A method for detecting polyA tails in mRNA vaccines and its application

Abstract

The present invention relates to the field of pharmaceutical analysis technology, in particular to IPC G01N30, and more specifically to a method and application for detecting polyA tails in mRNA vaccines. The present invention provides a time-saving, labor-saving, and high-resolution method for detecting polyA tails in mRNA vaccines, comprising the following steps: S1: performing enzymatic digestion, magnetic bead purification, and elution recovery on mRNA in an mRNA vaccine solution to obtain polyA tail fragments; S2: detecting the poly A tail fragments using a liquid chromatography-mass spectrometer (LC-MS); the detection method of the present invention can detect single nucleotides and multiple oligonucleotide sequences, thereby accurately determining the polyA distribution range with high resolution; and the pretreatment method is simple, the detection time is short, the stability is greatly improved, and time is saved, and it can be used as a common characterization of mRNA vaccines.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis technology, in particular to IPC G01N30, and more specifically to a method for detecting polyA tails in mRNA vaccines and its application. Background Art

[0002] The word "vaccine" has become a national focus, with various innovative technologies being used in vaccine development. Among them, mRNA vaccines, as the most promising vaccine of the future, are expected to develop not only in common preventive vaccines but also in promising therapeutic vaccines.

[0003] In eukaryotes, mRNA, also known as messenger ribonucleic acid, undergoes a series of modifications after transcription, forming a specialized structure at the 5' end known as a cap and a poly(A) tail at the 3' end. During post-transcriptional modification in vivo, poly(A) polymerase gradually introduces 100-200 A bases onto the 3'-OH residue of RNA, influencing the stability of mature mRNA, translation initiation, and nuclear export.

[0004] According to literature, there are many methods for characterizing poly A tail length, such as HPLC, PCR, and next-generation sequencing. HPLC and PCR methods have low resolution. While next-generation sequencing offers the highest resolution and can detect the distribution of poly A, it requires RNA amplification, making the process cumbersome. Unlike these methods, mass spectrometry (MS) allows for direct detection, resolving individual nucleotides by molecular weight differences. It can also simultaneously detect multiple oligonucleotide sequences and has been adopted as a diagnostic platform.

[0005] Aibixin (Shanghai) Biotechnology Co., Ltd. has disclosed a poly A tail detection method for mRNA quality detection, which is based on endonuclease and LC-MS platform for poly A tail detection, but the resolution of the poly A tail detection results obtained by the solvent and operation is low. Summary of the Invention

[0006] In order to solve the problems in the prior art, the first aspect of the present invention provides a time-saving, labor-saving, high-resolution method for detecting polyA tails in mRNA vaccines, comprising the following steps:

[0007] S1: Enzyme digestion, magnetic bead purification, and elution recovery of the mRNA in the mRNA vaccine solution to obtain polyA tail fragments;

[0008] S2: The Poly A tail fragment is detected using a liquid chromatography-mass spectrometer (LC-MS); wherein the mobile phase of the liquid chromatography in the LC-MS consists of an aqueous phase A and an organic phase B; the phase A comprises hexafluoroisopropanol, N,N-diisopropylethylamine, and water; the phase B comprises pure methanol; and the mass spectrometer scan adopts a negative ion mode.

[0009] Preferably, the enzymatic cleavage step is to use RNase T1 enzyme to enzymatically cleave the mRNA in the mRNA vaccine solution.

[0010] Preferably, the specific steps of the enzymatic cleavage are heating the mRNA vaccine solution at 90-100°C for 3-8 minutes, then placing it on ice and rapidly cooling it to room temperature; then adding 10x RNase H buffer and RNase T1, mixing well, and reacting at 35-40°C for 3-3.5 hours.

[0011] Preferably, the volume of the mRNA vaccine solution is 100 to 150 μL.

[0012] Preferably, the magnetic bead purification step is to wash the magnetic beads with a binding buffer, then add the mRNA vaccine solution after enzyme cleavage treatment, collect the magnetic beads, and discard the liquid.

[0013] Preferably, the ratio of the magnetic beads to mRNA is 1 g:(100-500); more preferably, it is 1 g:300 pmol.

[0014] Preferably, the binding buffer comprises 30-50 v / v% 40-60 mM Tris-HCl, 20-30 v / v% 3-8 M LiCl, 1-3 v / v% 3-8 M EDTA, and 17-49 v / v% water; further preferably, it comprises 40 v / v% 50 mM Tris-HCl, 25 v / v% 5 M LiCl, 2 v / v% 0.1 M EDTA, and 33 v / v% water.

[0015] Preferably, the elution and recovery step is to successively add a washing buffer solution, ammonium acetate and a 75% by mass methanol solution to the purified magnetic beads, and collect the filtrate after elution to obtain the polyA fragment.

[0016] Preferably, the washing buffer comprises 20-30 v / v% 40-60 mM Tris-HCl, 1-5 v / v% 2-8 M LiCl, 0.1-3 v / v% 0.01-1 M EDTA, and 62-78.9 v / v% water; further preferably, it comprises 25 v / v% 50 mM Tris-HCl, 3 v / v% 5 M LiCl, 1 v / v% 0.1 M EDTA, and 69 v / v% water.

[0017] Preferably, the temperature of the methanol solution with a mass fraction of 75% is 80°C.

[0018] In the present invention, by selecting a binding buffer of 30-50 v / v% 40-60 mM Tris-HCl, 20-30 v / v% 3-8 M LiCl, 1-3 v / v% 3-8 M EDTA, 17-49 v / v% water and a washing buffer of 20-30 v / v% 40-60 mM Tris-HCl, 1-5 v / v% 2-8 M LiCl, 0.1-3 v / v% 0.01-1 M EDTA, 62-78.9 v / v% water, while improving the purity of the polyA tail, reducing the impact on subsequent operations, and improving the sensitivity and resolution of LC-MS result determination. The inventors found that the commonly used buffer for eluting the poly tail is 0.1 M ammonium acetate solution, but when 0.1 M ammonium acetate solution is used to elute the magnetic beads bound to the polyA tail in the mRNA vaccine, there is noise in the resulting spectrum, and the results cannot clearly show the distribution of the polyA tail.

[0019] In a preferred embodiment, the method for detecting polyA tails in mRNA vaccines comprises the following specific steps:

[0020] Step M1: Enzyme digestion: Heat 100-150 μL of mRNA vaccine solution at 90-100°C for 3-8 minutes, then place on ice and quickly cool to room temperature; then add 15-25 μL of 10x RNase H buffer and 5-15 μL of RNase T1 enzyme, mix well, and react at 37°C for 3 hours;

[0021] Step M2: Magnetic bead purification: Pipette the magnetic bead solution into a centrifuge tube, place it on a magnetic rack and let it stand, then discard the liquid; remove the centrifuge tube from the magnetic rack, add binding buffer, place it on the magnetic rack and let it stand, discard the liquid, and repeat twice; add 100-200µL binding buffer and the mRNA vaccine solution treated in step M1 to the washed magnetic beads, incubate at room temperature with shaking for 10-20 minutes, place it on a magnetic rack to collect the magnetic beads, and discard the liquid;

[0022] Step M3: Elution and recovery: Add 100-200 µL of washing buffer to the magnetic beads obtained in step M2, place on a magnetic stand for 1-3 minutes, discard the liquid, and repeat twice; then add 100-200 µL of 100 mM ammonium acetate, place on a magnetic stand for 1-3 minutes, discard the liquid, and repeat three times; add 80.0-120.0 µL of 75% methanol heated to 80°C to the washed magnetic beads; incubate at 80°C for 5 minutes, place on a magnetic stand for 1-3 minutes, collect the liquid, and lyophilize at room temperature to obtain a lyophilized powder;

[0023] Step M4: Sample analysis: The lyophilized powder obtained in step M3 was reconstituted with 60.0-100.0 µL of 100 µM EDTA solution and analyzed by LC-MS. The aqueous phase comprised hexafluoroisopropanol, N,N-diisopropylethylamine, and water; the organic phase comprised pure methanol; and the mass spectrometer was scanned in negative ion mode.

[0024] In the present invention, 100 to 200 μL of 100 mM ammonium acetate is added for washing to further dissolve other impurities on the magnetic beads, thereby improving the purity of the polyA tail on the magnetic beads, increasing the sensitivity of the LC-MS detection method, and increasing the response rate of the polyA tail.

[0025] In the present invention, the purified polyA tail is lyophilized and then reconstituted before use, which improves the LC-MS response value and thus the sensitivity of LC-MS. The inventors have found that compared to rotary evaporation of methanol, lyophilization of the solution evaporates the methanol and water in the polyA solution, which not only collects high-purity polyA fragments but also maintains the chemical structure of the polyA fragments, thereby improving the LC-MS response value and thus the sensitivity of LC-MS.

[0026] Preferably, the EDTA in the M4 contains 1 wt % of methanol.

[0027] Preferably, when the Poly A tail fragment is detected by liquid chromatography-mass spectrometry LC-MS in S2, the operating steps in the liquid chromatography are as follows: separation is performed using a UPLC system, the analysis sample is placed in an 8°C automatic injector, the column temperature is 75°C, the flow rate is 300 μL / min, and the injection volume is 45 μL; the relevant liquid chromatography gradient is set as follows: 0-2min, phase B is maintained at 5%; 2-10min, phase B changes linearly from 5% to 50%; 10-10.5min, phase B changes linearly from 50% to 90%; 10.5-12.5min, phase B is maintained at 90%; 12.5-13.0min, phase B changes linearly from 90% to 5%; 13.0-15.0min, phase B is maintained at 5%.

[0028] Preferably, the phase A is composed of 2 v / v% hexafluoroisopropanol, 0.1 v / v% N,N-diisopropylethylamine and 97.9 v / v% water;

[0029] Preferably, the phase B is pure methanol.

[0030] In the present invention, by selecting hexafluoroisopropanol and N,N-diisopropylethylamine as the mobile phase A, the background is cleaner and the response is higher. The inventors have found that in the method of detecting polyA tails using LC-MS, the commonly used mobile phase A is 100mM triethylamine acetate, but the use of triethylamine acetate results in high background noise and low response, affecting the final detection results. The inventors speculate that the mixed solution of hexafluoroisopropanol and N,N-diisopropylethylamine has a weaker boiling point and acidity, is more easily volatilized in the electrospray ionization gas phase state, has less interference with the ionization of oligonucleotides, and is more compatible with electrospray mass spectrometry, resulting in reduced background noise and improved response rate in the detection results.

[0031] Preferably, when the poly A tail fragment is detected by liquid chromatography-mass spectrometry (LC-MS) in S2, the mass spectrometry operation steps are as follows:

[0032] Mass spectrometry analysis was performed using an Acquity H-Class Plus Xevo G2-XS mass spectrometer (Waters) in negative ion mode. The conditions were as follows: System setup; Mass range: 400 m / z–3000 m / z; Capillary voltage: 2.5 kV; Cone voltage: 80 V; Desolvation temperature: 350°C; Cone gas flow rate: 50 L / h; Desolvation flow rate: 700 L / h.

[0033] The second aspect of the present invention provides an application of a method for detecting polyA tails in mRNA vaccines, which is used to construct a method system and detection platform for detecting polyA tails in mRNA vaccines.

[0034] Beneficial effects

[0035] 1. In the present invention, by selecting hexafluoroisopropanol and N,N-diisopropylethylamine as phase A in the mobile phase, the background is cleaner and the response is higher.

[0036] 2. In the present invention, by selecting a binding buffer of 30-50 v / v% 40-60 mM Tris-HCl, 20-30 v / v% 3-8 M LiCl, 1-3 v / v% 3-8 M EDTA, and 17-49 v / v% water and a washing buffer of 20-30 v / v% 40-60 mM Tris-HCl, 1-5 v / v% 2-8 M LiCl, 0.1-3 v / v% 0.01-1 M EDTA, and 62-78.9 v / v% water, the purity of the poly A tail is improved while reducing the impact on subsequent operations, thereby improving the sensitivity and resolution of LC-MS result determination.

[0037] 3. In the present invention, the purified polyA tail is freeze-dried and then redissolved before use, which improves the response value of LC-MS and thus improves the sensitivity of LC-MS.

[0038] 4. The present invention can achieve the enrichment of polyA fragments through enzyme digestion, magnetic bead purification, and elution recovery, thereby improving sensitivity and solving the problem of being unable to detect due to low concentration;

[0039] 5. The present invention can realize the detection of single nucleotides through LC-MS detection and analysis, and can detect multiple oligonucleotide sequences, thereby accurately determining the distribution range of polyA with high resolution; and the pretreatment method is simple, the detection time is short, the stability is greatly improved, and time is saved, and it can be used as a common characterization of mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the detection result graph of the polyA tail obtained by LC-MS detection of the new coronavirus mRNA vaccine in Example 1;

[0041] Figure 2 This is a graph showing the detection results of the polyA tail obtained by LC-MS detection of tumor therapeutic mRNA vaccine in Example 2;

[0042] Figure 3 This is the detection result graph of the polyA tail obtained by detecting the new coronavirus mRNA vaccine using LC-MS in Comparative Example 1;

[0043] Figure 4 This is the detection result graph of the polyA tail obtained by detecting the new coronavirus mRNA vaccine using LC-MS in Comparative Example 2;

[0044] Figure 5 This is the detection result graph of the polyA tail obtained by using LC-MS to detect the new coronavirus mRNA vaccine in Comparative Example 3. DETAILED DESCRIPTION

[0045] Example 1

[0046] A first aspect of this embodiment provides a method for detecting polyA tails in mRNA vaccines, comprising the following steps:

[0047] Step S1: Enzyme digestion: Take 130µL of mRNA vaccine solution (mass concentration 0.1mg / mL, molar concentration 2.2pmol / µL) and heat it at 95℃ for 5min, then place it on ice and quickly cool it to 25℃; then add 20.0µL 10x RNase H buffer and 10.0µL RNase T1 (200KU / mL), mix well and react at 37℃ for 3h; the mRNA vaccine solution is the new coronavirus mRNA vaccine, which is sourced from Shanghai Microspectro Detection Technology Group Co., Ltd.

[0048] Step S2: Magnetic Bead Purification: Pipette 190.0µL of the 5mg / mL magnetic bead solution into a 1.5mL centrifuge tube, place it on a magnetic rack, and let it sit for 30 seconds before discarding the liquid. Remove the centrifuge tube from the magnetic rack, add 150.0µL of binding buffer, and place it on the magnetic rack for 1 minute. Discard the liquid and repeat twice. Add 150.0µL of binding buffer and the mRNA vaccine solution treated in step S1 to the washed magnetic beads. Incubate at 25°C with shaking for 15 minutes. Collect the beads on a magnetic rack and discard the liquid. The binding buffer consists of 40% v / v 50mM Tris-HCl, 25% v / v 5M LiCl, 2% v / v 0.1M EDTA, and 33% v / v water.

[0049] Step S3: Elution and Recovery: Add 150.0µL of wash buffer to the magnetic beads obtained in Step S2, place on a magnetic rack for 1 minute, and discard the liquid. Repeat twice. Then, add 150.0µL of 100mM ammonium acetate, place on a magnetic rack for 1 minute, and discard the liquid. Repeat three times. Add 100.0µL of 75% methanol heated to 80°C to the washed magnetic beads. Incubate at 80°C for 5 minutes, place on a magnetic rack for 1 minute, collect the liquid, and lyophilize at 25°C. The wash buffer is 25 v / v% 50mM Tris-HCl, 3 v / v% 5M LiCl, 1 v / v% 0.1M EDTA, and 69 v / v% water.

[0050] Step S4: Sample analysis: The lyophilized powder from step S3 was reconstituted by adding 80.0 µL of 100 µM EDTA (containing 1 wt% methanol) solution and analyzed by LC-MS. Figure 1 . Figure 1 The image shows a high-resolution and clear polyA mass spectrum, accurately detecting the length of the polyA at this molecular weight.

[0051] When the Poly A tail fragment is detected by liquid chromatography-mass spectrometry LC-MS in S4, the liquid chromatography operation steps are as follows: separation is performed using a UPLC system, the analysis sample is placed in an 8°C automatic sampler, the column temperature is 75°C, the flow rate is 300 μL / min, and the injection volume is 45 μL; the relevant liquid chromatography gradient is set as follows: 0-2 min, phase B is maintained at 5%; 2-10 min, phase B changes linearly from 5% to 50%; 10-10.5 min, phase B changes linearly from 50% to 90%; 10.5-12.5 min, phase B is maintained at 90%; 12.5-13.0 min, phase B changes linearly from 90% to 5%; 13.0-15.0 min, phase B is maintained at 5%.

[0052] The phase A is composed of 2 v / v% hexafluoroisopropanol, 0.1 v / v% N,N-diisopropylethylamine and 97.9 v / v% water;

[0053] The phase B is pure methanol.

[0054] When the poly A tail fragment is detected by liquid chromatography-mass spectrometry (LC-MS) in S4, the mass spectrometry operation steps are as follows:

[0055] Mass spectrometry analysis was performed using an Acquity H-Class Plus Xevo G2-XS mass spectrometer (Waters) in negative ion mode; the conditions were as follows: Project: System Settings; Mass range: 400 m / z–3000 m / z; Capillary voltage: 2.5 kV; Cone voltage: 80 V; Desolvation temperature: 350°C; Cone gas flow rate: 50 L / h; Desolvation flow rate: 700 L / h.

[0056] The second aspect of this embodiment provides an application of a method for detecting polyA tails in mRNA vaccines, which is applied to construct a method system and detection platform for detecting polyA tails in mRNA vaccines.

[0057] Example 2

[0058] The specific implementation of Example 2 is the same as that of Example 1, except that the mRNA vaccine used in step S1 is a tumor therapeutic mRNA vaccine from Shanghai Microspectra Detection Technology Group Co., Ltd. Figure 2 . Figure 2 The polyA map of the mRNA vaccine is neatly distributed with high resolution, and can accurately measure the distribution range and length of polyA.

[0059] Comparative Example 1

[0060] The specific implementation of Comparative Example 1 is the same as that of Example 1, except that the mobile phase A is triethylamine acetate, and its ratio is 0.6 v / v% acetic acid, 1.4 v / v% trimethylamine and 98 v / v% water. Figure 3 , Figure 3 The spectrum response is low, the distribution is disordered, there are many summed ion peaks, and the noise of the system interferes greatly with the target components of the spectrum, affecting the judgment of the results.

[0061] Comparative Example 2

[0062] The specific implementation of Comparative Example 2 is the same as that of Example 1, except that the samples in steps S2 and S3 are medium, no magnetic beads are used for purification, and the enzyme digestion is directly applied to the machine. Figure 4 , the response is low and the target peak cannot be found.

[0063] Comparative Example 3

[0064] The specific implementation of Comparative Example 3 is the same as that of Example 1, except that the operating steps in the liquid chromatography are as follows: UPLC system is used for separation, the analysis sample is placed in an 8°C autosampler, the column temperature is 75°C, the flow rate is 300 μL / min, and the injection volume is 45 μL; the relevant liquid chromatography gradient is set as follows: 0-2min, phase B is maintained at 50%; 2-10min, phase B changes linearly from 50% to 100%; 10-10.5min, phase B changes linearly from 100% to 50%; 10.5-15.0min, phase B is maintained at 50%. Results are shown in Figure 5 , the target compound was not eluted.

Claims

1. A method for detecting polyA tails in mRNA vaccines, characterized in that: The following steps are involved: S1: Enzyme digestion, magnetic bead purification, and elution recovery of the mRNA in the mRNA vaccine solution to obtain polyA tail fragments, specifically including: Step M1: Enzyme digestion: Heat 100-150 μL of mRNA vaccine solution at 90-100°C for 3-8 minutes, then place on ice and quickly cool to room temperature; then add 15-25 μL of 10x RNase H buffer and 5-15 μL of RNase T1 enzyme, mix well, and react at 37°C for 3 hours; Step M2: Magnetic bead purification: Pipette the magnetic bead solution into a centrifuge tube, place it on a magnetic stand and let it stand, then discard the liquid; remove the centrifuge tube from the magnetic stand, add binding buffer, place it on a magnetic stand and let it stand, discard the liquid, and repeat twice; add 100-200 μL of binding buffer and the mRNA vaccine solution treated in step M1 to the washed magnetic beads, incubate with shaking at room temperature for 10-20 minutes, place it on a magnetic stand to collect the magnetic beads, and discard the liquid; Step M3: Elution and recovery: Add 100-200 μL of washing buffer to the magnetic beads obtained in step M2, place on a magnetic stand for 1-3 minutes, discard the liquid, and repeat twice; then add 100-200 μL of 100 mM ammonium acetate, place on a magnetic stand for 1-3 minutes, discard the liquid, and repeat three times; add 80.0-120.0 μL of 75% methanol heated to 80°C to the washed magnetic beads; incubate at 80°C for 5 minutes, place on a magnetic stand for 1-3 minutes, collect the liquid, and lyophilize at room temperature to obtain a lyophilized powder; Step M4: Sample analysis: Add 60.0-100.0 μL of 100 μM EDTA solution to the lyophilized powder obtained in step M3 for reconstitution; S2: Detecting the Poly A tail fragment using a liquid chromatography-mass spectrometer (LC-MS); wherein the mobile phase of the liquid chromatography in the LC-MS is composed of an aqueous phase A and an organic phase B; the phase A comprises hexafluoroisopropanol, N,N-diisopropylethylamine, and water; the phase B comprises pure methanol; and the mass spectrometer scan is performed in negative ion mode; When the poly A tail fragment is detected by liquid chromatography-mass spectrometry LC-MS in S2, the operation steps of the liquid chromatography are as follows: separation is performed using a UPLC system, the analysis sample is placed in an 8°C autosampler, the column temperature is 75°C, the flow rate is 300 μL / min, and the injection volume is 45 μL; the relevant liquid chromatography gradient is set as follows: 0-2 min, phase B is maintained at 5%; 2-10 min, phase B changes linearly from 5% to 50%; 10-10.5 min, phase B changes linearly from 50% to 90%; 10.5-12.5 min, phase B is maintained at 90%; 12.5-13.0 min, phase B changes linearly from 90% to 5%; 13.0-15.0 min, phase B is maintained at 5%; When the Poly A tail fragment is detected by liquid chromatography-mass spectrometry LC-MS in S2, the mass spectrometry operation steps are as follows: mass spectrometry analysis is performed using an Acquity H-Class Plus Xevo G2-XS mass spectrometer in negative ion mode; the conditions are as follows: Item: System Settings; Mass Range: 400m / z~3000m / z; Capillary Voltage: 2.5kV; Cone Voltage: 80V; Desolvation Temperature: 350°C; Cone Gas Size: 50L / h; Desolvation Size: 700L / h.

2. The method for detecting polyA tails in mRNA vaccines according to claim 1, wherein The enzymatic cleavage step is to use RNase T1 enzyme to enzymatically cleave the mRNA in the mRNA vaccine solution.

3. The method for detecting polyA tails in mRNA vaccines according to claim 2, wherein The usage ratio of the magnetic beads and mRNA is 1 g: (100-500) pmol.

4. The method for detecting polyA tails in mRNA vaccines according to claim 3, wherein The binding buffer comprises 30-50 v / v% 40-60 mM Tris-HCl, 20-30 v / v% 3-8 M LiCl, 1-3 v / v% 3-8 M EDTA and 17-49 v / v% water.

5. The method for detecting polyA tails in mRNA vaccines according to claim 1, wherein The washing buffer comprises 20-30 v / v% 40-60 mM Tris-HCl, 1-5 v / v% 2-8 M LiCl, 0.1-3 v / v% 0.01-1 M EDTA and 62-78.9 v / v% water.

6. An application of the method for detecting polyA tails in mRNA vaccines according to any one of claims 1 to 5, characterized in that: A method system and detection platform for constructing polyA tail detection in mRNA vaccines.

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