Industrial purification method for improving integrity of self-replicating RNA (Ribonucleic Acid)

Abstract

The invention discloses an industrial purification method for improving the integrity of self-replicating RNA (Ribonucleic Acid), which comprises the following steps: (1) combining: mixing saRNA subjected to capping with a high-salt ion concentration buffer solution, and loading a sample to a affinity chromatographic column; (2) washing: washing the affinity chromatography column with a low-salt ion concentration buffer solution to remove impurities; and (3) eluting: eluting with DEPC (diethylpyrocarbonate) water, and collecting the eluent in the range of 150 mAU to 150 mAU, so as to obtain the saRNA product. According to the method, the purification process of the saRNA can be rapidly completed, the technological operation process is simple, convenient and rapid, the integrity of the saRNA is obviously improved by 8%-11.4%, the experimental process is easy to amplify, and the method has very strong technical advantages for the technological process for operating the long-fragment saRNA.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for industrial production, separation and purification of a self-replicating RNA stock solution. Background Art

[0002] Self-replicating RNA (saRNA) is based on the conventional mRNA sequence, with the addition of a self-replicating sequence element. It can use its own RNA sequence as a template to self-replicate and amplify, producing more saRNA copies. Compared with conventional mRNA, saRNA has a great advantage in dosage. It can achieve the same protein expression level as traditional mRNA with a very low dose, and the effect lasts longer. Studies have shown that in the same immune response, saRNA is hundreds or even thousands of times smaller in dose than traditional mRNA, so compared with conventional mRNA, the advantage is more obvious.

[0003] For large-scale preparation of mRNA, the current mainstream method in the industry is still chromatography. Based on the chemical specificity of the mRNA molecule itself, there are many optional chromatography methods, including reverse phase, ion exchange, hydrophobic and affinity chromatography, but each method has its advantages and disadvantages. In the selection of a specific chromatography method, it is necessary to comprehensively consider factors such as scalability, platformization, purification efficiency, recovery rate, and economy. Affinity, ion exchange and hydrophobic applications have good scalability and are also the most widely used purification methods. In particular, affinity chromatography is currently widely used for Poly (A) mRNA capture.

[0004] The affinity chromatography column is composed of a resin matrix, cross-linked poly(styrene-divinylbenzene, PS-DVB), functionalized with poly(dT) on the surface. The poly(dT) base pairs with the poly(A) tail of mRNA, forming hydrogen bonds and thereby capturing the mRNA. Oligo(dT) carries a negative phosphate charge, which repulses the negative phosphate charges on the poly(A) backbone. In high-salt buffer conditions, salt ions shield the electrostatic repulsion between the negatively charged backbone, allowing the dT to bind to the poly(A) tail. In low-salt / no-salt conditions, the negative charges of the two repel each other, eluting the mRNA. Summary of the Invention

[0005] The present invention aims to provide an industrial purification method that can effectively improve the integrity of saRNA stock solutions based on existing technologies. This method is particularly suitable for the purification of saRNAs with base numbers ranging from 6,000 to 15,000 nt. The present invention optimizes the conditions for saRNA affinity chromatography purification and screens for purification conditions that are particularly suitable for saRNA, effectively improving the integrity of saRNA.

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

[0007] An industrial purification method for improving the integrity of self-replicating RNA comprises the following steps:

[0008] (1) Binding: The capped saRNA is mixed with a high salt ion concentration buffer containing 1-0.5 M NaCl and loaded onto an affinity chromatography column;

[0009] (2) Washing: Washing the affinity chromatography column with a low salt ion concentration buffer to remove impurities; the low salt ion concentration buffer contains 0.1-0.05M NaCl;

[0010] (3) Elution: Use DEPC water for elution, collect the eluate in the range of 150mAU~max~150mAU, and obtain the saRNA product.

[0011] In step (1), preferably, the capped saRNA sample can be prepared by in vitro transcription and capping reaction of a saRNA linearized plasmid.

[0012] The purification method of the present invention is suitable for various saRNAs with a base number of less than 15,000 nt, especially around 10,000 nt (8,000 nt-15,000 nt) with a poly (A) tail. As long as the base type meets the requirements, there is no restriction on the specific saRNA.

[0013] The affinity chromatography column of the present invention is preferably filled with Oligo(dT)25 affinity filler.

[0014] The high salt ion concentration buffer of the present invention contains Tris and EDTA, and 0.8-0.7M NaCl; in a preferred embodiment, the high salt ion concentration buffer comprises 0.75M NaCl, 10mM Tris and 1mM EDTA, and its pH is 7.0-7.8 (preferably around 7.4).

[0015] The low salt ion concentration buffer of the present invention contains Tris and EDTA, and 0.08-0.07M NaCl. In a preferred embodiment, the low salt ion concentration buffer comprises 0.075M NaCl, 10mM Tris and 1mM EDTA, and its pH is 7.0-7.8 (preferably around 7.4).

[0016] In step (1), the sample retention time is 3-5 minutes.

[0017] Before loading the sample in step (1), the affinity chromatography column can be equilibrated with a high salt ion concentration buffer until the conductivity, UV (A 260 、A 280 ) and pH stable.

[0018] Before washing in step (2), the affinity chromatography column may be washed with a high salt ion concentration buffer until UV equilibrium is achieved.

[0019] In step (2), the affinity chromatography column is washed with a low salt ion concentration buffer until the conductivity and UV (A 260 、A 280 )balance.

[0020] The present invention optimizes the key process parameters of affinity chromatography and explores the salt ion concentration and washing ratio in the buffer system to improve the recovery rate and integrity of industrial production and reduce impurities and by-products in the saRNA stock solution.

[0021] Beneficial effects of the present invention:

[0022] 1) The saRNA chromatography purification process provided by the present invention can quickly complete the saRNA purification process, and the process operation process is simple and fast, meeting the requirements of the process;

[0023] 2) The saRNA chromatography purification process provided by the present invention significantly improves the integrity of saRNA (8%-11.4%). The integrity of the saRNA final product is 80.4%-83.9%, the purity is 78.51%-83.64%, and the average recovery rate is 57.7%, which has obvious process technology advantages.

[0024] 3) The saRNA chromatography purification process provided by the present invention has a simple operation process, strong operability, and the experimental process is easy to scale up, which has a strong technical advantage for the process of operating long-fragment saRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the capillary gel electrophoresis pattern of the RNA ladder microfluidic chip in Example 1 of the present invention;

[0026] Figure 2 This is the capillary gel electrophoresis pattern of the saRNA purification product on a microfluidic chip in Example 1 of the present invention;

[0027] Figure 3 This is the SEC-HPLC profile of the saRNA purification product in Example 1 of the present invention;

[0028] Figure 4 This is the capillary gel electrophoresis pattern of the saRNA purification product on a microfluidic chip in Example 2 of the present invention;

[0029] Figure 5 This is the SEC-HPLC profile of the saRNA purification product in Example 2 of the present invention;

[0030] Figure 6 This is the capillary gel electrophoresis pattern of the saRNA purification product on a microfluidic chip in Example 3 of the present invention;

[0031] Figure 7 This is the SEC-HPLC profile of the saRNA purification product in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0033] Example 1. Affinity chromatography experimental process of sequence 1 capped sample

[0034] 1.1 Preparation before chromatography experiment

[0035] 1) Affinity chromatography column: filled with Thermo Scientific TM Brand, POROS TM Oligo(dT)25 affinity medium.

[0036] 2) saRNA sample: A self-replicating saRNA sample (SEQ ID NO: 1) was synthesized by in vitro transcription from a linearized plasmid. SEQ ID NO: 1 is 10,000 nt in length, and this product is provided as an example only. The method of the present invention is applicable to any in vitro transcription product less than 15,000 nt in length, not limited to SEQ ID NO: 1. The in vitro transcription product of SEQ ID NO: 1 is provided as an example only.

[0037] 3) Chromatography Buffer:

[0038]

[0039] 1.2 Chromatography experiment process

[0040] 1) Before starting the experiment, check whether there are bubbles in the pipelines (Pump A, Pump B, Pump S). If there are bubbles, manually exhaust them.

[0041] 2) Connect the chromatography column and set the alarm pressure, column position and flow rate;

[0042] 3) Equilibrate the column with 75% Buffer A + 25% Buffer B until the conductivity and UV (A 260 、A 280 ), pH stability;

[0043] 4) Premix the capped sample with 2*Buffer A (2 times the concentration of Buffer A, the same below) to increase the sample salt concentration to 0.75M, and then perform the sample loading process. The sample retention time is about 3-5 minutes;

[0044] 5) After the system injection is completed, continue to flush the chromatography column with 75% Buffer A + 25% Buffer B until UV equilibrium is achieved;

[0045] 6) Then adjust the ratio of Buffer A (7.5%) and Buffer B (92.5%) to reduce the salt ion concentration so that the impurities are washed away, and rinse until the conductivity and UV (A 260 、A 280 ) until equilibrium is reached;

[0046] 7) Elute the sample with Elution Buffer and collect the eluate between 150 mAU and max and 150 mAU to obtain the saRNA product.

[0047] 1.3 LiCl purification process

[0048] Take 100 μL of the capped sample for LiCl purification as a control for chromatography purification. Add 60 μL of 7.5 M lithium chloride to the capped EP tube, mix thoroughly, and place in a -20°C refrigerator for 30 minutes. Then, centrifuge at 8000 rpm for 15 minutes, collect the precipitate, and wash it with 1 mL of 70% ethanol. Repeat this process once more, and finally dissolve it in 30 μL of enzyme-free water.

[0049] 1.4 Sample testing

[0050] 1) Recovery rate: The concentration of the collected saRNA samples was detected using qubit, and the recovery rate was then calculated;

[0051] 2) Integrity testing: PE Labchip was used to detect the integrity of the collected saRNA;

[0052] 3) Purity detection: SEC-HPLC was used to detect the purity of the collected saRNA.

[0053] 1.5 Experimental Results

[0054] 1) The recovery rate was calculated by Qubit detection of saRNA concentration and was 57.2%.

[0055] 2) Figure 1 It is the RNA Ladder integrity map; Figure 2This is the integrity profile of the saRNA purification product, with an integrity of 83.9%. The integrity of the same batch of samples after LiCl purification was 72.5%, an improvement of 11.4%;

[0056] 3) Figure 3 This is the SEC-HPLC profile of the saRNA purification product. The purity of the saRNA is 83.64%.

[0057] Example 2, affinity chromatography experimental process of sequence 2 capped sample

[0058] 2.1 Preparation before chromatography experiment

[0059] 1) Affinity chromatography column: filled with Thermo Scientific TM Brand, POROS TM Oligo(dT)25 affinity medium.

[0060] 2) saRNA sample: A self-replicating saRNA sample (SEQ ID NO: 2) was synthesized by in vitro transcription from a linearized plasmid; SEQ ID NO: 2 was 10,000 nt in length and was provided as an example only.

[0061] 3) Chromatography Buffer:

[0062]

[0063]

[0064] 2.2 Chromatography experiment process

[0065] 1) Before starting the experiment, check whether there are bubbles in the pipelines (Pump A, Pump B, Pump S). If there are bubbles, manually exhaust them.

[0066] 2) Connect the chromatography column and set the alarm pressure, column position and flow rate;

[0067] 3) Equilibrate the column with 75% Buffer A + 25% Buffer B until the conductivity and UV (A 260 、A 280 ), pH stability;

[0068] 4) Premix the capped sample with 2*Buffer A to increase the sample salt concentration to 0.75M, and then perform the sample loading process. The sample retention time is about 3-5 minutes.

[0069] 5) After the system injection is completed, continue to flush the chromatography column with 75% Buffer A + 25% Buffer B until UV equilibrium is achieved;

[0070] 6) Then adjust the ratio of Buffer A (7.5%) and Buffer B (92.5%) to reduce the salt ion concentration so that the impurities are washed away, and rinse until the conductivity and UV (A 260 、A 280 ) until equilibrium is reached;

[0071] 7) Elute the sample with Elution Buffer and collect the eluate between 150 mAU and max and 150 mAU to obtain the saRNA product.

[0072] 2.3 LiCl purification process

[0073] Take 100 μL of the capped sample for LiCl purification as a control for chromatography purification. Add 60 μL of 7.5 M lithium chloride to the capped EP tube, mix thoroughly, and place in a -20°C refrigerator for 30 minutes. Then, centrifuge at 8000 rpm for 15 minutes, collect the precipitate, and wash it with 1 mL of 70% ethanol. Repeat this process once more, and finally dissolve it in 30 μL of enzyme-free water.

[0074] 2.4 Sample testing

[0075] 1) Recovery rate: The concentration of the collected saRNA samples was detected using qubit, and the recovery rate was then calculated;

[0076] 2) Integrity testing: PE Labchip was used to detect the integrity of the collected saRNA;

[0077] 3) Purity detection: SEC-HPLC was used to detect the purity of the collected saRNA.

[0078] 2.5 Experimental Results

[0079] 1) The recovery rate was calculated by Qubit detection of saRNA concentration and was 55.28%.

[0080] 2) Figure 4 This is the integrity profile of the saRNA purification product, with an integrity of 83.8%. The integrity of the same batch of samples after LiCl purification was 74.5%, an improvement of 9.3%;

[0081] 3) Figure 5 The figure shows the SEC-HPLC profile of the purified saRNA product. The purity of the saRNA is 80.98%.

[0082] Example 3, affinity chromatography experimental process of sequence 3 capped sample

[0083] 3.1 Preparation before chromatography experiment

[0084] 1) Affinity chromatography column: filled with Thermo Scientific TM Brand, POROS TM Oligo(dT)25 affinity medium.

[0085] 2) saRNA sample: A self-replicating saRNA sample (SEQ ID NO: 3) was synthesized by in vitro transcription from a linearized plasmid; SEQ ID NO: 3 was 10,000 nt in length and was provided as an example only.

[0086] 3) Chromatography Buffer:

[0087]

[0088] 3.2 Chromatography experiment process

[0089] 1) Before starting the experiment, check whether there are bubbles in the pipelines (Pump A, Pump B, Pump S). If there are bubbles, manually exhaust them.

[0090] 2) Connect the chromatography column and set the alarm pressure, column position and flow rate;

[0091] 3) Equilibrate the column with 75% Buffer A + 25% Buffer B until the conductivity and UV (A 260 、A 280 ), pH stability;

[0092] 4) Premix the capped sample with 2*Buffer A to increase the sample salt concentration to 0.75M, and then perform the sample loading process. The sample retention time is about 3-5 minutes.

[0093] 5) After the system injection is completed, continue to flush the chromatography column with 75% Buffer A + 25% Buffer B until UV equilibrium is achieved;

[0094] 6) Then adjust the ratio of Buffer A (7.5%) and Buffer B (92.5%) to reduce the salt ion concentration so that the impurities are washed away, and rinse until the conductivity and UV (A 260 、A 280 ) until equilibrium is reached;

[0095] 7) Elute the sample with Elution Buffer and collect the eluate between 150 mAU and max and 150 mAU to obtain the saRNA product.

[0096] 3.3 LiCl purification process

[0097] Take 100 μL of the capped sample for LiCl purification as a control for chromatography purification. Add 60 μL of 7.5 M lithium chloride to the capped EP tube, mix thoroughly, and place in a -20°C refrigerator for 30 minutes. Then, centrifuge at 8000 rpm for 15 minutes, collect the precipitate, and wash it with 1 mL of 70% ethanol. Repeat this process once more, and finally dissolve it in 30 μL of enzyme-free water.

[0098] 3.4 Sample testing

[0099] 1) Recovery rate: The concentration of the collected saRNA samples was detected using qubit, and the recovery rate was then calculated;

[0100] 2) Integrity testing: PE Labchip was used to detect the integrity of the collected saRNA;

[0101] 3) Purity detection: SEC-HPLC was used to detect the purity of the collected saRNA.

[0102] 3.5 Experimental Results

[0103] 1) The recovery rate was calculated by Qubit detection of saRNA concentration and was 60.64%.

[0104] 2) Figure 6 This is the integrity profile of the saRNA purification product, with an integrity of 80.4%. The integrity of the same batch of samples after LiCl purification was 71.8%, an improvement of 8.6%;

[0105] 3) Figure 7 The SEC-HPLC profile of the saRNA purification product shows a purity of 78.51%.

Claims

1. An industrial purification method for improving the integrity of self-replicating RNA, characterized in that The steps include: (1) Binding: The capped saRNA is mixed with a high salt ion concentration buffer containing 1-0.5 M NaCl and loaded onto an affinity chromatography column; (2) Washing: Washing the affinity chromatography column with a low salt ion concentration buffer to remove impurities; the low salt ion concentration buffer contains 0.1-0.05 M NaCl; (3) Elution: Use DEPC water for elution and collect the eluate in the range of 150 mAU~max~150 mAU to obtain saRNA product.

2. The method according to claim 1, characterized in that The number of bases in the self-replicating RNA is less than 15,000 nt, preferably between 8,000 nt and 15,000 nt.

3. The method according to claim 1, characterized in that The affinity chromatography column is filled with Oligo(dT)25 affinity filler.

4. The method according to claim 1, characterized in that The high salt ion concentration buffer contains Tris and EDTA; the low salt ion concentration buffer contains Tris and EDTA.

5. The method according to claim 1, characterized in that The high salt ion concentration buffer comprises 0.75 M NaCl, 10 mM Tris and 1 mM EDTA, and has a pH of 7.0-7.

8.

6. The method according to claim 1, characterized in that In step (1), the sample retention time is 3-5 minutes.

7. The method according to claim 1, characterized in that The low salt ion concentration buffer comprises 0.075 M NaCl, 10 mM Tris and 1 mM EDTA, and has a pH of 7.0-7.

8.

8. The method according to claim 1, characterized in that Before loading the sample in step (1), use a high salt ion concentration buffer to equilibrate the affinity chromatography column until the conductivity, UV (A 260 、A 280 ) and pH stable.

9. The method according to claim 1, characterized in that Before washing in step (2), the affinity chromatography column is first flushed with a high salt ion concentration buffer until UV equilibrium is achieved.

10. The method according to claim 1, characterized in that In step (2), the affinity chromatography column is washed with a low salt ion concentration buffer until the conductivity and UV (A 260 、A 280 )balance.

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