Oligonucleotide analysis method

Abstract

This oligonucleotide analysis method includes: an application step for applying a matrix and a sample containing an oligonucleotide onto a plate; an acquisition step for irradiating the sample applied to the plate with laser light to perform mass spectrometry and acquiring a mass spectrum in a MALDI mass spectrometer; and a determination step for determining that the sample may contain an impurity if a peak other than a peak derived from the oligonucleotide is observed in the acquired mass spectrum. The intensity of the laser light is an intensity at which it is possible to suppress an in-source decomposition of the oligonucleotide, and the matrix includes an acetophenone-based compound.

Description

Oligonucleotide Analysis Methods

[0001] The present invention relates to a method for analyzing oligonucleotides.

[0002] In recent years, synthetic oligonucleotides have been increasingly used in medical applications. Examples include ribozymes, nucleic acid aptamers, antisense oligonucleotides, and RNA interference (RNAi), which are known as nucleic acid drugs. Synthetic oligonucleotides are primarily synthesized on solid supports by sequential reactions using phosphoramidite nucleic acid monomers.

[0003] JP 2016-057219 A

[0004] Although oligonucleotide synthesis methods have been optimized to a level where purification is unnecessary for use as PCR primers, etc., more advanced purification and analysis are required for pharmaceutical applications. Long-chain DNA, modified DNA, backbone variants, etc., in particular, are prone to unreacted coupling or side reactions during synthesis. Therefore, synthetic oligonucleotide reaction mixtures may contain mainly incomplete oligonucleotides or incomplete oligonucleotides. Furthermore, impurities may include incompletely or incorrectly deprotected fragments and stereoisomers with partial chirality.

[0005] For the analysis of such synthetic oligonucleotide reaction mixtures, high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) is mainly used (for example, Patent Document 1).

[0006] However, when analyzing using HPLC or other methods, separating each component takes time, leaving room for improvement in terms of throughput. Furthermore, because stereoisomers often contain minor components with similar properties to the main component, the separation mode must be changed depending on the type of impurity. Therefore, compared to general small molecule drugs, the workload for analysis is higher, which is also an issue.

[0007] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for analyzing oligonucleotides that can easily detect impurities.

[0008] As a result of extensive research, the present inventors have discovered that impurities contained in an oligonucleotide to be analyzed can be easily detected by performing MALDI mass spectrometry using a predetermined matrix and setting the laser intensity to a predetermined level, and have thus completed the present invention.

[0009] A first aspect of the present invention relates to a method for analyzing oligonucleotides, comprising: a coating step of coating a sample containing the oligonucleotide and a matrix onto a plate; an acquisition step of performing mass analysis by irradiating the sample coated on the plate with laser light in a MALDI mass spectrometer to obtain a mass spectrum; and a determination step of determining that the sample may contain impurities when a peak other than a peak derived from the oligonucleotide is observed in the obtained mass spectrum, wherein the intensity of the laser light is such that in-source decay of the oligonucleotide is suppressed, and the matrix contains an acetophenone-based compound.

[0010] According to the present invention, it is possible to provide a method for analyzing oligonucleotides that can easily detect impurities.

[0011] FIG. 1 shows a mass spectrum obtained by analyzing an oligonucleotide using a MALDI mass spectrometer. The horizontal axis shows the m / z value, and the vertical axis shows the detection intensity. FIG. 2 shows a mass spectrum obtained by analyzing an oligonucleotide using a MALDI mass spectrometer. The horizontal axis shows the m / z value, and the vertical axis shows the detection intensity. FIG. 3 shows a mass spectrum obtained by analyzing an oligonucleotide using a MALDI mass spectrometer. The horizontal axis shows the m / z value, and the vertical axis shows the detection intensity. FIG. 4 shows a mass spectrum obtained by analyzing an oligonucleotide using a MALDI mass spectrometer. The horizontal axis shows the m / z value, and the vertical axis shows the detection intensity. FIG. 5 shows a mass spectrum obtained by analyzing an oligonucleotide using a MALDI mass spectrometer. The horizontal axis shows the m / z value, and the vertical axis shows the detection intensity.

[0012] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0013] <<Method for Analyzing Oligonucleotides>> A first aspect of the present invention is a method for analyzing oligonucleotides, comprising: a coating step of coating a sample containing the oligonucleotide and a matrix onto a plate; an acquisition step of performing mass analysis by irradiating the sample coated on the plate with laser light in a MALDI mass spectrometer to obtain a mass spectrum; and a determination step of determining that the sample may contain impurities when a peak other than a peak derived from the oligonucleotide is observed in the obtained mass spectrum, wherein the intensity of the laser light is such that in-source decay of the oligonucleotide is suppressed, and the matrix contains an acetophenone-based compound.

[0014] <Coating Step> In this step, a sample containing oligonucleotides and a matrix are coated onto a plate.

[0015] In this embodiment, the sample to be analyzed contains an oligonucleotide. "Oligonucleotide" refers to a nucleotide polymer in which 2 to 50 identical or different nucleotides are linked via phosphodiester bonds, phosphorodithioate bonds, or the like. The oligonucleotide typically comprises deoxyribonucleotides and ribonucleotides as building blocks. Examples of nucleobases contained in the deoxyribonucleotides and ribonucleotides include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). The nucleobase may be modified with a methyl group, a methoxyethyl group, or the like. Alternatively, the oligonucleotide may be a cross-linked nucleic acid known as a locked nucleic acid, or a morpholino nucleic acid. The oligonucleotide may typically be single-stranded or double-stranded. The oligonucleotide may also have another compound, such as a sugar such as GalNAc, bound to its terminus.

[0016] In this embodiment, the sample is not particularly limited as long as it contains the target oligonucleotide, and may be in the form of a solution or a solid. In one aspect of this embodiment, the sample is preferably an aqueous solution.

[0017] The term "matrix" refers to a compound that supports the ionization of the oligonucleotide in MALDI mass spectrometry. In this embodiment, the matrix includes an acetophenone-based compound. Here, "acetophenone-based compound" refers to a compound having the chemical structure of acetophenone, and examples thereof include acetophenone, hydroxyacetophenone (HAP), dihydroxyacetophenone (DHAP), and trihydroxyacetophenone (THAP). In this embodiment, the acetophenone-based compound preferably includes at least one selected from the group consisting of hydroxyacetophenone, dihydroxyacetophenone, and trihydroxyacetophenone. As long as the effects of the present invention are achieved, the matrix may also include compounds other than acetophenone-based compounds.

[0018] Conventionally, when oligonucleotides are subjected to MALDI mass spectrometry, the analysis has been carried out using a matrix (e.g., 3-HPA) that is prone to in-source decay. This is because sequence information of the oligonucleotide can be determined by mass spectrometry of the in-source decayed fragments (sometimes referred to as "ion series"). Therefore, the idea of ​​deliberately suppressing in-source decay to perform MALDI mass spectrometry of oligonucleotides has never been considered. The present inventor, unconstrained by conventional common technical knowledge, performed MALDI mass spectrometry of oligonucleotides using a matrix containing an acetophenone compound and a laser beam of a predetermined intensity, as described below. He found that the appearance of "peaks derived from in-source decayed fragments" was suppressed, enabling the easy detection of impurities contained in the oligonucleotide to be analyzed, and thus completed the present invention.

[0019] In one aspect of this embodiment, the matrix preferably further contains ammonium citrate, which makes it possible to suppress salt addition such as sodium addition.

[0020] The method for applying the sample and the matrix to the plate is not particularly limited, and any known method can be used, such as mixing an aqueous solution of the sample and a solution of the matrix, dropping the mixture onto the plate, and then evaporating the solvent by natural drying or the like.

[0021] In one aspect of this embodiment, the coating step preferably includes coating a solution containing the sample and the matrix on the plate on which the matrix microcrystals have been coated. This improves the sensitivity and quantitation of mass spectrometry. Examples of methods for coating the matrix microcrystals on the plate include the following: First, a solution in which the matrix is ​​dissolved in an organic solvent (e.g., methanol, ethanol, etc.) is dripped onto the plate. Then, the organic solvent is evaporated by natural drying to produce the matrix microcrystals on the plate. Instead of dripping the matrix solution onto the plate, the solution may be sprayed onto the plate by a sprayer. The sprayer is not particularly limited as long as it can spray the matrix uniformly, and may be an airbrush or an atomizer. Instead of the sprayer, a dedicated device for spraying the matrix (e.g., iMLayer AERO, manufactured by Shimadzu Corporation) or a matrix vapor deposition device (e.g., iMLayer, manufactured by Shimadzu Corporation) may be used.

[0022] <Acquisition Step> In this step, mass analysis is performed by irradiating the sample applied to the plate with laser light in a MALDI mass spectrometer, and a mass spectrum is acquired.

[0023] In this embodiment, the MALDI mass spectrometer is not particularly limited as long as it is an apparatus capable of performing mass spectrometry by matrix-assisted laser desorption / ionization. An example of the MALDI mass spectrometer is the benchtop MALDI-TOF MS "MALDI-8030" (manufactured by Shimadzu Corporation).

[0024] In this embodiment, the laser light irradiated onto the sample is preferably ultraviolet laser light. The intensity of the laser light is an intensity capable of suppressing in-source decay of the oligonucleotide. Here, the term "intensity capable of suppressing in-source decay" does not only mean "intensity capable of completely preventing in-source decay," but also includes the concept of "intensity at which some in-source decay may occur." In one aspect of this embodiment, the intensity of the laser light is preferably an intensity capable of suppressing in-source decay of the oligonucleotide when a matrix containing an acetophenone compound is used. "In-source decay" refers to all fragmentation that occurs in the ionization chamber simultaneously with or immediately after ionization.

[0025] In one aspect of this embodiment, the intensity of the laser light is preferably slightly higher than the threshold at which the oligonucleotide ionizes. The "threshold at which the oligonucleotide ionizes" can also be understood as the intensity value at which the oligonucleotide ions are detected when the intensity of the laser light is gradually increased. Note that when the intensity of the laser light is equal to or higher than the threshold, the ions can be detected regardless of where the laser light is applied to the sample spot on the plate. In another aspect of this embodiment, the intensity of the laser light can also be understood as being slightly higher than the threshold at which the oligonucleotide ionizes when a matrix containing an acetophenone-based compound is used.

[0026] For example, when using a benchtop MALDI-TOF MS "MALDI-8030" (manufactured by Shimadzu Corporation) as the MALDI mass spectrometer, the "intensity slightly higher than the threshold for ionization of oligonucleotides" can be determined by the following procedure. First, determine the lowest laser power at which ions begin to be emitted, then increase the laser power to an extreme level to measure and search for a peak that appears to be generated by in-source decay. Finally, determine the threshold by reducing the laser power to a level that suppresses the in-source decay peak while still obtaining a stable mass spectrum. Alternatively, determine the threshold by gradually increasing the laser power from a low laser power at which ions are not detected and searching for the lowest laser power at which the S / N ratio of the oligonucleotide peak is stable. The set value for the laser light intensity can also be determined for other MALDI mass spectrometers using a similar method.

[0027] The position to be irradiated with the laser light is not particularly limited and may be anywhere within the sample spot on the plate.

[0028] The range of mass spectrum (m / z range) to be acquired is not particularly limited as long as the peak of the oligonucleotide and the peak of impurities can be detected, and may be, for example, 1 to 20,000. For the purpose of excluding peaks derived from the matrix, the range may be 500 to 20,000 or 1,000 to 20,000.

[0029] In this step, when a peak other than the peak attributable to the oligonucleotide is observed in the acquired mass spectrum, it is determined that the sample may contain an impurity. In one aspect of this embodiment, when a peak other than the peak attributable to the oligonucleotide is observed, it may be determined that the sample contains an impurity.

[0030] In this embodiment, "peaks derived from oligonucleotides" refer to peaks derived from the oligonucleotides that are the main target of measurement (the oligonucleotides that are the main product or the main component). Examples of peaks derived from the oligonucleotides include peaks of the full-length oligonucleotides and peaks of the oligonucleotides that have undergone in-source degradation. In one aspect of this embodiment, the peaks derived from the oligonucleotides preferably include peaks of the full-length oligonucleotides and peaks of the oligonucleotides that have undergone in-source degradation. The peaks of the full-length oligonucleotides can be identified from the molecular weight of the oligonucleotides. The peaks of the oligonucleotides that have undergone in-source degradation can be identified by MALDI mass spectrometry of a standard sample of the oligonucleotide (preferably with a purity of 90% or more) under conditions that allow in-source degradation to occur (for example, the conditions of the comparative example described below).

[0031] In this embodiment, the "peaks other than those derived from the oligonucleotide" are peaks derived from impurities. Therefore, when such peaks are observed, it can be determined that the sample may contain impurities. The impurities may be deletions or additions of the oligonucleotide.

[0032] Examples of the deletion product of the oligonucleotide include a deletion product in which 1 to 14 bases are deleted, and preferably a deletion product in which 1 to 9 bases are deleted. The deletion site of the deletion product is not particularly limited, but the deletion is usually at the 5'-end or 3'-end. In some cases, only the base portion of the nucleotide is deleted.

[0033] Examples of the adducts of the oligonucleotide include adducts in which a protecting group (e.g., a dimethoxytrityl group (DMTr group)) remains at the 5'-terminus, adducts in which an acetyl group used for capping remains at the 5'-terminus, and adducts in which 1 to 3 bases have been added, or impurities generated by the capping reaction acting on internal nucleotide residues.

[0034] <Other Steps> <Impurity Quantification Step> In one aspect of this embodiment, the method for analyzing the oligonucleotide preferably further comprises an impurity quantification step of quantifying the impurities based on the intensity of peaks other than the peak derived from the oligonucleotide. The method for quantifying the impurities is not particularly limited, and known methods can be used. Examples of such methods include the absolute calibration curve method, the internal standard method, and relative quantification relative to the main product.

[0035] The method for analyzing oligonucleotides according to this embodiment has been described above. In this analysis method, MALDI mass spectrometry is performed under conditions that suppress in-source decay, as compared to conventional analysis methods. Therefore, impurities contained in the oligonucleotide to be analyzed can be easily detected. The method for analyzing oligonucleotides according to this embodiment can also be used for quality control of nucleic acid drugs.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0037] <Experiment 1: Investigation of Matrix Components> The following experiment was carried out to investigate the influence of matrix components on the analysis of oligonucleotides by MALDI mass spectrometry. First, an aqueous solution of Mipomersen (concentration: 0.1 mg / mL) was prepared as a sample.

[0038] Next, the following two types of aqueous solutions were prepared as matrix solutions: (1) 3-HPA (3-hydroxypicolinic acid, final concentration: 40 mg / mL) and ACD (Ammonium Citrate Dibasic, final concentration: 40 mM) in a 50% aqueous solution of acetonitrile; and (2) THAP (trihydroxyacetophenone) (final concentration: 40 mg / mL) and ACD (final concentration: 40 mM) in a 50% aqueous solution of MeOH.

[0039] A mixture was obtained by mixing the sample and the matrix solution ((1) or (2)) at a volume ratio of 1:1. 1.5 μL of the mixture was dropped onto a MALDI plate and air-dried. The MALDI plate was then set in a tabletop MALDI-TOF MS instrument (product name: MALDI-8030, manufactured by Shimadzu Corporation), and MALDI mass spectrometry was performed under the following measurement conditions. The laser light intensity described below is an intensity that can suppress in-source decay of oligonucleotides when a matrix containing THAP (an acetophenone-based compound) is used. Measurement conditions Ion mode: negative ion linear mode Laser light intensity: variable depending on the matrix, slightly exceeding the threshold value Delayed extraction: optimized to 7000 Da

[0040] The mass spectrum obtained by MALDI mass spectrometry is shown in Figure 1. The range indicated by "x50" in Figure 1 shows peaks with peak intensities expanded 50 times. From the results in Figure 1, when 3-HPA was used, in-source decay (ISD) peaks were observed in the range where the peak intensities were expanded 50 times. On the other hand, when THAP was used, almost no peaks were observed in this range. From these results, it was found that when an acetophenone compound such as THAP is used as a matrix, mass spectrometry can be performed with in-source decay suppressed.

[0041] <Experiment 2: Mass spectrometry of oligonucleotides containing impurities> Based on the results of Experiment 1, the following experiment was performed to examine whether impurities contained in samples can be detected when THAP is used as a matrix. First, the following three types of aqueous solutions were prepared as samples. Here, "impurities" refers to a one-base deleted oligonucleotide (one base deleted on the 3'-end) and a three-base deleted oligonucleotide (three bases deleted on the 3'-end). (1) Mipomersen (concentration 0.1 mg / mL), impurities (each 10% by mass based on the Mipomersen contained in the sample). (2) Nusinersen (concentration 0.1 mg / mL), impurities (each 10% by mass based on the Nusinersen contained in the sample). (3) Inotersen (concentration 0.1 mg / mL), impurities (each 10% by mass based on the Inotersen contained in the sample).

[0042] Information on the oligonucleotides contained in the above samples is shown in Table 1.

[0043] The following aqueous solutions were prepared as matrix solutions: THAP (final concentration: 10 mg / mL), ACD (final concentration: 40 mM) in 70% MeOH aqueous solution.

[0044] A 0.2 μL solution of THAP in methanol (concentration: 10 mg / mL) was dropped onto a MALDI plate to form a membrane of microcrystals. A 1.5 μL mixture of the sample and the matrix solution (volume ratio: 1:1) was then dropped onto the MALDI plate on which the microcrystals had formed, and the plate was allowed to air-dry (coating step). The MALDI plate was then placed in a benchtop MALDI-TOF MS instrument (product name: MALDI-8030, manufactured by Shimadzu Corporation), and MALDI mass spectrometry was performed under the same measurement conditions as in Experiment 1, obtaining a mass spectrum (acquisition step). The results are shown in Figure 2.

[0045] From the results in Figure 2, peaks other than those derived from each oligonucleotide were observed for all of Mipomersen, Nusinersen, and Inotersen, and it was found that these peaks were all peaks of the one-base deleted (N-1) and three-base deleted (N-3) corresponding to each oligonucleotide (impurity peaks) (determination step).

[0046] <Experiment 3: Detectable Concentration of Impurities> The following experiment was conducted to investigate the maximum concentration of impurities that can be detected in a sample. First, the following aqueous solutions were prepared as samples. Here, "impurities" refers to oligonucleotides with one base deletion and three base deletions. Mipomersen (concentration 0.1 mg / mL), impurities (0 to 10% by mass based on the Mipomersen contained in the sample).

[0047] The following aqueous solutions were prepared as matrix solutions: THAP (final concentration: 10 mg / mL), ACD (final concentration: 40 mM) in 70% MeOH aqueous solution.

[0048] Using each of the prepared samples and matrix solutions, MALDI mass spectrometry was performed in the same manner as in Experiment 2, and mass spectra were obtained. The results are shown in Figures 3 and 4. The ranges marked "enlarged 10x" and "enlarged 20x" in Figures 3 and 4 indicate peaks with their peak intensities enlarged 10x or 20x. The results in Figures 3 and 4 indicated that impurities could be detected in the range of 0.5% to 10% by mass. Some peaks overlapped with those of in-source decay fragments, but their intensities were greater than those of the corresponding peaks in the mass spectrum when the impurity concentration was 0% by mass. This suggested that these peaks also contained peaks derived from impurities. In other words, comparing the mass spectrum obtained from a standard oligonucleotide (an oligonucleotide containing no impurities) with the mass spectrum obtained from the measurement sample suggested that it was possible to determine whether an impurity with the same molecular weight as the in-source decay fragment was present.

[0049] Experiment 4: Analysis of Crude Synthetic Oligonucleotides In Experiments 2 and 3, mass spectrometry was performed on samples containing known impurities. Similar mass spectrometry was performed on crude synthetic oligonucleotides to investigate what impurities would be detected. Nusinersen was first synthesized using a solid-phase synthesis system using a standard phosphoramidite method to obtain a crude product. An aqueous solution of the resulting crude product (concentration: 0.5 mg / mL) was prepared and used as a sample.

[0050] The following aqueous solutions were prepared as matrix solutions: THAP (final concentration: 10 mg / mL), ACD (final concentration: 40 mM) in 70% MeOH aqueous solution.

[0051] A 400 μL solution of THAP in methanol (concentration: 10 mg / mL) was sprayed onto a MALDI plate to form microcrystals. A 1.5 μL mixture of the sample and matrix solution (volume ratio: 1:1) was then dropped onto the MALDI plate on which the microcrystals had formed, and the plate was allowed to air dry (application step). The MALDI plate was then placed in a benchtop MALDI-TOF MS instrument (product name: MALDI-8030, manufactured by Shimadzu Corporation), and MALDI mass spectrometry was performed under the same measurement conditions as in Experiment 1, obtaining a mass spectrum (acquisition step). The results are shown in Figure 5.

[0052] In FIG. 5, the peak of full-length Nusinase ([M−H] － , [M-2H] ２－ In addition to the N-X series (N-1 to N-15) deletions at the 5' end, the N-X series (N-1 to N-15) was detected in the low molecular weight region. Furthermore, a group of deletions at the 3' end was also detected, albeit weakly. Peaks likely to represent impurities derived from capping and impurities with remaining protecting groups were also observed in the high molecular weight region. These results demonstrate that the method for analyzing oligonucleotides according to the present invention can easily detect impurities. Although the crude oligonucleotide synthesized in this study did not contain nucleotide adducts, it is known that adducts may also be produced depending on the synthesis conditions. Considering that N-1 deletions were detected at 0.5% by mass or more, it is believed that N+1 adducts can also be detected in a similar concentration range.

[0053] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0054] (Item 1) A method for analyzing oligonucleotides according to one embodiment includes: a coating step of coating a sample containing the oligonucleotide and a matrix onto a plate; an acquisition step of performing mass analysis by irradiating the sample coated on the plate with laser light in a MALDI mass spectrometer to obtain a mass spectrum; and a determination step of determining that the sample may contain impurities when peaks other than those attributable to the oligonucleotides are observed in the obtained mass spectrum, wherein the intensity of the laser light is sufficient to suppress in-source decay of the oligonucleotides, and the matrix contains an acetophenone-based compound. The method for analyzing oligonucleotides according to item 1 enables easy detection of impurities.

[0055] (Item 2) In the method for analyzing oligonucleotides according to item 1, the acetophenone-based compound includes at least one compound selected from the group consisting of hydroxyacetophenone, dihydroxyacetophenone, and trihydroxyacetophenone. According to the method for analyzing oligonucleotides according to item 2, in-source decay is further suppressed, thereby improving the detection sensitivity of impurities.

[0056] (Item 3) In the method for analyzing an oligonucleotide according to item 1 or 2, the impurity is a deletion or adduct of the oligonucleotide. According to the method for analyzing an oligonucleotide according to item 3, the analysis method is preferably used for detecting the deletion or adduct of the oligonucleotide.

[0057] (Item 4) In the method for analyzing an oligonucleotide according to any one of Items 1 to 3, the peaks derived from the oligonucleotide include a peak of the full-length oligonucleotide and a peak of the oligonucleotide that has undergone in-source degradation.

[0058] (Item 5) In the method for analyzing oligonucleotides according to any one of Items 1 to 4, the coating step includes coating a solution containing the sample and the matrix onto the plate on which microcrystals of the matrix have been coated. The method for analyzing oligonucleotides according to Item 5 improves the detection sensitivity and quantitation of impurities.

[0059] (Item 6) In the method for analyzing oligonucleotides according to any one of Items 1 to 5, the matrix further contains ammonium citrate. According to the method for analyzing oligonucleotides according to Item 6, addition of sodium ions to the oligonucleotides is suppressed, thereby improving detection accuracy.

[0060] (Item 7) In the method for analyzing oligonucleotides according to any one of Items 1 to 6, the impurities are quantified based on the intensity of peaks other than the peaks derived from the oligonucleotides. The method for analyzing oligonucleotides according to Item 7 enables the quantification of impurities.

[0061] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0062] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

Claims

1. 1. A method for analyzing oligonucleotides, comprising: a coating step of coating a sample containing the oligonucleotide and a matrix onto a plate; an acquisition step of performing mass analysis by irradiating the sample applied to the plate with laser light in a MALDI mass spectrometer to acquire a mass spectrum; a determining step of determining that the sample may contain impurities when a peak other than a peak derived from the oligonucleotide is observed in the acquired mass spectrum, the intensity of the laser light is such that in-source degradation of the oligonucleotide is suppressed; A method for analyzing oligonucleotides, wherein the matrix contains an acetophenone-based compound.

2. The intensity of the laser light is higher than the threshold for ionization of the oligonucleotide, 2. The method for analyzing oligonucleotides according to claim 1, wherein the threshold value is determined by first determining the lowest laser light intensity at which ions of the oligonucleotide begin to be emitted, then increasing the laser light intensity from the determined lowest laser light intensity and measuring to identify peaks resulting from in-source decay of the oligonucleotide, and finally reducing the laser light intensity to a level at which the mass spectrum can be stably obtained while suppressing the peaks resulting from the in-source decay.

3. The intensity of the laser light is higher than the threshold for ionizing the oligonucleotide, The method for analyzing oligonucleotides described in claim 1, wherein the threshold value is determined by gradually increasing the intensity of the laser light from a low intensity at which the ions of the oligonucleotide are not detected, and finding the lowest intensity of the laser light at which the S / N ratio of the peak derived from the oligonucleotide is stable.

4. The method for analyzing an oligonucleotide according to claim 1 , wherein the acetophenone-based compound includes at least one selected from the group consisting of hydroxyacetophenone, dihydroxyacetophenone, and trihydroxyacetophenone.

5. The method for analyzing an oligonucleotide according to claim 1 , wherein the impurity is a deletion or an adduct of the oligonucleotide.

6. The method for analyzing oligonucleotides according to claim 1 , wherein the peaks derived from the oligonucleotides include a peak of the full-length oligonucleotide and a peak of the oligonucleotides subjected to in-source degradation.

7. The method for analyzing oligonucleotides described in any one of claims 1 to 3, wherein the coating step includes coating a solution containing the sample and the matrix onto the plate on which microcrystals of the matrix have been coated.

8. The method for analyzing oligonucleotides according to claim 1 , wherein the matrix further comprises ammonium citrate.

9. The method for analyzing an oligonucleotide according to any one of claims 1 to 3, further comprising an impurity quantification step of quantifying the impurities based on the intensity of peaks other than the peak derived from the oligonucleotide.