A method for characterizing plasmid quality

Abstract

This invention belongs to the field of plasmid and mRNA quality control technology, specifically disclosing a method for characterizing plasmid quality. Existing plasmid quality control systems cannot effectively predict mRNA product quality and do not focus on the purity of the PolyA tail. This invention establishes a positive correlation between the purity of the PolyA region in the plasmid and the quality of the mRNA product by detecting its purity, thus achieving early mRNA quality prediction. The prediction method includes the following steps: plasmid sample enzyme digestion, analysis of PolyA fragment purity using capillary gel electrophoresis, and substituting the PolyA fragment purity into a correlation curve to obtain mRNA purity. Based on this, this invention effectively shortens the production cycle, reduces costs, and provides a new evaluation dimension for plasmid quality control by screening plasmids with acceptable PolyA purity for mRNA production.

Description

Technical Field

[0001] This invention relates to the field of plasmid and mRNA quality control technology, and specifically to a method for characterizing plasmid quality. Background Technology

[0002] In the development of vaccines and therapeutics using mRNA as an API (active pharmaceutical ingredient), plasmids, i.e. DNA templates, serve as the starting material for in vitro transcription (IVT) to prepare mRNA, and their quality has a crucial impact on the purity, integrity, and translation efficiency of the final mRNA product.

[0003] Currently, the industry's quality control of supercoiled plasmids used for mRNA production mainly relies on a series of routine detection indicators, including but not limited to:

[0004] Appearance: Visually inspect whether it is a colorless and clear liquid;

[0005] Purity: The A260 / A280 ratio is determined by UV-Vis spectrophotometry and is typically between 1.8 and 2.0.

[0006] Concentration: determined by A260 absorbance, or high performance liquid chromatography (HPLC), or agarose gel electrophoresis (AGE), etc.

[0007] Plasmid structure verification: Restriction enzyme digestion was used to confirm whether the band size met the theoretical expectations;

[0008] Supercoil ratio: Evaluated using ion exchange chromatography-high performance liquid chromatography (IEC-HPLC), typically requiring a minimum of 90%;

[0009] Residue detection includes host cell proteins (HCP), host cell DNA, host cell RNA, and bacterial endotoxins.

[0010] Sequence accuracy: Sanger sequencing confirmed that the plasmid sequence was consistent with the theoretical sequence.

[0011] Microbial limits and pH value: These were determined using membrane filtration and pH metering, respectively.

[0012] Although the aforementioned detection methods are widely used in plasmid quality control, they often fail to effectively predict key quality attributes of the mRNA product transcribed from the plasmid, such as mRNA purity. Therefore, existing technologies have significant limitations in comprehensively evaluating plasmid quality, making it difficult to identify plasmid batches that may lead to poor mRNA product quality at an early stage.

[0013] Furthermore, the structural integrity and purity of specific functional regions in plasmids (such as the PolyA tail) have not yet been incorporated into routine plasmid quality assessment systems. However, this region plays a crucial role in mRNA stability and translation efficiency, and its quality can directly affect the performance of the final mRNA product.

[0014] Therefore, there is an urgent need in this field to develop a plasmid characterization method that can more accurately and earlier predict the quality of mRNA products, so as to improve the controllability of plasmid production, reduce development costs, and ensure the consistency and efficacy of the final mRNA drug. Summary of the Invention

[0015] The purpose of this invention is to provide a plasmid quality characterization method that can more accurately and earlier predict the quality of mRNA products. In particular, by detecting the purity of the PolyA region in the plasmid and establishing its correlation with the purity of mRNA, the method can effectively predict and control the quality of mRNA products and provide a reliable basis for plasmid strain screening and production process optimization.

[0016] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0017] In a first aspect, the present invention provides a method for characterizing plasmid quality, comprising the following steps:

[0018] Obtain a plasmid sample to be tested, wherein the plasmid contains a PolyA region;

[0019] The purity of the PolyA region in the plasmid was detected;

[0020] The quality of the plasmid is evaluated based on the purity of the PolyA region; wherein, the purity of the PolyA region is positively correlated with the purity of the mRNA product transcribed from the plasmid, therefore, the higher the purity of the PolyA region, the higher the purity of the mRNA product transcribed from the plasmid, and the better the plasmid quality.

[0021] Furthermore, the method for detecting the purity of the PolyA region in the plasmid is as follows: the plasmid sample is digested with enzymes to obtain a DNA fragment containing the PolyA region; the digestion products are analyzed using capillary gel electrophoresis to obtain the purity of the PolyA fragment.

[0022] Furthermore, when the purity of plasmid PolyA is ≥85%, the plasmid is determined to be suitable for high-quality mRNA production.

[0023] Secondly, the present invention provides a method for predicting the quality of mRNA products, comprising the following steps:

[0024] Obtain plasmid samples for mRNA transcription;

[0025] Measure the purity of the PolyA region in the plasmid;

[0026] The purity of the mRNA product transcribed from the plasmid is predicted based on the purity of the PolyA region, and the purity of the PolyA region is positively correlated with the purity of the mRNA product transcribed from the plasmid.

[0027] In practical mRNA product quality prediction, plasmids with gradient PolyA region purity are first prepared; then, they are converted into mRNA and their purity is detected, establishing standard curves for PolyA purity and RNA purity. In subsequent applications, for the same plasmid sample, only its PolyA purity needs to be detected and substituted into the standard curve to obtain the corresponding mRNA purity, thus achieving the purpose of predicting mRNA product quality. Therefore, this method can effectively reduce the production cycle and cost of qualified plasmids, thereby accelerating the production of mRNA drugs and improving drug quality.

[0028] Furthermore, the method for detecting the purity of the PolyA region in the plasmid is as follows: the plasmid sample is digested with enzymes to obtain a DNA fragment containing the PolyA region; the digestion products are analyzed using capillary gel electrophoresis to obtain the purity of the PolyA fragment.

[0029] In a preferred embodiment, the purity of the PolyA region and the purity of the mRNA product transcribed from the plasmid satisfy a linear regression equation y = 0.645x + 0.3168, where x is the purity of plasmid PolyA, y is the predicted mRNA purity, and the correlation coefficient R² > 0.94.

[0030] Thirdly, the present invention provides a method for screening plasmid strains, comprising the following steps:

[0031] Provide multiple candidate plasmid strains;

[0032] The purity of the PolyA region in the plasmids produced by each candidate plasmid strain was determined.

[0033] Based on the purity of the PolyA region, target plasmid strains capable of producing high-quality mRNA products were screened.

[0034] Furthermore, the method for detecting the purity of the PolyA region in the plasmid is the same as above, and the screening criterion is to select strains corresponding to plasmids with PolyA purity ≥ 85%.

[0035] Fourthly, this invention provides the application of the above-mentioned methods for characterizing plasmid quality, predicting mRNA product quality, or screening plasmid strains in the preparation of products using mRNA transcribed from the plasmid as the active substance. These methods pre-determine the mRNA quality by detecting the purity of the PolyA region in the plasmid, providing reliable plasmids and plasmid strains as raw materials for the preparation of high-quality mRNA.

[0036] Furthermore, the products using mRNA as the active substance include personalized mRNA tumor vaccines and universal mRNA tumor vaccines.

[0037] The present invention has the following beneficial effects:

[0038] 1. Improved predictive ability for mRNA product quality. This invention establishes a direct correlation between key functional regions of plasmids and the quality of the final mRNA product by detecting the purity of the PolyA region in the plasmid. It can predict key quality attributes of the mRNA product in the early stages of plasmid production, solving the defect of existing quality control systems that are disconnected from the final mRNA product quality results.

[0039] 2. It shortens the production cycle of qualified plasmids and reduces development costs. By eliminating plasmid batches with insufficient PolyA purity in the early screening stage, it avoids subsequent ineffective production inputs, effectively compresses the plasmid production cycle, and thus accelerates the overall development process of mRNA drugs.

[0040] 3. This invention provides a new evaluation dimension for plasmid quality control. It overcomes the limitations of traditional detection methods, proposing a novel strategy for plasmid quality evaluation linked to mRNA purity, and providing scientific support for the development of quality standards for plasmid templates during mRNA product production. Attached Figure Description

[0041] Figure 1 The correlation curves between plasmid PolyA purity and mRNA purity in the examples demonstrate the linear relationship and regression analysis results between the two.

[0042] Figure 2 The flowchart in this example illustrates the introduction of PolyA purity detection into the plasmid production process.

[0043] Figure 3 The example demonstrates the effectiveness of incorporating PolyA purity detection into the plasmid production process to detect mRNA purity. Detailed Implementation

[0044] The existing quality control methods for supercoiled plasmids used in mRNA production have the following limitations:

[0045] Lack of effective predictability for mRNA product quality: Existing plasmid quality assessment systems (such as appearance, A260 / A280 ratio, concentration, enzyme digestion verification, supercoiling ratio, and residue detection) can control the basic properties of the plasmid itself, but cannot effectively predict the key quality attributes (such as mRNA purity) of the mRNA product transcribed from the plasmid. This leads to a disconnect between plasmid quality assessment and the final mRNA product quality results, making it impossible to identify plasmid batches that may lead to substandard mRNA products in the early stages of production.

[0046] The correlation between plasmid functional region integrity and mRNA quality has not been adequately addressed: Current technologies do not incorporate the purity or structural integrity of specific key functional regions (such as the PolyA tail) into routine plasmid quality assessment systems. These regions have a critical impact on mRNA stability, translation efficiency, and the quality of the final product; defects in these regions can directly lead to a decrease in mRNA product purity.

[0047] Low efficiency in screening and cost control during plasmid production: Due to the lack of plasmid indicators that can predict the quality of mRNA products in advance, reliance on traditional indicators in plasmid strain screening and production process control may lead to long production cycles, high costs, and an inability to accurately guarantee the quality consistency of the final product.

[0048] To address this, this invention introduces the PolyA region purity index into plasmid quality detection and establishes a positive correlation between it and the purity of mRNA products. This enables early prediction of mRNA product quality and provides a reliable quality control basis for plasmid strain screening and mRNA product production process optimization.

[0049] This invention is applicable to the detection of plasmids with different polyA lengths. The commonly used polyA length range is approximately 50-150, all of which are within the scope of this invention.

[0050] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.

[0051] Example 1: Small-scale preparation of plasmids

[0052] 1. Pick a single colony containing the plasmid sample (polyA structure is 99 consecutive adenine nucleotides) and inoculate it into 50 mL of LB medium (containing the corresponding antibiotic, such as ampicillin 100 μg / mL), and incubate at 37℃ and 220 rpm for 16 h with shaking.

[0053] 2. Take 40 mL of the culture medium, centrifuge at 8000 rpm for 5 min to collect the bacterial cells, add 4 mL of Solution I (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 μg / mL RNase A (Thermo Fisher, catalog number EN0531)) to resuspend the bacterial cells, and incubate on ice for 5 min;

[0054] 3. Add 8 mL of Solution II (0.2 M NaOH, 1% SDS), gently invert and mix 5 times, and let stand at room temperature for 2 min;

[0055] 4. Add 6 mL of pre-cooled Solution III (3M KAc pH5.5), mix vigorously by inverting 10 times, and incubate on ice for 10 min;

[0056] 5. Centrifuge at 12000 rpm for 10 min, take the supernatant and add an equal volume of isopropanol, let stand at -20℃ for 30 min;

[0057] 6. Centrifuge at 12000 rpm for 15 min, collect the precipitate, wash twice with 75% ethanol, vacuum dry for 5 min, and then dissolve in 50 μL of enzyme-free water to obtain the plasmid solution.

[0058] 7. Enzyme digestion: The enzyme digestion system (20 μL) includes: 10 μL of crude plasmid extract, 1 μL of EcoRI (NEB catalog number R0101S), 1 μL of Xbal (NEB catalog number R0145S), 2 μL of 10× enzyme digestion buffer (NEB Buffer 2.1, catalog number B7201), and enzyme-free water to a final volume of 20 μL.

[0059] Incubate at 8.37℃ for 2 hours, then add 1 μL of 0.5M EDTA solution to terminate the enzyme digestion reaction;

[0060] 9. Sample pretreatment: After filtering the enzyme digestion product through a 0.22μm filter membrane (Millipore, catalog number SLGP033RB), dilute it 10-fold with dilution buffer 1×TE (10mM Tris-HCl (pH 8.0): Sigma, catalog number T1503; 10×TE stock solution is autoclaved and diluted before use). The resulting sample can then be analyzed for polyA purity by capillary electrophoresis.

[0061] Example 2: Analysis of polyA purity using capillary electrophoresis

[0062] Step 1: Preparation of reagent kits and consumables

[0063] Core components: Agilent DNF 905 Complete Kit (catalog number: DNF-905-0500, including gel, dye, buffer, marker and ladder, etc.); mineral oil (Sigma, catalog number M8410).

[0064] Consumables: 96-well plate / 8-tube strip, RNase-free / DNase-free pipette tips, 1.5 mL centrifuge tubes, mineral oil.

[0065] Instrument: Agilent 5200 Fragment Analyzer. The capillary array was confirmed to be properly installed and the detector was functioning correctly.

[0066] Step 2: Reagent preparation

[0067] (1) Gel-dye mixture (prepared and used immediately):

[0068] Take 10 mL of DNF-905 gel, add 10 μL of DNF-600 intercalating dye, vortex mix, and then sonicate to degas for 5 min to avoid residual air bubbles.

[0069] (2) Preparation of buffer solution:

[0070] Inlet buffer: Dilute 5× stock solution to 1× with ultrapure water, mix thoroughly and degas.

[0071] Capillary buffer: Dilute 5× stock solution to 1×, degas and use.

[0072] (3) Marker and Ladder Preparation:

[0073] The marker is ready to use; dispense directly into 96-well plates, 10 μL per well, seal with mineral oil, and store at 4°C. Dilute the ladder to the working concentration according to the instructions, and bring to volume with 1× TE, avoiding repeated freeze-thaw cycles.

[0074] Step 3: Instrument and Software Preparation

[0075] (1) Power-on and self-test:

[0076] Turn on the 5200 host and control software, and perform an instrument self-test (check capillary position, buffer level, and detector status).

[0077] Set the solution volume in the software (Utilities → Solution Levels) to ensure that the gel and buffer solution are sufficient.

[0078] (2) Capillary pretreatment (new capillary or long-unused):

[0079] Wash with 1× capillary buffer for 5 min each time, then wash with gel-dye mixture for 5 min each time to remove air bubbles and improve separation efficiency.

[0080] (3) Pallet configuration:

[0081] Buffer tray (drawer B): Add 150 μL of 1× inlet buffer to A1-A12.

[0082] Marker tray (drawer M): Add 10 μL of marker to A1-A12 and seal with mineral oil.

[0083] Sample tray (drawer S): Place samples and ladders in sequence, with the 96-well plate A1 facing left and back.

[0084] Step 4: Method Setup and Execution (Software)

[0085] (1) Create a new method (Method → ​​New):

[0086]

[0087] (2) Queue settings:

[0088] Add sample location, ladder location, and marker location; set the analysis name and save path.

[0089] (3) Start running:

[0090] After confirming that the tray position is correct, click Start Run. The instrument will automatically perform sample injection and separation, and monitor the electrophoresis pattern in real time.

[0091] Step 5: Data Analysis and Instrument Post-processing

[0092] (1) Data analysis:

[0093] After the operation is completed, the software automatically calls the DNF-905 analysis template to perform fragment size calculation and qualitative evaluation.

[0094] Verify Ladder resolution: Adjacent bands with R ≥ 0.8 are considered acceptable; otherwise, re-optimize injection / separation parameters.

[0095] Results output: Export electrophoresis pattern, fragment size distribution table, and label abnormal peaks (such as impurities and primer dimers).

[0096] Purity calculation: The purity of polyA is calculated using the main peak area normalization method, and the percentage of the main peak area of ​​polyA to the total peak area is used as the purity of polyA.

[0097] (2) Instrument post-processing:

[0098] After the run, rinse the capillary with 1× capillary buffer for 5 min, then rinse with the storage solution and seal to prevent the capillary from drying out.

[0099] Empty and clean the buffer / marker / sample tray, and let it air dry before use.

[0100] Example 3: mRNA preparation and purity detection

[0101] Using the plasmid prepared in Example 1 as a template, mRNA was prepared by in vitro transcription (IVT) as follows:

[0102] Step 1: Plasmid linearization

[0103] (1) The linearization system (50 μL) includes: 10 μg of circular plasmid template, BspQI enzyme (NEB, catalog number: R0712L; to make the BspQI activity unit to plasmid mass ratio reach more than 1 U / μg), 5 μL of 10×bspQI reaction buffer (NEB, catalog number: B7204S), and enzyme-free water to make up to 50 μL;

[0104] (2) Incubate at 50±1℃ for 1 hour to complete linearization and then set aside for use;

[0105] Step 2: IVT reaction and mRNA purity detection

[0106] (1) IVT reaction

[0107] The transcription system (50 μL as an example) includes linearization of the circular plasmid prepared in Example 1 using BspQI.

[0108] Template 2 μg, 10× transcription buffer 5 μL (NEB, catalog number: B0332S), rNTP mixture (2.5 Mm each, NEB, catalog number: N0405L) 10 μL, T7 RNA polymerase 2 Ml (NEB, catalog number: M0251L), RNase inhibitor 1 Ml (ThermoFisher, catalog number: EO0381), enzyme-free water to 50 μL;

[0109] After incubation at 37℃ for 2.5 h, add DNase I (1 U / μg plasmid, NEB, catalog number: M0303L) and incubate at 37℃ for 30 min to remove the template plasmid;

[0110] (2) mRNA was purified using an RNA purification kit (column type, Thermo Fisher, catalog number: K0731, containing lysis buffer, wash buffer I, wash buffer II, adsorption column, collection tube, etc.):

[0111] Add an equal volume of lysis buffer to the transcription product, mix thoroughly, transfer to an adsorption column, and centrifuge at 12,000 rpm for 1 min.

[0112] Add 500 μL of washing solution I, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0113] Add 500 μL of washing solution II, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat the washing once;

[0114] Centrifuge the empty column at 12000 rpm for 2 min to remove residual washing solution, add 30 μL of enzyme-free water, let stand at room temperature for 2 min, then centrifuge at 12000 rpm for 1 min, collect the eluent, and obtain purified mRNA.

[0115] (3) mRNA purity detection

[0116] The chromatography column was a Thermo Fisher Scientific DNAPac RP column (4 μm, 2.1 × 100 mm, Thermo Fisher Scientific, catalog number: 063387), with a column temperature of 65 °C and a flow rate of 0.35 mL / min.

[0117] Mobile phase A consists of 50 mM dibutylammonium acetate (TCI America, catalog number: D1247) + 100 mM triethylammonium acetate (Sigma-Aldrich, 90358), and mobile phase B consists of 50% acetonitrile + 50 mM dibutylammonium acetate + 100 mM triethylammonium acetate.

[0118] Gradient elution program: 0–1.5 min, 25% B (hold); 1.5–4.5 min, 25%–50% B (gradient); 4.5–19 min, 50%–56% B (gradient); 19–19.5 min, 56%–100% B (gradient); 19.5–24.5 min, 100% B (hold); 24.5–25 min, 100%–25% B (gradient); 25–30 min, 25% B (hold).

[0119] The detection wavelength was 260 nm, and the injection volume was 2 μg mRNA (dissolved in enzyme-free water).

[0120] The mRNA purity is determined by the percentage of the main mRNA peak (MP) area to the total peak area. Attention is also paid to the presence of a late elution peak (LP, corresponding to lipid-mRNA adducts), and the LP peak area percentage should be ≤1%.

[0121] Each sample was tested in parallel three times, and the relative standard deviation (RSD) was calculated. An RSD ≤ 3% was considered acceptable, and a purity ≥ 80% was considered to meet the quality requirements. The retention time of the main peak was also recorded for qualitative confirmation.

[0122] Example 4: Correlation between POLYA in plasmid template and mRNA purity

[0123] Plotting PolyA purity (%) in the plasmid on the x-axis and mRNA purity (%) on the y-axis, the results are as follows: Figure 1 As shown, mRNA purity exhibits a significant linear increase with increasing PolyA purity, with a linear regression equation of y = 0.645x + 0.3168 (R² = 0.9461). This indicates a strong positive correlation between PolyA purity and mRNA purity in the plasmid, suggesting that a predictive model for mRNA purity can be established based on PolyA purity in the plasmid. When PolyA purity is ≥85%, the corresponding mRNA purity is above 85%, indicating that plasmids with PolyA purity ≥85% can be used for high-quality mRNA production.

[0124] Figure 2 This paper demonstrates a key control point in the plasmid production process, specifically the introduction of PolyA purity detection during the PCB (cell bank for research) screening stage. The process includes steps such as plasmid template synthesis, transfection, small-scale plasmid extraction, strain transfection, quality control, RCB amplification, and plasmid production. The PolyA purity indicator is introduced as an optional indicator in the plasmid quality control stage for screening dominant clones.

[0125] Example 5

[0126] mRNA purity was predicted using polyA purity of RCB stage plasmids. The table below shows polyA purity and mRNA purity data for different sequences. Among them, sequences 1005, 1010, B16F10 and 1001 have the same mRNA length, 5'URT, 3'URT and polyA structure, but contain different target gene sequences.

[0127]

[0128] The polyA purity and mRNA purity in this embodiment are compared with... Figure 1 Compared to (e.g.) Figure 3 As shown in the figure, testing polyA purity at the RCB stage can effectively predict mRNA purity. The results show that, as predicted in Example 4, the polyA purity and mRNA purity at the RCB stage are similar. Figure 1 The linear relationship.

[0129] Comparative Example 1

[0130] This comparative study tested plasmid purity (the sum of the ratios of supercoiled monomers and supercoiled polymers). Comparing the plasmid purity and mRNA purity test results in the table below, it can be seen that high plasmid purity does not correspond to high mRNA purity, indicating that plasmid purity cannot accurately predict mRNA purity.

[0131]

[0132] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A method for characterizing plasmid quality, characterized in that, Includes the following steps: Obtain a plasmid sample to be tested, wherein the plasmid contains a PolyA region; The purity of the PolyA region in the plasmid was detected; The quality of the plasmid is evaluated based on the purity of the PolyA region.

2. The method for characterizing plasmid quality according to claim 1, characterized in that, The method for detecting the purity of the PolyA region in the plasmid is as follows: the plasmid sample is digested with enzymes to obtain a DNA fragment containing the PolyA region; the digestion products are analyzed by capillary gel electrophoresis to obtain the purity of the PolyA fragment.

3. The method for characterizing plasmid quality according to claim 1, characterized in that, When the purity of plasmid PolyA is ≥85%, the plasmid is considered suitable for high-quality mRNA production.

4. A method for predicting the quality of mRNA products, characterized in that, Includes the following steps: Obtain plasmid samples for mRNA transcription; Measure the purity of the PolyA region in the plasmid; The purity of the mRNA product transcribed from the plasmid is predicted based on the purity of the PolyA region, and the purity of the PolyA region is positively correlated with the purity of the mRNA product transcribed from the plasmid.

5. The method for predicting mRNA product quality according to claim 4, characterized in that, The method for detecting the purity of the PolyA region in the plasmid is as follows: the plasmid sample is digested with enzymes to obtain a DNA fragment containing the PolyA region; the digestion products are analyzed by capillary gel electrophoresis to obtain the purity of the PolyA fragment.

6. The method for predicting mRNA product quality according to claim 5, characterized in that, The purity of the PolyA region and the purity of the mRNA product transcribed from this plasmid satisfy a linear regression equation y = 0.645x + 0.3168, where x is the purity of plasmid PolyA and y is the predicted mRNA purity, with a correlation coefficient R² > 0.

94.

7. A method for screening plasmid-containing bacterial strains, characterized in that, Includes the following steps: Provide multiple candidate plasmid strains; The purity of the PolyA region in the plasmids produced by each candidate plasmid strain was determined. Based on the purity of the PolyA region, target plasmid strains capable of producing high-quality mRNA products were screened.

8. The method for screening plasmid strains according to claim 7, characterized in that, The screening criterion is to select strains corresponding to plasmids with a PolyA purity of ≥85%.

9. The application of the plasmid quality characterization method according to any one of claims 1-3, the method for predicting mRNA product quality according to any one of claims 4-6, or the plasmid strain screening method according to any one of claims 7-8 in the preparation of products using mRNA transcribed from the plasmid as the active substance.

10. The application according to claim 9, characterized in that, The products using mRNA as the active substance include personalized mRNA tumor vaccines and universal mRNA tumor vaccines.

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