Freeze-drying concentration method of mRNA (messenger Ribonucleic Acid) capable of maintaining mRNA

Abstract

The invention provides a method for effectively concentrating mRNA, mRNA-LNP and saRNA and maintaining the integrity of RNA, the advantages of freeze-drying concentration are adopted, and conditions such as freeze-drying time, freeze-drying initial temperature and freeze-drying temperature are optimized, so that the mRNA-LNP is effectively concentrated, the mRNA concentration is improved, the influence of concentration operation on the integrity of mRNA is reduced, and the method is suitable for large-scale industrial production. The problem that in the preparation and analysis process of an mRNA-LNP preparation, an effective technology is needed for improving the mRNA-LNP concentration is solved. The method is simple and rapid to operate, and has small influence on RNA integrity. By providing a universal and reliable RNA concentration means, the invention aims to overcome the key links of the RNA-LNP preparation from research and development to industrial production and quality detection, and provides key support for the progress of the whole biotechnology industry chain.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a method for lyophilizing and concentrating mRNA and its formulations while maintaining mRNA integrity. Background Technology

[0002] The successful development of mRNA COVID-19 vaccines has significantly enhanced the application prospects of mRNA technology in disease prevention and treatment. This therapy boasts advantages such as high efficacy, short development cycle, and good safety; however, its application is limited by the inherent instability of mRNA—its susceptibility to nuclease degradation, which affects the integrity of the drug's efficacy. Currently, lipid nanoparticles (LNPs) have been proven to be effective carriers for delivering mRNA. Notably, self-amplified mRNA (saRNA), as a novel type of mRNA, retains traditional mRNA delivery methods (such as saRNA-LNP) while carrying a replicase gene that enables the RNA sequence to self-amplify, achieving protein expression levels comparable to traditional mRNA at lower doses, demonstrating stronger immunostimulatory potential and clinical value. However, the low-dose advantage of saRNA is a double-edged sword, posing extremely high requirements for RNA stability detection: the lower the concentration, the greater the detection difficulty and the higher the challenge to the limit of quantitation. Currently, methods for detecting the integrity and stability of mRNA-LNP (especially saRNA-LNP) are very limited, making it difficult to meet the needs of research and quality control. Therefore, establishing a reliable concentration technology that can perfectly maintain the integrity of RNA while increasing the concentration of the formulation is crucial for overcoming current technological bottlenecks and promoting the development of mRNA therapy.

[0003] RNA integrity testing is a crucial part of RNA stability assessment. Even when most physicochemical data remain consistent, subtle changes in mRNA integrity can still affect transcription efficiency and biological activity. As the terminology suggests, integrity refers to the multidimensional integrity of mRNA, making it one of the few indicators that can systematically assess mRNA quality. Especially for low-concentration samples, the tiny fragments produced by mRNA degradation (particularly saRNA, which is inherently low-dose) often hinder accurate assessment of RNA integrity. Therefore, finding suitable methods for precise integrity testing is urgently needed for these low-dose, highly active mRNAs.

[0004] Given the inherent chemical instability of mRNA, a biomolecule, it is easily degraded by high temperatures and enzymes in the air. Traditional drug concentration methods, including boiling, rotary evaporation, and reverse osmosis, all involve high temperatures or prolonged exposure to air, making them unsuitable. Furthermore, current modern concentration techniques often involve vacuum centrifugation, which, while improving efficiency compared to traditional methods, still requires significant time and temperature control is difficult. Therefore, finding a suitable concentration method is crucial. Vacuum freeze-drying involves freezing wet materials or solutions into a solid state at low temperatures (-10℃ to -50℃), then sublimating the water directly into a gaseous state under vacuum (1.3–13 Pa), ultimately dehydrating the material. After the sample becomes a freeze-dried powder, reconstitution with a small amount of enzyme-free water can create a high-concentration solution. This drying technique can be converted into a concentration technique for converting low-concentration solutions into high-concentration solutions.

[0005] Freeze-drying effectively separates solvent water molecules from solute molecules, and the process avoids the high-temperature environment of traditional concentration methods, seemingly making it an ideal method for mRNA concentration. However, the freeze-drying process involves pressure changes, and samples may face localized high-pressure environments during this concentration process. Whether the integrity of mRNA will decrease under such conditions remains to be scientifically investigated. This invention relates to the development of a freeze-drying-based concentration method, aiming to achieve efficient concentration of saRNA, mRNA, and mRNA-LNP solutions, with a focus on verifying and ensuring the stability of mRNA structures. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for concentrating saRNA, mRNA, and mRNA-LNP solutions without altering RNA integrity. By using lyophilization, the large solute mRNA in the solution is safely separated from the small solvent water molecules. Combined with specific sample processing and detection procedures, and optimization of various key factors (such as lyophilization time, initial sample temperature, and demulsification method of the LNP solution), this method solves the technical problem of effectively concentrating saRNA, mRNA, and mRNA-LNP solutions without altering mRNA integrity. It effectively increases the concentration of low-concentration saRNA, mRNA, and mRNA-LNP solutions, providing reliable methodological support for the integrity detection of mRNA formulations, especially low-concentration formulations.

[0007] To achieve the above objectives, the present invention employs the following solution: On one hand, the present invention provides an effective method for concentrating saRNA, mRNA, and mRNA-LNP solutions. The concentration method employs lyophilization to increase the concentration of the original solution, and includes the following steps, as can be seen... Figure 1 : Place appropriate amounts of saRNA solution, mRNA solution and mRNA-LNP solution into a freeze dryer respectively; Furthermore, the freeze-drying concentration method for mRNA, including saRNA, and its LNP formulation proposed in this invention differs from other freeze-drying preparation methods. This invention concentrates existing low-concentration mRNA solutions and optimizes the freeze-drying concentration process to ensure that the integrity of the mRNA is not damaged.

[0008] (2) After the sample from step (1) is freeze-dried into freeze-dried powder under suitable freeze-drying conditions (freeze-drying time, initial sample temperature, freeze-drying temperature), it is reconstituted with a small amount of enzyme-free water according to the experimental purpose. (3) After reconstituted saRNA and / or mRNA samples from step (2), incubate at 70°C for 2 minutes, then incubate at 4°C for 5 minutes. Finally, use the Agilent 5300 fragment analysis system or the Guangding Bio Qsep100 fully automated nucleic acid and protein analysis system to detect the integrity of saRNA and mRNA samples respectively. Two detection instruments are used because saRNA is more prone to degradation due to its longer transcriptional sequence compared to mRNA. Therefore, the Agilent 5300, which has a higher throughput, is used to detect the integrity of saRNA.

[0009] (4) The mRNA-LNP sample from step (2) needs to be demulsified before its integrity is tested. The demulsification steps are as follows: Accurately transfer 100 μL of sample into a 1.5 mL centrifuge tube, add 900 μL of ammonium acetate isopropanol solution (60 mM), and vortex at 14000 g for 5 minutes. Remove the supernatant. Add another 1 mL of ammonium acetate isopropanol solution and vortex at 14000 g for 5 minutes. Remove the supernatant. Finally, rinse with 70% ethanol solution and vortex at 14000 g for 5 minutes. Remove the supernatant. Redissolve the precipitate with enzyme-free water. Incubate the sample at 70°C for 2 minutes, then at 4°C for 5 minutes. Finally, use the Qsep100 fully automated nucleic acid and protein analysis system to test the integrity of the sample.

[0010] In some implementations, the lyophilization and concentration efficiency of saRNA, mRNA, and mRNA-LNP is affected by the lyophilization temperature. Too low a temperature leads to longer cooling times, increasing the time cost of concentration operations, while too high a temperature results in poorer lyophilization and difficulty in complete lyophilization. Currently, the detection principle for mRNA integrity is based on capillary electrophoresis, thus requiring a certain sample concentration. Too high a concentration can cause detection overload, while too low a concentration results in excessive instrument noise and reduced accuracy. Therefore, this method sets the sample detection concentration range at 5-50 μg / mL based on the instrument's detection capability.

[0011] Furthermore, in step (1), the freeze-drying time for freeze-drying concentration should be 22-36 h.

[0012] The lyophilization time should not be too short, as it is difficult to completely freeze-dry the sample. If the un-lyophilized sample is reconstituted, it will be difficult to control the volume, and the dilution factor will be uncontrollable. The lyophilization time should not be too long either, as mRNA is easily degraded by enzymes. If the lyophilization time is too long, the sample will be more likely to come into contact with enzymes in the air, and the mRNA will be degraded, resulting in a decrease in integrity. It is generally recommended that the lyophilization time be around 24 hours.

[0013] Furthermore, in step (1), the initial temperature of the sample should be -80℃ to 25℃.

[0014] The lower the initial temperature of the sample, the lower the molecular activity, and the lower the rate of mRNA degradation by airborne enzymes during the pre-freezing stage. When the initial temperature of the sample is above 25°C, mRNA is easily degraded by airborne enzymes, thus greatly reducing its integrity. In practice, it is recommended that the initial temperature of the sample be below -30°C, as numerous studies have demonstrated that the mRNA degradation rate is almost negligible at this temperature.

[0015] The reason for incorporating saRNA concentration in this invention is that, under normal circumstances, saRNA has a higher molecular weight and a faster degradation rate compared to similar mRNAs, and its concentration is also lower in practical applications, making a suitable method for concentration even more urgent for detection. This invention can concentrate saRNA without affecting its integrity, demonstrating the universality and effectiveness of this invention for RNA concentration and potentially solving the challenges of detecting various RNA integrity issues.

[0016] Furthermore, in step (4), the impact of the mRNA-LNP demulsification method on integrity is evaluated using Ttiton-X100 and ammonium acetate isopropanol solution. Based on the experimental results, ammonium acetate isopropanol solution is used to demulsify all mRNA-LNPs.

[0017] Furthermore, to compare the effects of lyophilization concentration and commonly used concentration methods on integrity, in step (1), mRNA-LNP test samples of the same volume concentration were placed in a Colparmer benchtop vacuum centrifuge (Chicago, USA) and a rotary evaporator, respectively, and adjusted to appropriate parameters and concentrated by the same factor. The concentrate was demulsified as described above and incubated at 70°C for 2 minutes, then incubated at 4°C for 5 minutes. Finally, the integrity of the sample was detected using the Qsep100 fully automated nucleic acid and protein analysis system.

[0018] In steps (1) to (4), the temperature of the sample should be kept below 4°C. Specifically, the sample can be placed on crushed ice during the operation to reduce the activity of nucleases in the air around the sample and reduce the error caused by sample degradation. Attached Figure Description

[0019] Figure 1 Summary of freeze-drying concentration and integrity testing procedures; Figure 2 The effect of different concentrations of lyophilization and concentration on mRNA integrity (control group: mRNA fraction without concentration treatment). Figure 3 The effect of different demulsification methods on the integrity of mRNA in mRNA-LNP (control group is naked mRNA that has not undergone concentration treatment). Figure 4 The effect of different concentrations of lyophilization and concentration on the integrity of mRNA in mRNA-LNP (control group is mRNA-LNP fraction that has not undergone concentration treatment). Figure 5 The effect of different concentration methods on the integrity of mRNA in mRNA-LNP (control group is mRNA-LNP component without concentration treatment). Figure 6 The effect of different concentrations of freeze-drying concentration on the integrity of saRNA (control group: saRNA fraction without concentration treatment). Figure 7 Original mRNA solution integrity detection spectra (from top to bottom, the corresponding concentrations are 50 μg / mL, 10 μg / mL and 2 μg / mL); Figure 8 Original mRNA integrity detection spectra in mRNA-LNP solution (from top to bottom, the corresponding concentrations are 50 μg / mL, 10 μg / mL and 2 μg / mL respectively). Figure 9 Original mRNA-LNP concentration spectra obtained by different concentration methods (from top to bottom: lyophilized concentration, vacuum centrifugation concentration, and rotary evaporation concentration). Figure 10 The original spectra of saRNA solutions with different concentrations for integrity detection: (A) is 75 μg / mL saRNA, (B) is 15 μg / mL saRNA, and (C) is 3 μg / mL saRNA. Figure 11 Comparison of original electrophoretic patterns before and after freeze-drying and concentration. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] In the field of biotechnology, the effective concentration of mRNA formulations is crucial for their formulation development and industrial production. Lyophilization is a common technique for solvent removal in the laboratory, and it is well known that solvent removal is an effective means of concentration. However, the lyophilization process is often accompanied by significant pressure and temperature changes, which may damage the integrity of the mRNA, making it difficult to meet the requirements of formulation development and industrial production. Therefore, this embodiment proposes a lyophilization concentration method for mRNA solutions that does not affect the integrity of the mRNA, as detailed below: Take appropriate amounts of 2 μg / mL and 10 μg / mL mRNA solutions and place them in 1 mL vials. First, freeze the samples at -80℃ or -30℃ until completely frozen. Then, place them in a lyophilizer and set the lyophilization temperature to -35℃ for 22 h. Reconstitute the lyophilized powder, incubate the resulting sample at 70℃ for 2 minutes, then at 4℃ for 5 minutes. Finally, use the Qsep100 fully automated nucleic acid and protein analysis system to check the sample integrity. The results are as follows: Figure 2 As shown.

[0024] Combined with original electrophoresis Figure 7 At a concentration of 2 μg / mL, the small peak area meant that even slight signal changes and small fragments resulting from degradation could interfere with the accuracy of integrity testing. Therefore, a concentration method was employed. The concentrated mRNA at concentrations of 10 and 50 μg / mL achieved integrity levels of 80.0% and 83.0%, respectively. While the peak area significantly increased, the integrity remained relatively consistent with the control group (P ≤ 0.001). Before formally performing lyophilization concentration, special attention must be paid to the following: the sample to be concentrated must first be stored in a -80°C freezer before being loaded into the lyophilizer. This step is crucial because a lower initial sample temperature reduces molecular activity, thereby slowing down the degradation rate of mRNA by atmospheric enzymes during the pre-freezing stage. Conversely, when the initial sample temperature exceeds 25°C, mRNA is more susceptible to degradation by atmospheric enzymes, leading to a significant decrease in its integrity. Therefore, extreme caution must be exercised when lyophilizing fragile mRNA to protect its sensitive components during the pre-freezing stage. This also highlights the need for researchers to closely monitor temperature control during the lyophilization concentration process to minimize mRNA degradation caused by temperature fluctuations.

[0025] Demulsifying LNPs is a prerequisite for detecting the integrity of mRNA in mRNA-LNPs. This invention has developed two methods for demulsifying LNPs: demulsification with Ttiton-X100 solution and demulsification with ammonium acetate isopropanol solution.

[0026] Ttiton-X100 demulsification: Take 20 μL of mRNA-LNP solution, add 2 μL of 10% Ttiton-X100 solution, and incubate for 3 minutes. The resulting sample is incubated at 70℃ for 2 minutes, then at 4℃ for 5 minutes. Finally, the integrity of the sample is checked using the Qsep100 fully automated nucleic acid and protein analysis system.

[0027] Demulsification with ammonium acetate isopropanol solution: Accurately transfer 100 μL of the test sample into a 1.5 mL centrifuge tube, add 900 μL of ammonium acetate isopropanol solution (60 mM), and extract by vortexing at 14000 g for 5 minutes. Remove the supernatant. Repeat the above steps once more by adding 1 mL of ammonium acetate isopropanol solution. Finally, rinse with 70% ethanol solution and repeat the above steps. Redissolve the precipitate with enzyme-free water, incubate the sample at 70℃ for 2 minutes, then incubate at 4℃ for 5 minutes. Finally, check the sample integrity using the Qsep100 fully automated nucleic acid and protein analysis system.

[0028] The results are as follows Figure 3 As shown, both Triton-X100 and ammonium acetate isopropanol solution can effectively demulsify LNPs, but their mechanisms of action and effects differ. Triton-X100, as a nonionic surfactant, dissolves the lipid structure of the outer layer of LNPs, thereby releasing the internal mRNA. Isopropanol, as a commonly used organic reagent, dissolves the lipid components in LNPs, causing the hydrophilic mRNA to precipitate, which can then be purified by centrifugation. The comparison revealed that ammonium acetate isopropanol solution has a superior demulsification effect. This is because Triton-X100, while dissolving lipids, does not separate and precipitate the mRNA, but rather dissolves both in the solution, leading to lower subsequent detection results. The mRNA obtained by demulsification with ammonium acetate isopropanol solution has higher integrity, indicating that demulsification with ammonium acetate isopropanol solution has less impact on integrity; therefore, ammonium acetate isopropanol solution was used for demulsification in subsequent tests.

[0029] Take appropriate amounts of 2 μg / mL and 10 μg / mL mRNA-LNP solutions and place them in 1 mL vials. First, freeze the samples at -80℃ or -30℃ until completely frozen. Then, place them in a lyophilizer and set the lyophilization temperature to -35℃ for 22 h. Remove the lyophilized powder and reconstitute it with a small amount of solvent to obtain a concentrated solution. Accurately transfer 100 μL of the concentrated solution to a 1.5 mL centrifuge tube, add 900 μL of ammonium acetate isopropanol solution (60 mM), and vortex at 14000 g for 5 min. Remove the supernatant. Repeat the above steps once more by adding 1 mL of ammonium acetate isopropanol solution. Finally, rinse with 70% ethanol solution and repeat the above steps. Reconstitute the precipitate with enzyme-free water. Incubate the resulting sample at 70℃ for 2 minutes, then at 4℃ for 5 minutes. Finally, use the Qsep100 fully automated nucleic acid and protein analysis system to check the sample integrity. The results are as follows: Figure 4 As shown.

[0030] Based on the original electrophoresis pattern 8, at a concentration of 2 μg / mL, similar to the description in Example 1, it was impossible to accurately integrate and quantify the integrity. However, after concentration, the integrity at concentrations of 10 and 50 μg / mL was 71.3% and 72.0%, respectively, even exceeding the 69.7% and 67.0% of the control group. This phenomenon may be due to the protective effect of the LNP vector on mRNA—preventing significant loss of mRNA integrity during lyophilization and concentration. Simultaneously, compared to the previously described naked mRNA, the integrity of mRNA-LNP decreased after demulsification. This decrease may be attributed to two main factors: First, the shear stress and physical forces generated by vortexing and oscillation during demulsification promote non-enzymatic degradation of mRNA. Second, the Qsep instrument uses agarose gel and other matrices as molecular sieves; residual salt ions and ethanol during demulsification can cause electroosmotic interference, thus affecting the final signal response intensity.

[0031] Take an appropriate amount of 2 μg / mL mRNA-LNP solution and place it into a 1 mL vial, a 1.5 mL centrifuge tube, and a rotary evaporator flask, respectively. The lyophilization concentration step follows the specific steps of Experiment Example 3; the vacuum centrifugation concentration step is performed at room temperature, and the concentrate is removed when it reaches one-fifth of the original volume; the rotary evaporation step is performed under water bath conditions, and heating is stopped when the concentrate reaches one-third of the original volume. The remaining liquid is then concentrated to one-fifth of the original volume using residual heat, and the rotary evaporation is stopped. After demulsification of the above concentrates according to the above methods, they are incubated at 70℃ for 2 minutes, then at 4℃ for 5 minutes. Finally, the integrity of the samples is detected using the Qsep100 fully automated nucleic acid and protein analysis system. The results are as follows: Figure 5 As shown.

[0032] Among commonly used concentration methods, neither vacuum centrifugation nor rotary evaporation can achieve the following two points compared to lyophilization: 1. Precise quantification of the concentration factor; 2. Preservation of the integrity of temperature-sensitive samples. The second point is the primary reason for the significant difference in mRNA integrity in mRNA-LNP: compared to the 71.3% integrity achieved by lyophilization, the integrity of vacuum centrifugation is only 44.2%, while rotary evaporation even results in peak splitting due to high temperatures, making precise integration and quantification of integrity impossible. Figure 9 As shown. Therefore, compared with commonly used concentration methods, lyophilized mRNA concentration has many advantages, and can protect the integrity of mRNA from damage.

[0033] Because the Qsep100 cannot accurately quantify and separate low concentrations of saRNA, and given the easy degradation of saRNA, this invention uses an Agilent 5300 for integrity testing when verifying the integrity of saRNA. Take appropriate amounts of 3 μg / mL and 15 μg / mL saRNA solutions and place them in 1 mL vials. First, freeze the samples at -80℃ or -30℃ until completely frozen. Then, place them in a lyophilizer and set the lyophilization temperature to -35℃ for 22 hours. Reconstitute the lyophilized powder. Take 2 μL of the concentrated sample and add 22 μL of diluted RNA marker (15 nt, Agilent, catalog number DNF-369-0004), and mix thoroughly. Detect RNA purity using an Agilent 5300 analyzer. The running procedure is as follows: pre-run at 8.0 kV for 30 seconds, followed by injection at 5.0 kV for 4 seconds, and finally separation at 4.0 kV for 90 minutes. The results are as follows: Figure 6 As shown.

[0034] The integrity of saRNA after lyophilization and concentration was 82.7% and 80.6%, respectively. These results confirm that the concentration process suitable for mRNA is also suitable for saRNA, and can achieve ideal results. Even for the more unstable saRNA, the data showed a small standard deviation (SD) value, indicating reduced batch-to-batch variability. This observation is supported by... Figure 10 Further support. Specifically, Figure 10 Figure C shows the raw map of saRNA integrity analysis at a concentration of 3 μg / mL. As shown in the figure, saRNA integrity is almost undetectable at this concentration—this is precisely the core challenge addressed in this study. Therefore, as... Figure 5 As shown, we labeled this dataset as "Not Applicable (NA)" because it could not be integrated. However, after concentration processing, the electrophoretic patterns of the two concentrations obtained were consistent with... Figure 10Similar to 10B, this study validates the effectiveness and practicality of our method. It is noteworthy that there are two key reasons for including saRNA in this study: first, under normal conditions, saRNA transcripts are longer and degrade at a higher rate than their mRNA counterparts; second, saRNA is typically present at lower concentrations in practical applications, making the development of suitable detection and concentration methods more challenging. This study demonstrates the feasibility of this method.

Claims

1. A method for lyophilizing concentration of mRNA and its formulation maintaining the integrity of mRNA, characterized in that, The concentration method adopts a freeze-drying method to improve the concentration of saRNA, mRNA and mRNA-LNP preparations, and comprises the following steps: (1) placing appropriate amounts of saRNA solution, mRNA solution and mRNA-LNP solution in a freeze dryer, respectively; (2) freeze-drying the saRNA sample, mRNA sample and mRNA-LNP sample of step (1) respectively under conditions comprising setting a freeze-drying time, setting an initial sample temperature and setting a freeze-drying temperature to form freeze-dried powder, and reconstituting with a small amount of enzyme-free water; (3) detecting the sample integrity with an analytical system to evaluate the influence of concentration on the integrity of saRNA, mRNA and mRNA-LNP, wherein, before the detection of the mRNA-LNP sample, a surfactant is used to demulsify to release the mRNA from the nanoparticles; The concentration method according to claim 1, wherein in step (2), the freeze-drying time is set to 22-36 h.

2. The concentration method according to claim 1, characterized in that, In step (2), the set concentration of saRNA, mRNA and mRNA-LNP is 5-50 μg / mL.

3. The concentration method of claim 1, wherein, In step (2), the initial sample temperature is -80℃ to 25℃.

4. The concentration method of claim 1, wherein, In step (2), the initial sample temperature is -80℃ or -30℃.

5. The concentration method of claim 1, wherein, In step (2), the freeze-drying temperature is -45℃ to -25℃.

6. The concentration method of claim 1, wherein, In step (1) and steps (1)-(3), the sample temperature is kept below 4℃.

7. The concentration method of claim 1, wherein, In step (3), the surfactant is a Triton-100 solution or an ammonium acetate isopropyl alcohol solution.

8. The concentration method of claim 1, wherein, In step (3), the demulsification method is as follows: 100 μL of the mRNA-LNP sample is precisely transferred into a 1.5 mL centrifuge tube, 900 μL of an ammonium acetate isopropyl alcohol solution with a concentration of 60 mM is added, and the sample is vortexed and oscillated at a centrifugal force of 14000 g for 5 min, and the supernatant is removed; 1 mL of the ammonium acetate isopropyl alcohol solution is added again, and the above steps are repeated once, and finally the sample is rinsed with a 70% ethanol solution, and the above steps are repeated. The obtained precipitate is reconstituted with enzyme-free water and detected.

9. The concentration method of claim 1, wherein, In step (3), the preparation method of the ammonium acetate isopropyl alcohol solution with a concentration of 60 mM is as follows: 0.185 g of ammonium acetate is weighed, 40 mL of isopropyl alcohol is added to dissolve, and the mixture is uniformly mixed to obtain the solution.

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