miR-34a.

[0154] Table 4 Analysis of recombinant miR-34a by NanoC18 Figure 5 Chromatogram corresponding parameters

[0155]

[0156] Through the above examples, by optimizing the mobile phase, pH value, elution conditions, column temperature and other conditions, and screening different types of chromatographic columns from different manufacturers, the recombinant small RNA liquid phase analysis method of the embodiment was finally established, and the practicality of the liquid phase analysis method was further tested subsequently.

[0157] Example 7

[0158] Accuracy and precision test of quantitative analysis method of recombinant small RNA

[0159] Using the liquid phase analysis method in Example 6, different concentrations of recombinant miR-34a solutions were selected as samples: 2.50, 5.00, 10.00, 25.00, 50.00, 100.00, 250.00, 500.00, 1000.00, 1200.00 μg / mL, and a linear regression equation between the recombinant miR-34a solution and the chromatographic peak area was established to obtain a linear equation. Detection limit: 2.5 μg / mL; Quantification limit: 10 μg / mL; Linear range: 10-1200 μg / mL; Linear curve: y=7108.9x-384444 (R 2 =0.9997).

[0160] Recombinant miR-34a test products (concentrations of 25.00, 100.00 and 1000.00 μg / mL) were selected, and the injection volume was 10 μL. After the injection was completed, the peak area was determined, and the accuracy (accuracy describes the closeness of the measured value of the analyte to the true concentration, expressed as the percentage of the measured value to the true value) and precision (precision refers to the consistency between the measured values ​​under the same conditions, when the analyte is repeatedly measured multiple times, expressed as the relative standard deviation percentage, RSD%) were calculated. The quality control samples were analyzed six times on the same day to determine the intra-day accuracy and precision. The process was repeated three times on different days to determine the inter-day accuracy and precision. The results are shown in Table 5. The liquid phase analysis met the requirements in terms of accuracy and precision of analyzing and testing recombinant small RNA.

[0161] Table 5 Intra-day / inter-day accuracy and precision test results First day Intra-day accuracy and precision test results:

[0162]

[0163] The results of the accuracy and precision test on the second day:

[0164]

[0165] The results of the intraday accuracy and precision test on the third day:

[0166]

[0167] Results of daytime accuracy and precision test:

[0168]

[0169] Example 8

[0170] Short-term storage stability test of recombinant small RNA

[0171] The recombinant miR-34a test sample (concentration of 1000.00 μg / mL) was selected and stored at 4°C and 25°C for 24, 48, and 72 h, respectively. 10 μL was injected under the chromatographic conditions of Example 6. Each sample was injected 6 times, the peak area was measured, and the storage stability at 4°C and 25°C was calculated. The results are shown in Figure 7 As shown, at 4°C ( Figure 7 A), 1000.00 μg / mL, after 72 hours of storage, the concentration dropped to 96.45% of the original value. Figure 7B is the stability of storage at 25℃. After 72h of storage at 1000.00μg / mL, the concentration decreased to 95.13% of the original. After 72h of storage at 4℃ and 25℃, the concentration of the test sample hardly decreased, indicating that the recombinant microRNA has good stability. When the samples were stored for the same time, the content decreased slightly more at 25℃ than at 4℃. Therefore, recombinant miR-34a is more stable when stored at 4℃ than at 25℃.

[0172] Example 9

[0173] Purity testing of recombinant small RNA

[0174] Purity determination of different batches of recombinant miR-34a: Take 4 different batches of recombinant miR-34a and dilute them with sterile DEPC water to the linear range of the standard curve. Take 10 μL of the diluted test sample and inject it under the chromatographic conditions of Example 6. The chromatograms of different batches of recombinant miR-34a are as follows: Figure 8 As shown, the content and purity of recombinant miR-34a in different batches were calculated according to the peak area normalization method (Table 6). The results showed that the purity of recombinant miR-34a in different batches was basically the same, ranging from 95.053% to 97.908%, and all achieved a high purity.

[0175] Table 6 Results of content and purity detection of recombinant miR-34a in different batches

[0176]

[0177] Example 10

[0178] Determination of retention time of recombinant small RNA with different molecular weights

[0179] To verify the universality of the liquid chromatography analysis method, recombinant RNAs of different fragment sizes were taken: MSA (108nt), recombinant miR-34a (180nt), and recombinant MGA (Malachite green aptamer, MGA) (240nt), and diluted with sterile DEPC water to within the linear range of the standard curve. 10 μL of the diluted test sample was injected under the liquid chromatography conditions of Example 6. The chromatograms of recombinant small RNA analysis of different fragment sizes are shown in the following figure. Fig. 9 As shown, the proposed liquid phase method is observed for the separation of recombinant RNA of different sizes.

[0180] According to the chromatogram of recombinant small RNA analysis, it can be seen that the peak time of recombinant small RNAs of different fragment sizes is different. The peak time of recombinant MSA is 13.88min, the peak time of recombinant miR-34a is 15.17min, and the peak time of recombinant MGA is 15.66min. The experimental results show that the peak time of recombinant small RNAs of different fragment sizes is different, and the smaller the fragment, the earlier the peak time. The established liquid phase analysis method can achieve baseline separation between MSA and recombinant miR-34a, and between MSA and recombinant MGA; recombinant miR-34a and recombinant MGA cannot achieve baseline separation.

[0181] The present invention screened different chromatographic columns based on the structure and physicochemical properties of recombinant small RNA, taking recombinant miR-34a as an example, and found that the nano-micro C18 chromatographic column is suitable for analyzing recombinant small RNA, with good separation and excellent peak shape. An HPLC analysis method for recombinant small RNA was established, and methodological verification was performed. The results of the relevant embodiments show that the liquid phase analysis method of the present invention has high accuracy, high precision and good stability, and all indicators meet the requirements. The recombinant small RNA has a good linear relationship in the range of 10-1200 μg / mL, and can be used as a quantitative analysis method for recombinant small RNA. The liquid chromatography analysis method of the present invention can be used to determine the stability of recombinant small RNA under different conditions, detect and analyze the purity of different batches of multiple recombinant small RNAs, and other properties of recombinant small RNAs. It is a method specifically used to analyze recombinant small RNAs.

[0182] In the research related to RNA drugs, especially in clinical research, the corresponding RNA raw materials must be of high purity and can be accurately quantified to reduce the off-target toxicity and potential danger caused by the interference of endogenous RNA or impurities. Biosynthetic small RNA technology is expected to provide a solution to the problem of raw materials required for RNA research and the development of new RNA therapies, thereby promoting the research and development of RNA drugs, but there is no method for detecting and analyzing the relevant properties of recombinant small RNA. The present invention obtains a chromatographic column with high column efficiency, good separation degree, excellent peak shape, and suitable for recombinant small RNA through a series of screening and optimization of chromatographic conditions such as mobile phase, pH value, elution gradient, column temperature, etc., and then establishes a set of liquid phase analysis methods for recombinant small RNA, and analyzes the stability of recombinant small RNA, the purity of different batches of multiple recombinant small RNAs, and the retention time of recombinant small RNAs of different fragment sizes by this method, verifying the accuracy, precision and stability of the present invention-a liquid phase analysis method for recombinant small RNA, and all indicators meet the analysis requirements. The present invention provides a quantitative and purity detection method for the production of RNA raw materials for enterprises, and at the same time provides a reference method for quantitative analysis of the in vivo pharmacokinetic process of recombinant small RNA.

[0183] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention shall still fall within the protection scope of the technical solution of the present invention.

Claims

1. A high performance liquid phase detection method for recombinant small RNA determination, characterized in that: The following steps are involved: S1. Prepare a recombinant small RNA test solution of a certain concentration and provide a blank sample solution; S2. Prepare a mobile phase, wherein the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a triethylammonium acetate solution, and the mobile phase B is a mixed solution of the triethylammonium acetate solution and acetonitrile in a volume ratio of (70-80): (20-30); S3, using a C18 chromatographic column to perform gradient elution on the recombinant small RNA test solution and the blank sample solution, setting the flow rate to 0.8-1.2 mL / min, the column temperature to 40-55° C., and recording the chromatogram; S4. Select recombinant small RNA test solutions of different concentrations for repeated elution, record the chromatogram, and calculate the content, purity, separation, short-term storage stability, accuracy and precision of different batches of the recombinant small RNA test solutions according to the chromatogram.

2. The high performance liquid phase detection method for recombinant small RNA determination according to claim 1, characterized in that: In step S1, the blank sample solution is DEPC water; The recombinant small RNA test sample comprises a tRNA scaffold, a miRNA precursor sequence and a target small RNA sequence, and the target small RNA sequence is miRNA, siRNA, shRNA or RNA aptamer.

3. The high performance liquid phase detection method for recombinant small RNA determination according to claim 2, characterized in that: The tRNA scaffolds include human serine tRNA, human glutamate tRNA, human cysteine ​​tRNA, human leucine tRNA, human lysine tRNA, human glutamine tRNA, human tyrosine tRNA and bacterial methionine tRNA.

4. The high performance liquid phase detection method for recombinant small RNA determination according to claim 1, characterized in that: The concentration of the recombinant small RNA test solution is 2.50-1200.00 μg / mL.

5. The high performance liquid phase detection method for recombinant small RNA determination according to any one of claims 1 to 4, characterized in that: In step S2, the concentration of mobile phase A is 90-110 mM and the pH is 7.

0.

6. The high performance liquid phase detection method for recombinant small RNA determination according to claim 5, characterized in that: In step S3, the C18 chromatographic column has a length of 150 mm, 200 mm or 300 mm, and an inner diameter of 5 μm; In step S4, the gradient elution conditions are: the content of mobile phase B in the mobile phase is increased from 20% to 80% within 0-30 min, the content of mobile phase B in the mobile phase is reduced from 80% to 20% within 30-35 min, and the content of mobile phase B in the mobile phase is maintained at 20% within 35-45 min.

7. The high performance liquid phase detection method for recombinant small RNA determination according to claim 1, characterized in that: The recombinant small RNA sample to be tested is a small RNA produced by biological recombination; the mobile phase B is a triethylamine acetate solution and acetonitrile in a volume ratio of 80:20, and the concentration of the mobile phase A is 100 mM; The C18 chromatographic column is nano-C18, and the flow rate during elution is 0.9 / min and the column temperature is 45°C.

8. Use of the high performance liquid phase detection method for determining recombinant small RNA according to any one of claims 1 to 7 in the quality detection and quantitative analysis of recombinant RNA products produced by microbial fermentation.

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