Method for analyzing morpholine ring-containing oligonucleotide

Abstract

This method for analyzing a morpholine ring-containing oligonucleotide is configured such that a morpholine ring-containing oligonucleotide contained in a sample is analyzed with a matrix-assisted laser desorption / ionization mass spectrometer using a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2',4',6'-trihydroxyacetophenone (THAP). This method uses a matrix suitable for the ionization of molecules of a morpholine ring-containing oligonucleotide by the MALDI method, and it is thus possible to more reliably measure the molecular weight of a morpholine ring-containing oligonucleotide in mass spectrometry using the MALDI method.

Description

Method for analyzing morpholine ring-containing oligonucleotides

[0001] The present invention relates to a method for analyzing an oligonucleotide having a morpholine ring.

[0002] Oligonucleotides called morpholino nucleic acids, which have a morpholine ring instead of the sugar moiety found in DNA's deoxyribose or RNA's ribose (hereafter referred to as morpholine ring-containing oligonucleotides), are used in nucleic acid medicines. Morpholino nucleic acids are artificially synthesized nucleic acids and belong to the antisense nucleic acid category of nucleic acid medicines. Morpholino nucleic acids have attracted attention as third-generation antisense nucleic acids that overcome the challenges of conventional antisense nucleic acids (specificity, stability, cytotoxicity, etc.).

[0003] Whether or not the target morpholino nucleic acid has been synthesized as designed is generally confirmed by measuring the molecular weight of the morpholino nucleic acid contained in the synthesis reaction product by gel electrophoresis or by measuring it using LC-MS, which is a combination of a liquid chromatograph (LC) and a mass spectrometer (MS).

[0004] In gel electrophoresis, multiple components contained in a sample are separated by moving them through a gel under the action of an electric field. The mobility (migration distance) of each component varies depending on the molecular size, charge, and structure, so the molecular weight of each component is determined from the migration distance. In gel electrophoresis, to accurately separate the multiple components contained in a sample, it is necessary to increase the migration distance of each component and increase the difference in migration distance between components, which is time-consuming. Another problem is that because the molecular weight of each component is determined from the migration distance, only approximate values ​​of the molecular weight can be determined.

[0005] On the other hand, LC-MS involves separating multiple components in a sample using LC and measuring the m / z values ​​of the multivalent ions generated from each component using MS. Specifically, in MS, each component is converted into charged droplets in an electric field, for example, using electrospray ionization (ESI), generating multiple types of multivalent ions from the charged droplets, and measuring the m / z values ​​of these multivalent ions. The molecular weight of each component is then calculated from the m / z values ​​of the multivalent ions derived from each component using deconvolution processing. In LC-MS, target components in a sample are separated and purified using LC, and then their m / z values ​​are measured using MS, thereby obtaining accurate molecular weight values ​​for the target components. However, this requires accurate separation of the target component from other components using LC, which requires time and effort to determine conditions such as the type of column and mobile phase used in LC. Furthermore, ESI generally detects component molecules in a sample as multiple types of multivalent ions, resulting in complex peaks in the mass spectrum, necessitating software (analysis tools) capable of high-precision peak analysis.

[0006] In recent years, attempts have been made to analyze nucleic acids such as DNA and RNA using a mass spectrometer (MALDI-MS) that utilizes the matrix-assisted laser desorption / ionization (MALDI) method (Patent Document 1). In the MALDI method, a sample for analysis (sample / matrix mixed crystal) is prepared by mixing the sample with an ionization aid called a matrix, and then laser light is irradiated onto the sample to rapidly vaporize and ionize the component molecules in the sample for analysis. In MALDI-MS, the component molecules are mainly divided into [M+H] +These compounds are detected as singly charged ions such as . This simplifies the mass spectrum, making it easy to select peaks from the mass spectrum that correspond to the target component. Furthermore, the molecular weight of the target component can be determined directly from the m / z value of the peak (without deconvolution). Furthermore, by optimizing the matrix type and sample preparation method for the target component, MALDI-MS can increase the intensity of the ion peak of the target component molecule and suppress the ion peaks of contaminants. Therefore, MALDI-MS can sometimes provide information about the molecular weight of the target component in a sample without separating the target component using LC in LC-MS.

[0007] International Publication No. 2023 / 234374

[0008] As described above, MALDI-MS can detect ion peaks of various target molecules with high sensitivity. Therefore, the ability to measure the molecular weight of morpholino nucleic acids and other morpholine-containing oligonucleotides using MALDI-MS is significant for the development of new nucleic acid medicines.

[0009] The problem to be solved by the present invention is to enable the molecular weight of a morpholine ring-containing oligonucleotide to be measured more reliably using a mass spectrometer utilizing the MALDI method (MALDI-MS).

[0010] The method for analyzing morpholine ring-containing oligonucleotides according to the present invention, which has been developed to solve the above problems, uses a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2',4',6'-trihydroxyacetophenone (THAP) and analyzes morpholine ring-containing oligonucleotides contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer.

[0011] According to the method for analyzing morpholine ring-containing oligonucleotides of the present invention, a matrix suitable for ionizing morpholine ring-containing oligonucleotide molecules by the MALDI method is used, and therefore the molecular weight of the morpholine ring-containing oligonucleotide can be accurately measured by mass spectrometry using the MALDI method.

[0012] FIG. 1 shows a mass spectrum of viltolarsen in Example 1. FIG. 2 shows a mass spectrum of another viltolarsen in Example 1. FIG. 3 shows a mass spectrum of viltolarsen and a photograph of sample / matrix mixed crystals on a sample plate in Example 2. FIG. 4 shows a mass spectrum of viltolarsen in Example 3. FIG. 5 shows a mass spectrum of another viltolarsen in Example 3. FIG. 6 shows a mass spectrum of casimersen in Example 4. FIG. 7 shows a mass spectrum of casimersen and a photograph of sample / matrix mixed crystals on a sample plate in Example 5. FIG. 8 shows a mass spectrum of golodirsen and a photograph of sample / matrix mixed crystals in Example 6. FIG. 9 shows a mass spectrum of viltolarsen in Example 7. FIG. 10 shows a mass spectrum of viltolarsen in Example 8. FIG. 11 shows a mass spectrum of casimersen in Example 9.

[0013] In the method for analyzing morpholine ring-containing oligonucleotides according to the present invention, a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2',4',6'-trihydroxyacetophenone (THAP) is used, and the morpholine ring-containing oligonucleotides contained in a sample are analyzed by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS).

[0014] To ionize target component molecules using the MALDI method, it is important to use an appropriate matrix depending on the characteristics of the target component and the analytical purpose. However, because the ionization mechanism by the MALDI method has not yet been fully elucidated, it is difficult to theoretically identify a matrix suitable for each substance, especially for substances that have not previously been measured using MALDI-MS, such as morpholine ring-containing oligonucleotides. In contrast, the present invention was made based on the discovery that morpholine ring-containing oligonucleotide molecules can be more reliably ionized by the MALDI method by using a mixture of 3-HPA and THAP, which have previously been known as MALDI matrices, rather than using them alone. According to the present invention, the molecular weight of morpholine ring-containing oligonucleotides can be more reliably measured by MALDI-MS.

[0015] Hereinafter, an embodiment of the analytical method according to the present invention will be specifically described. In the analytical method, an analytical sample (sample / matrix mixture) containing the mixed matrix and the sample is prepared, the analytical sample is subjected to mass analysis using MALDI-MS, and from the obtained mass spectrum, ions (specifically, [M+H]) that give molecular weight information of the morpholine ring-containing oligonucleotide contained in the sample are extracted. + , [MH] - (where M is a molecule corresponding to the morpholine ring-containing oligonucleotide, and H is a hydrogen atom)) peaks are detected. The method for analyzing morpholine ring-containing oligonucleotides according to this embodiment can be used, for example, when synthesizing a morpholine ring-containing oligonucleotide, to confirm whether the target morpholine ring-containing oligonucleotide is contained in a synthesis reaction product. Alternatively, it can be used to confirm whether the synthesis reaction product contains impurities other than the target morpholine ring-containing oligonucleotide (for example, impurities obtained by removing one or more bases from the target morpholine ring-containing oligonucleotide).

[0016] (Morpholine Ring-Containing Oligonucleotide) The morpholine ring-containing oligonucleotide contained in the sample is not particularly limited as long as it contains a morpholine ring as a constituent unit. For example, an oligonucleotide having a structure represented by chemical formula (1) as a constituent unit can be mentioned.

[0017]

[0018] In chemical formula (1), "Base" represents a nucleic acid base. Examples of nucleic acid bases include adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), and modified bases thereof. The base length of the morpholine ring-containing oligonucleotide is not particularly limited and may be from several to several tens of bases (e.g., 20 to 30 bases).

[0019] The morpholine ring-containing oligonucleotide may have a hydroxy group (OH group) bound to the 5' carbon of the 5'-terminal nucleotide, as shown in chemical formula (2). The morpholine ring-containing oligonucleotide may also have a structure containing an alkylene glycol group or a polyalkylene glycol group bound to the 5' carbon of the 5'-terminal nucleotide. Hereinafter, in descriptions common to alkylene glycol groups and polyalkylene glycol groups, both will be referred to collectively as "(poly)alkylene glycol group." Examples of (poly)alkylene glycol groups include (poly)ethylene glycol groups and (poly)propylene glycol groups. For example, a structure containing an ethylene glycol group or a polyethylene glycol (PEG) group, as shown in chemical formula (3), may be bound. In chemical formula (3), n is an integer of 1 or greater, indicating that n ethylene oxide groups are repeatedly bound. The number of repeating units of the (poly)alkylene glycol group (the value of n in chemical formula (3)) is not particularly limited, but is preferably approximately 1 to 5, and more preferably 3. Examples of morpholino oligonucleotides containing a morpholino group include morpholino nucleic acids such as viltolarsen, eteplirsen, golodirsen, and casimersen, which are used as nucleic acid drugs.

[0020]

[0021]

[0022] (Mixed Matrix) The mixed matrix contains 3-hydroxypicolinic acid (3-HPA) and 2',4',6'-trihydroxyacetophenone (THAP). The volume ratio of 3-HPA to THAP in the mixed matrix (3-HPA:THAP) is not particularly limited. If it is known that a hydroxy group is bonded to the 5' carbon at the 5' end of the morpholine ring-containing oligonucleotide contained in the sample, the fragment ion peaks in the mass spectrum are suppressed and the [M+H] + or [MH] - The ratio of 3-HPA to THAP is preferably 5:1 to 2:1, since peaks of [M+H] are detected. + or [MH] - From the viewpoint of highly sensitive detection of the peak of [M+H], the ratio of 3-HPA to THAP is preferably 3:1 to 2:1, and particularly preferably 3:1. On the other hand, when it is known that a structure containing a (poly)alkylene glycol group as shown in chemical formula (3) is bound to the 5'-end of the morpholine ring-containing oligonucleotide contained in the sample, [M+H] + or [MH] - From the viewpoint of improving the detection sensitivity of the peak, the ratio of 3-HPA:THAP is preferably 3:1 to 1:5, more preferably 3:1 to 1:3, and particularly preferably 1:2 to 1:3.

[0023] (Preparation of analytical samples) Examples of methods for preparing analytical samples include the on-target method and the pre-mix method. The on-target method involves preparing a sample solution containing a sample and a matrix solution containing a mixed matrix, and then dropping the sample solution and the matrix solution onto the wells of a MALDI-MS sample plate and drying them. The pre-mix method involves mixing the sample solution and the matrix solution to prepare a sample / matrix mixed solution, and then dropping the sample / matrix mixed solution onto the wells of the sample plate and drying them. By using the above method, sample / matrix mixed crystals are formed on the wells of the sample plate as analytical samples.

[0024] Since morpholine ring-containing oligonucleotides are water-soluble, it is preferable to use water as the solvent for the sample solution. In terms of the solubility of 3-HPA and THAP, it is preferable to use water or a mixture of water and an organic solvent (e.g., acetonitrile) as the solvent for the matrix solution. In terms of ease of evaporation, it is particularly preferable to use a mixture of water and an organic solvent.

[0025] The matrix solution may further contain a matrix additive, such as diammonium hydrogen citrate (ACD). There are several types of ammonium salts of citric acid, depending on the number of ammonium ions bound to the citrate ion. However, it is preferable to use a salt in which two ammonium ions are bound to one citrate ion as the matrix additive.

[0026] The concentration of the matrix additive in the matrix solution is preferably 10 mM or more and 100 mM or less, more preferably 30 mM or more and 90 mM or less. This allows the [M+H] of the morpholine ring-containing oligonucleotide to be determined from the mass spectrum obtained by MALDI-MS measurement. + or [MH] - The peak can be detected with high sensitivity.

[0027] Alternatively, analytical samples can be prepared by forming a thin film of THAP matrix on the wells of a sample plate and then dropping a sample solution and a matrix solution onto the thin film. Alternatively, analytical samples can be prepared by forming a thin film on the wells and then dropping a sample / matrix mixed solution onto the thin film. In these methods, a sample / matrix mixed crystal, which is the analytical sample, is formed on the thin film formed on the wells of the sample plate.

[0028] When the matrix contains THAP, simply dropping the sample solution and matrix solution, or the sample / matrix mixed solution, onto the wells of a sample plate can result in the formation of sample / matrix mixed crystals locally on the wells. Even if the sample / matrix mixed crystals form over the entire well, there may be areas (called sweet spots) where the sample is abundant and suitable for detecting peaks derived from the sample. In this case, the ionization efficiency of the sample component molecules varies depending on the position on the well of the sample plate where the laser light is irradiated.

[0029] In contrast, forming a thin film of THAP on the wells of a sample plate allows for the formation of relatively uniform sample / matrix mixed crystals over the entire well. It has been confirmed that the formation of a relatively uniform sample / matrix mixed crystal over the wells is achieved by forming a thin film of THAP, not only when the matrix contained in the sample / matrix mixed crystals is a mixed matrix consisting of 3-HPA and THAP, but also when the matrix is ​​THAP alone.

[0030] A thin film of THAP can be formed, for example, by dropping a thin film-forming solution containing THAP onto the wells of a sample plate and drying it. Because the solvent evaporates easily by natural drying, it is preferable to use an organic solvent alone or a solvent containing a high proportion of organic solvent (e.g., 50% or more) as the solvent for the thin film-forming solution containing THAP. Among these, 100% acetonitrile is preferred as the solvent for the thin film-forming solution in terms of THAP solubility and ease of solvent evaporation. To ensure the reliable formation of a thin film of THAP on the wells and to form uniform sample / matrix mixed crystals, the concentration of THAP contained in the thin film-forming solution is preferably 4 mg / mL or more but not more than the saturation concentration. The saturation concentration is the maximum concentration at which THAP dissolves in the solvent at room temperature (25°C), for example. The concentration of THAP contained in the thin film-forming solution is determined by the [M+H] of the morpholine ring-containing oligonucleotide. + or [MH] -The concentration is preferably 40 to 100 mg / mL, since this allows the peak to be detected with higher sensitivity.

[0031] (Mass Spectrometer) A MALDI-MS can have any configuration as long as it ionizes a sample using the MALDI method. For example, a MALDI-MS can be a MALDI-ITMS, which combines a MALDI ion source with an ion trap mass spectrometer (ITMS). An ITMS is a mass spectrometer with an ion trap for capturing ions generated in an ion source. An ITMS can also be a mass spectrometer that ejects ions trapped in the ion trap in ascending order of mass-to-charge ratio (m / z) using the mass separation function of the ion trap itself and detects the ions with a detector located outside the ion trap. Alternatively, an ITMS can be a mass spectrometer that simultaneously ejects ions trapped in the ion trap from the ion trap, separates the ejected ions according to their mass-to-charge ratio (m / z) using a mass separator (e.g., a time-of-flight mass separator) located outside the ion trap, and detects the ions with a detector located outside the ion trap. The radio frequency voltage applied to the ion trap to capture ions can be a sinusoidal or rectangular wave voltage. An ITMS that traps ions using a square-wave voltage is called a digital ion trap mass spectrometer (DITMS). Alternatively, a MALDI-MS may be a MALDI-TOFMS, which combines a MALDI ion source with a time-of-flight mass spectrometer (TOFMS).

[0032] MALDI-MS systems are typically equipped with a raster scan measurement function. In raster scan measurements, mass spectral data are acquired by irradiating multiple measurement points across the entire well with a laser a predetermined number of times in a predetermined order. The final mass spectral data is then obtained by integrating all the acquired mass spectral data. Therefore, even if sample / matrix mixed crystals are formed locally on the well of a sample plate or if a sweet spot is formed in the sample / matrix mixed crystals that are formed across the entire well, raster scan measurements can still obtain mass spectral data that include peaks derived from the components in the sample. This allows for the acquisition of objective (unbiased) mass spectral data, rather than arbitrary ones. Furthermore, raster scan measurements allow for the acquisition of reproducible mass spectral data that are not affected by the skill level of the operator.

[0033] The analytical method for morpholine ring-containing oligonucleotides according to this embodiment will be explained below with reference to examples, but these are merely examples and the present invention is not limited thereto.

[0034] [1] Preparation of sample solution A 20 pmol / μL aqueous solution of viltolarsen (MedChemExpress, 5'-CCTCCGGTTCTGAAGGTGTTC-3': SEQ ID NO: 1, 21 bases long, MW 6924.82) was prepared as a sample solution. The chemical structural formula of viltolarsen is shown in chemical formula (4). In the formula, B(n) indicates the nth base from the 5'-end (B(21) indicates the 21st base). A hydroxyl group (OH group) is bonded to the 5'-position carbon of the 5'-terminal nucleotide.

[0035]

[0036] [2] Preparation of Matrix Solutions. Diammonium hydrogen citrate (ACD) was dissolved in 50% acetonitrile (ACN) aqueous solution to a concentration of 70 mM to prepare an ACD solution. 3-hydroxypicolinic acid (3-HPA), 2,4-dihydroxyacetophenone (DHAP), 2',4',6'-trihydroxyacetophenone (THAP) monohydrate, or 6-aza-2-thiothymine (ATT) was dissolved in the ACD solution to a concentration of 40 mg / mL to prepare four matrix ACD solutions (3-HPA solution, DHAP solution, THAP solution, and ATT solution). The 3-HPA solution and the DHAP solution were mixed at a 1:1 (v / v) ratio to prepare a mixed matrix ACD solution containing 3-HPA and DHAP (3-HPA / DHAP (1:1) solution). Four mixed-matrix ACD solutions containing 3-HPA and THAP were prepared by mixing 3-HPA and THAP solutions at 1:1, 1:3, 3:1, or 5:1 (v / v) ratios: 3-HPA / THAP (1:1), 3-HPA / THAP (1:3), 3-HPA / THAP (3:1), and 3-HPA / THAP (5:1). A mixed-matrix ACD solution containing DHAP and THAP (DHAP / THAP (30:1)) was prepared by mixing DHAP and THAP solutions at 30:1 (v / v).

[0037] In the following description, both the single-matrix ACD solution and the mixed-matrix ACD solution are referred to as matrix solutions. In Example 1, 10 matrix solutions were prepared: 3-HPA solution, DHAP solution, THAP solution, ATT solution, 3-HPA / DHAP (1:1) solution, 3-HPA / THAP (1:1) solution, 3-HPA / THAP (1:3) solution, 3-HPA / THAP (3:1) solution, 3-HPA / THAP (5:1) solution, and DHAP / THAP (30:1) solution.

[0038] [3] Preparation of analytical samples: The sample solution prepared in [1] was mixed with one of the 10 matrix solutions prepared in [2] at a 1:1 (v / v) ratio to prepare a sample / matrix mixed solution. 1 μL of the sample / matrix mixed solution was dropped onto a well of a sample plate (SUS plate) and dried to prepare a sample / matrix mixed crystal for analysis.

[0039] [4] Mass spectrometry The sample plate carrying the sample / matrix mixed crystal was subjected to MALDI-DITMS (Shimadzu Corporation, MALDImini TM -1) and measurements were performed in positive mode. Mass spectrum data was obtained by raster scan measurement to enable as objective an evaluation as possible of conditions such as the matrix. The raster scan measurement conditions were 25 measurement points, with each measurement point irradiated with the laser four times. The laser power was set to an optimum value depending on the sample being analyzed.

[0040] Figure 1 shows the mass spectra of viltolarsen in nine matrices (3-HPA, DHAP, THAP, ATT, 3-HPA / DHAP (1:1), 3-HPA / THAP (1:1), 3-HPA / THAP (1:3), 3-HPA / THAP (3:1), and DHAP / THAP (30:1)). The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z) (m / z 2000 to m / z 18000), and the vertical axis represents the relative intensity (%int) (the same applies to the following graphs). The detected [M+H] + The peak intensities (mV) of the compounds are shown on the mass spectrum.

[0041] As shown in Figure 1, the mass spectrum data showed that the [M+H] of viltolarsen was + The peaks of [M+H] were detected, but the highest peak intensity was observed when the 3-HPA / THAP (3:1) solution was used. +When the matrix solutions of DHAP, THAP, 3-HPA / DHAP (1:1), 3-HPA / THAP (1:1), 3-HPA / THAP (1:3), and DHAP / THAP (30:1) were used, the [M+H] peaks in the mass spectrum were + On the lower mass-to-charge side of the peak of [M+H] + In contrast, when 3-HPA, ATT, and 3-HPA / THAP (3:1) matrix solutions were used, the peaks derived from the fragment ions were suppressed in the mass spectrum.

[0042] Next, [M+H] with the highest peak intensity + The mass spectra obtained using a 3-HPA / THAP (3:1) solution, in which peaks derived from fragment ions were detected and peaks derived from fragment ions were suppressed, were compared with those obtained using a 3-HPA / THAP (5:1) solution containing a mixed matrix with a higher ratio of 3-HPA than the 3-HPA / THAP (3:1) solution. Figure 2 shows the mass spectra of viltolarsen obtained using a 3-HPA / THAP (3:1) solution and a 3-HPA / THAP (5:1) solution. Note that Figures 1 and 2 were measured on different days. The mass spectrum in Figure 2 shows the [M+H] peaks in raster scan measurements. + In raster scan measurements, the peak detection status is shown in parentheses. In raster scan measurements, the peak detection status is [M+H] at a sensitivity where the S / N (signal / noise ratio) is greater than 2 (S / N>2). + The peak detection status is expressed as a numerical value [NS / TNS], where NS is the number of measurement points where the peak was detected and TNS is the total number of measurement points (25 in this example). A larger NS / TNS value means that a uniform sample / matrix mixed crystal was formed over the entire well.

[0043] As shown in Figure 2, the [M+H] peak of viltolarsen was observed with a higher intensity when using the 3-HPA / THAP (3:1) solution than when using the 3-HPA / THAP (5:1) solution. +Peaks derived from fragment ions were detected. Furthermore, peaks derived from fragment ions were suppressed when both the 3-HPA / THAP (3:1) and 3-HPA / THAP (5:1) solutions were used. However, the peak detection status values ​​shown in Figure 2 confirmed the problem of uneven or non-uniform formation of sample / matrix mixed crystals on the wells when both the 3-HPA / THAP (3:1) and 3-HPA / THAP (5:1) solutions were used.

[0044] Although there are problems as described above, the 3-HPA / THAP (3:1) solution or the 3-HPA / THAP (5:1) solution is effective in obtaining the [M+H] of viltolarsen, a morpholino nucleic acid with an OH group attached to the 5' carbon atom at the 5' end, from the mass spectrum. + It was found that the 3-HPA / THAP (3:1) solution was a suitable matrix solution for detecting the peaks of β-HPA and β-HPA.

[0045] [1] Preparation of sample solution As in Example 1, an aqueous solution containing 20 pmol / μL of viltolarsen was prepared as a sample solution.

[0046] [2] Preparation of matrix solution A 3-HPA / THAP (3:1) solution was prepared using the same procedure as in Example 1. In addition, two types of thin film solutions (3-HPA-2 solution and THAP-2 solution) were prepared by mixing 3-HPA or THAP with 100% ACN to a concentration of 40 mg / mL.

[0047] [3] Preparation of analytical samples: The sample solution prepared in [1] and the matrix solution prepared in [2] were mixed at a 1:1 (v / v) ratio to prepare a sample / matrix mixed solution. 1 μL of this sample / matrix mixed solution was dropped onto a well of a sample plate (SUS plate) and dried to prepare a sample / matrix mixed crystal, which was the analytical sample. Alternatively, 0.5 μL of the thin film solution (3-HPA-2 solution or THAP-2 solution) prepared in [2] was dropped onto a well of a sample plate (SUS plate) and dried to form a thin matrix film. Then, 1 μL of the sample / matrix mixed solution was dropped onto the thin film and dried to prepare a sample / matrix mixed crystal.

[0048] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 1, in the same mode and under the same conditions.

[0049] Figure 3 shows the mass spectrum of viltolarsen when using a 3-HPA / THAP (3:1) solution and a photograph of the sample / matrix mixed crystals on a sample plate, arranged vertically. Figure 3 (a) shows the case where no thin film was formed during preparation of the analytical sample, (b) shows the case where a thin film of 3-HPA was formed, and (c) shows the case where a thin film of THAP was formed. The numbers next to the photographs indicate the [M+H] values ​​in the raster scan measurement. + The peak detection status (NS / TNS) is shown.

[0050] In Figure 3, when the analytical sample was prepared without forming a thin film (Figure 3(a)), [M+H] + Although a peak was detected, the NS / TNS ratio was 18 / 25, suggesting that the sample / matrix mixed crystals were not uniformly formed on the wells. In contrast, when a thin film of THAP was formed on the wells of the sample plate before preparing the analytical sample (Figure 3(c)), it was thought that uniform sample / matrix mixed crystals were formed on the entire wells. As a result, [M+H] peaks were detected with good sensitivity at all measurement points. + On the other hand, when the analytical sample was prepared by forming a thin film of 3-HPA (Figure 3(b)), the peak of [M+H] was detected. + No peak was detected.

[0051] From the above, when a sample / matrix mixed solution was prepared using a 3-HPA / THAP (3:1) solution and then dropped onto the THAP thin film to prepare an analytical sample, a uniform sample / matrix mixed crystal was formed over the entire well, and the [M+H] of viltolarsen was + It was confirmed that the peaks could be detected with good reproducibility.

[0052] [1] Preparation of sample solution As in Example 1, an aqueous solution containing 20 pmol / μL of viltolarsen was prepared as a sample solution.

[0053] [2] Preparation of matrix solution A 3-HPA / THAP (3:1) solution was prepared using the same procedure as in Example 1. Eight types of thin film solutions were prepared by mixing THAP with 100% ACN to various concentrations (1, 4, 10, 20, 40, 70, 100 mg / mL, and the saturated concentration at room temperature (25°C)).

[0054] [3] Preparation of analytical samples Analytical samples were prepared in the same manner as in Example 2.

[0055] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 1, in the same mode and under the same conditions.

[0056] Figure 4(f) shows the mass spectrum of viltolarsen obtained when an analytical sample was prepared using a 3-HPA / THAP (3:1) solution without forming a thin film. Figures 4(a)-(e) and 5(a)-(d) show the mass spectra of viltolarsen obtained when a thin film of THAP was formed using a thin film solution containing THAP at various concentrations and an analytical sample was prepared on the thin film. Detected [M+H] + Peak intensity (mV) and [M+H] in raster scan measurement + The peak detection status (NS / TNS) is shown next to the mass spectrum. Note that Figures 4 and 5 show data measured on different days.

[0057] As shown in Figures 4(b) to 4(e) and Figures 5(a) to 5(d), when analytical samples were formed on thin films formed using thin film solutions with THAP concentrations of 4 mg / mL or higher, [M+H] was obtained with good sensitivity at all measurement points. + In contrast, when a thin film solution with a THAP concentration of 1 mg / mL was used (Fig. 4(a)) and when no thin film was formed (Fig. 4(f)), the peak of [M+H] was detected with good sensitivity. + The percentage of measurement points where peaks were detected was low.

[0058] In addition, in Figure 4, when the THAP concentration of the thin film solution was 40 mg / mL, the peak intensity was the highest [M+H] + In addition, in Figure 5, when the THAP concentration of the thin film solution was 40 to 100 mg / mL, the peak of [M+H] was detected with higher peak intensity. + The peak was detected.

[0059] From the above, when a thin film of THAP was formed using a thin film solution containing THAP at a concentration of 4 mg / mL or more but below the saturated concentration, the [M+H] peak of viltolarsen was observed uniformly and with sufficient peak intensity throughout the sample / matrix mixed crystal. + It was confirmed that the peak of [M+H] of viltolarsen was detected with higher peak intensity, especially when the thin film of THAP was formed using a thin film solution containing THAP at a concentration of 40 to 100 mg / mL. + It was confirmed that a peak was detected.

[0060] [1] Preparation of sample solution A 20 pmol / μL aqueous solution of the morpholino nucleic acid casimersen (MedChemExpress, 5'-CAATGCCATCCTGGAGTTCCTG-3': SEQ ID NO: 2, 22 bases long, MW 7584) was prepared as the sample solution. The chemical structural formula of casimersen is shown in chemical formula (5). Note that B(n) in the formula indicates the nth base from the 5'-end (B(22) indicates the 22nd base). A structure containing a PEG group linked to three ethylene oxides is bound to the 5'-position carbon of the 5'-terminal nucleotide.

[0061]

[0062] [2] Preparation of matrix solutions Four types of matrix solutions (3-HPA solution, DHAP solution, THAP solution, and 3-HPA / THAP (1:3) solution) were prepared using the same procedure as in Example 1. In addition, a matrix solution (DHAP / THAP (1:1) solution) was prepared by mixing DHAP solution and THAP solution at a 1:1 (v / v) ratio.

[0063] [3] Preparation of analytical samples Analytical samples were prepared in the same manner as in Example 1.

[0064] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 1, in the same mode and under the same conditions.

[0065] Figure 6 shows the mass spectra of casimersen when using various matrix solutions (3-HPA, DHAP, THAP, 3-HPA / THAP (1:3), DHAP / THAP (1:1)). The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z) (m / z 2000 to m / z 18000), and the vertical axis represents the relative intensity (%int) (the same applies to the following graphs). The detected [M+H] + The peak intensities (mV) are shown on the mass spectrum.

[0066] Regardless of the matrix solution used, the [M+H] of casimersen was observed in the mass spectrum. + Among them, the peak with the highest intensity was [M+H] when 3-HPA / THAP (1:3) solution was used. + The peak was detected.

[0067] From the above, the [M+H] of casimersen, a morpholino nucleic acid with a structure containing a PEG group attached to the 5' carbon of the 5' end, + The optimal matrix for detecting the peak was found to be 3-HPA / THAP (1:3).

[0068] [1] Preparation of sample solution As in Example 4, an aqueous solution containing 20 pmol / μL of casimersen was prepared as a sample solution.

[0069] [2] Preparation of matrix solutions Five types of matrix solutions (3-HPA solution, THAP solution, ATT solution, 3-HPA / THAP (1:3) solution, and 3-HPA / THAP (3:1) solution) were prepared using the same procedure as in Example 1. A matrix solution for thin film formation (THAP-2 solution) was prepared using the same procedure as in Example 2.

[0070] [3] Preparation of analytical samples Analytical samples were prepared in the same manner as in Example 2.

[0071] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 1, in the same mode and under the same conditions.

[0072] Figure 7 shows the mass spectra of casimersen when using various matrix solutions (THAP, 3-HPA / THAP (3:1), 3-HPA / THAP (1:3)) and photographs of the sample / matrix mixed crystals on a sample plate. The mass spectrum and photographs shown in the left half of Figure 7 are for a sample prepared for analysis without a thin film of THAP, while the mass spectrum and photographs shown in the right half are for a sample prepared for analysis with a thin film of THAP. Detected [M+H] + The peak intensity values ​​(mV) are shown on the mass spectrum.

[0073] When either matrix solution was used, the sample / matrix mixed crystals were more uniformly formed on the wells when a thin film was formed (right half) than when no thin film was formed (left half). Furthermore, when the THAP solution and the 3-HPA / THAP (1:3) solution were used as the matrix solution, the [M+H] concentration was higher when a thin film was formed (right half) than when no thin film was formed (left half). + Among them, when a thin film was formed and a 3-HPA / THAP (1:3) solution was used, the highest peak intensity was observed for [M+H]. + In Example 1, in which a morpholino nucleic acid having an OH group bound to the 5' carbon at the 5' end was analyzed, 3-HPA / THAP (3:1) was found to be the optimal matrix, but the results in this example were different.

[0074] Figure 8 shows the mass spectra of casimersen when using various matrix solutions (THAP, ATT, 3-HPA / THAP (1:3)). The mass spectrum on the left half of Figure 8 shows the case where the analytical sample was prepared without forming a thin film, while the mass spectrum on the right half shows the case where the analytical sample was prepared with forming a thin film of THAP. Detected [M+H] + The peak intensity (mV) of [M+H] is shown on the mass spectrum, and the [M+H] peak intensity (mV) of [M+H] is shown next to it. + 7 and 8 show the values ​​of the peak detection status NS / TNS. Note that Figures 7 and 8 show data measured on different days.

[0075] When any matrix solution was used, the [M+H] concentration was higher when a thin film was formed (right half) than when no thin film was formed (left half). + 7 and 8, when a thin film was formed using THAP and a THAP solution or a matrix solution containing THAP was used, the peak intensity of [M+H] was high. + Among them, the peak intensity of [M+H] was highest when the 3-HPA / THAP (1:3) solution was used. + The peak was detected.

[0076] From the above, the [M+H] of casimersen, a morpholino nucleic acid with a structure containing a PEG group attached to the 5' carbon of the 5' end, + The optimal matrix for detecting the [M+H] peak is 3-HPA / THAP (1:3). By preparing the analytical sample by dropping the sample / matrix mixed solution after forming a thin film of THAP, a more uniform and higher peak intensity of [M+H] can be obtained. + It was confirmed that a peak was detected.

[0077] [1] Preparation of Sample Solution A 20 pmol / μL aqueous solution of the morpholino nucleic acid golodirsen (MedChemExpress, 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3': SEQ ID NO: 3, 25 bases long, MW 8647) was prepared as the sample solution. The chemical structural formula of golodirsen is shown in Chemical Formula (6). Note that B(n) in the formula indicates the nth base from the 5'-end (B(25) indicates the 25th base). A structure containing a PEG group linked to three ethylene oxide groups is bound to the 5'-position carbon of the 5'-terminal nucleotide.

[0078]

[0079] [2] Preparation of matrix solutions Three types of matrix solutions (3-HPA solution, THAP solution, and 3-HPA / THAP (1:3) solution) were prepared using the same procedure as in Example 1. A thin film solution (THAP-2 solution) was prepared using the same procedure as in Example 2.

[0080] [3] Preparation of analytical samples Analytical samples were prepared in the same manner as in Example 2.

[0081] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 1, in the same mode and under the same conditions.

[0082] Figure 9 shows the mass spectra of golodirsen and photographs of the sample / matrix mixed crystals when using various matrix solutions (3-HPA, THAP, 3-HPA / THAP (1:3)). The mass spectrum and photograph on the left side of Figure 9 show the case where the analytical sample was prepared without forming a thin film, while the mass spectrum and photograph on the right side show the case where the analytical sample was prepared with forming a thin film of THAP. The horizontal axis of the mass spectrum shows the mass-to-charge ratio (m / z) (m / z 2000 to m / z 18000), and the vertical axis shows the relative intensity (%int). The detected [M+H] + The peak intensities (mV) are shown on the mass spectrum.

[0083] In Figure 9, when the 3-HPA / THAP (1:3) solution was used, the peak intensity of [M+H] was particularly high. +Among them, when a thin film was formed and a 3-HPA / THAP (1:3) solution was used, the peak of [M+H] was detected with the highest peak intensity. + The peak was detected.

[0084] From the above, the [M+H] of golodirsen, a morpholino nucleic acid with a structure containing a PEG group attached to the 5' carbon of the 5' end, + The optimal matrix for detecting the [M+H] peak is the mixed matrix 3-HPA / THAP (1:3). By preparing the analytical sample by dropping the sample / matrix mixed solution after forming a thin film of THAP, it is possible to obtain a more uniform and higher peak intensity [M+H] peak. + It was confirmed that a peak was detected.

[0085] The results of Examples 1 to 6 demonstrate that the use of the mixed matrix 3-HPA / THAP enables appropriate analysis of morpholine ring-containing oligonucleotides by MALDI-MS. Furthermore, for morpholine ring-containing oligonucleotides having a hydroxyl group bonded to the 5' carbon at the 5' end, a mixed matrix 3-HPA / THAP with a 3-HPA:THAP ratio of 5:1 to 2:1, particularly 3:1, is used. For morpholine ring-containing oligonucleotides having a structure containing a (poly)alkylene glycol group such as PEG bonded to the 5' carbon at the 5' end, a mixed matrix 3-HPA / THAP with a 3-HPA:THAP ratio of 3:1 to 1:5, particularly 1:3, is used. This suppresses the appearance of fragment ion peaks in the mass spectrum obtained by MALDI-ITMS measurement, while providing a [M+H] + Furthermore, for all morpholine ring-containing oligonucleotides, forming a thin film of THAP on the wells of a sample plate and then forming a sample / matrix mixed crystal on the thin film resulted in the formation of a relatively uniform sample / matrix mixed crystal over the entire well, resulting in a more highly sensitive and reproducible [M+H] peak in raster scan measurements. + It was found that the peaks could be detected.

[0086] [1] Preparation of sample solution As in Example 1, an aqueous solution containing 20 pmol / μL of viltolarsen was prepared as a sample solution.

[0087] [2] Preparation of matrix solutions Matrix solutions (THAP solution, 3-HPA / THAP (3:1) solution, 3-HPA / THAP (1:1) solution, and 3-HPA / THAP (1:3) solution) were prepared using the same procedure as in Example 1. THAP was mixed with 100% ACN to a concentration of 40 mg / mL to prepare a thin film solution (THAP-2 solution).

[0088] [3] Preparation of analytical samples: The sample solution prepared in [1] and the matrix solution prepared in [2] were mixed at a 1:1 (v / v) ratio to prepare a sample / matrix mixed solution. 1 μL of this sample / matrix mixed solution was dropped onto a well of a sample plate (SUS plate) and dried to prepare an analytical sample (sample / matrix mixed crystal). Alternatively, 0.3 μL of the thin film solution (THAP-2 solution) prepared in [2] was dropped onto a well of the sample plate and dried to form a thin matrix film. 1 μL of the sample / matrix mixed solution was then dropped onto the thin film and dried to prepare an analytical sample (sample / matrix mixed crystal).

[0089] [4] Mass spectrometry The sample plate carrying the analytical samples was inserted into a MALDI-TOFMS (Shimadzu Corporation, MALDI-8030), and raster scan measurement was performed in positive mode. The raster scan conditions were the same as in Example 1. Furthermore, the same laser power was used for all analytical samples.

[0090] Figure 10 shows the mass spectra of viltolarsen obtained using MALDI-TOFMS with various matrices (THAP, 3-HPA / THAP (3:1), 3-HPA / THAP (1:1), and 3-HPA / THAP (1:3)). The left half of Figure 10 shows the mass spectrum when the analytical sample was prepared without forming a thin film, and the right half shows the mass spectrum when the analytical sample was prepared with forming a thin film of THAP. The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z) (m / z 5000 to m / z 10000), and the vertical axis represents the relative intensity (%int). The detected [M+H] + The peak intensities (mV) are shown on the mass spectrum.

[0091] In the mass spectrum of Figure 10, when the 3-HPA / THAP (3:1) solution was used, the peak intensity was particularly high [M+H] + Among them, when a thin film was formed and a 3-HPA / THAP (3:1) solution was used, the peak of [M+H] was detected with the highest peak intensity. + The peak was detected.

[0092] From the above, the [M+H] of viltolarsen, a morpholino nucleic acid with a hydroxyl group attached to the 5' carbon at the 5' end, + The optimal matrix for detecting the [M+H] peak is a mixed matrix 3-HPA / THAP (3:1). By preparing the analytical sample by dropping the sample / matrix mixed solution after forming a thin film of THAP, the mass spectrum obtained by MALDI-TOFMS measurement shows a more uniform and higher peak intensity of [M+H]. + It was confirmed that a peak was detected.

[0093] [1] Preparation of sample solution As in Example 1, an aqueous solution containing 20 pmol / μL of viltolarsen was prepared as a sample solution.

[0094] [2] Preparation of matrix solutions Eleven types of matrix solutions (3-HPA / THAP (10:1) solution, 3-HPA / THAP (5:1) solution, 3-HPA / THAP (4:1) solution, 3-HPA / THAP (3:1) solution, 3-HPA / THAP (2:1) solution, 3-HPA / THAP (1:1) solution, 3-HPA / THAP (1:2) solution, 3-HPA / THAP (1:3) solution, 3-HPA / THAP (1:4) solution, 3-HPA / THAP (1:5) solution, and 3-HPA / THAP (1:10) solution) were prepared using the same procedure as in Example 1.

[0095] [3] Preparation of analytical sample: The sample solution prepared in [1] and the matrix solution prepared in [2] were mixed at a 1:1 (v / v) ratio to prepare a sample / matrix mixed solution. 1 μL of the sample / matrix mixed solution was then dropped onto a well of a sample plate (SUS plate) and dried to prepare an analytical sample (sample / matrix mixed crystal).

[0096] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 7 under the same mode and conditions.

[0097] Figure 11 shows the mass spectra of viltolarsen obtained by MALDI-TOFMS using various matrix solutions. The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z) (m / z 4000 to m / z 16000), and the vertical axis represents the relative intensity (%int). The detected [M+H] + The peak intensities (mV) are shown on the mass spectrum.

[0098] In the mass spectrum of Figure 11, when the 3-HPA / THAP (5:1) solution, the 3-HPA / THAP (4:1) solution, the 3-HPA / THAP (3:1) solution, or the 3-HPA / THAP (2:1) solution was used, the peak intensity of [M+H] was particularly high. + Among them, the peaks of [M+H] were detected with the highest peak intensity when using the 3-HPA / THAP (3:1) solution and the 3-HPA / THAP (2:1) solution. + The peak was detected.

[0099] From the above, the [M+H] of viltolarsen, a morpholino nucleic acid with an OH group attached to the 5' carbon at the 5' end, + The preferred mixed matrices for detecting the peaks of [M+H] were 3-HPA / THAP (3:1) or 3-HPA / THAP (2:1). By using these mixed matrices, the peaks of [M+H] were detected with higher intensity in the mass spectrum obtained by MALDI-TOFMS. + It was confirmed that a peak was detected.

[0100] [1] Preparation of sample solution As in Example 4, an aqueous solution containing 20 pmol / μL of casimersen was prepared as a sample solution.

[0101] [2] Preparation of matrix solutions Five types of matrix solutions (3-HPA / THAP (1:2) solution, 3-HPA / THAP (1:3) solution, 3-HPA / THAP (1:4) solution, 3-HPA / THAP (1:5) solution, and 3-HPA / THAP (1:10) solution) were prepared using the same procedure as in Example 1.

[0102] [3] Preparation of analytical samples Analytical samples were prepared in the same manner as in Example 8.

[0103] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 7 under the same mode and conditions.

[0104] Figure 12 shows the mass spectra of casimersen obtained by MALDI-TOFMS using various matrices. The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z) (m / z 4000 to m / z 16000), and the vertical axis represents the relative intensity (%int). The detected [M+H] + The peak intensities (mV) are shown on the mass spectrum.

[0105] In the mass spectrum of Figure 12, when 3-HPA / THAP (1:2) solution, 3-HPA / THAP (1:3) solution, 3-HPA / THAP (1:4) solution, or 3-HPA / THAP (1:5) solution was used, [M+H] peaks were observed with similar peak intensities. +On the other hand, when the 3-HPA / THAP (1:10) solution was used, the peak of [M+H] + The peak intensity decreased.

[0106] From the above, the [M+H] of casimersen, a morpholino nucleic acid with a structure containing a PEG group attached to the 5' carbon of the 5' end, + The preferred mixed matrices for detecting the peaks were 3-HPA / THAP (1:2), 3-HPA / THAP (1:3), 3-HPA / THAP (1:4), and 3-HPA / THAP (1:5). Furthermore, when mixed matrices of 3-HPA and THAP with a mixing ratio of 3-HPA to THAP ranging from 1:2 to 1:5 were used, the [M+H] peaks were detected with similar peak intensities in the mass spectra obtained by MALDI-TOFMS. + It was confirmed that a peak was detected.

[0107] [1] Preparation of sample solution As in Example 4, an aqueous solution containing 20 pmol / μL of casimersen was prepared as a sample solution.

[0108] [2] Preparation of matrix solutions Five types of matrix solutions (3-HPA / THAP (5:1) solution, 3-HPA / THAP (4:1) solution, and 3-HPA / THAP (3:1) solution) were prepared using the same procedure as in Example 1.

[0109] [3] Preparation of analytical samples Analytical samples were prepared in the same manner as in Example 8.

[0110] [4] Mass spectrometry Measurements were carried out using the same apparatus as in Example 7 under the same mode and conditions.

[0111] Figure 13 shows the mass spectra of casimersen obtained by MALDI-TOFMS using various matrix solutions. The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z) (m / z 4000 to m / z 16000), and the vertical axis represents the relative intensity (%int). The peak intensity (mV) of the detected [M+H]+ is also shown on the mass spectrum.

[0112] In the mass spectrum of FIG. 13, when the 3-HPA / THAP (3:1) solution was used, the peak intensity of [M+H] was higher. + On the other hand, when the 3-HPA / THAP (4:1) solution or the 3-HPA / THAP (5:1) solution was used, the peak of [M+H] + The peak intensity decreased.

[0113] From the above, the [M+H] of casimersen, a morpholino nucleic acid with a structure containing a PEG group attached to the 5' carbon of the 5' end, + The preferred mixed matrix for detecting the peak was 3-HPA / THAP (3:1). When a mixed matrix of 3-HPA and THAP with a mixing ratio of 3-HPA to THAP in the range of 4:1 to 5:1 was used, the mass spectrum obtained by MALDI-TOFMS measurement showed a peak of [M+H] + It was confirmed that the peak intensity of

[0114] Aspects It will be apparent to those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0115] (Item 1) A method for analyzing a morpholine ring-containing oligonucleotide according to one embodiment of the present invention uses a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2',4',6'-trihydroxyacetophenone (THAP) and analyzes the morpholine ring-containing oligonucleotide contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer.

[0116] According to the method for analyzing morpholine ring-containing oligonucleotides according to the first aspect, morpholine ring-containing oligonucleotides can be appropriately analyzed by measurement using a mass spectrometer (MALDI-MS) that utilizes the MALDI method. Specifically, the [M+H] of the morpholine ring-containing oligonucleotide can be determined from the mass spectrum obtained by measurement using MALDI-MS. + or [MH] - The peak can be detected with high sensitivity.

[0117] (Item 2) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 2 is the method for analyzing a morpholine ring-containing oligonucleotide according to Item 1, wherein the morpholine ring-containing oligonucleotide has a hydroxy group bonded to the 5' carbon of the 5'-terminal nucleotide, and the mixed ratio of 3-HPA and THAP in the mixed malic acid is 2:1 to 5:1.

[0118] (Item 3) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 3 is the method for analyzing a morpholine ring-containing oligonucleotide according to Item 1, wherein the morpholine ring-containing oligonucleotide has a structure containing an alkylene glycol group or a polyalkylene glycol group bound to the 5'-position carbon of the 5'-terminal nucleotide, and the mixing ratio of 3-HPA and THAP in the mixed matrix is ​​1:5 to 3:1.

[0119] According to the method for analyzing a morpholine ring-containing oligonucleotide according to the second or third aspect, the morpholine ring-containing oligonucleotide can be analyzed appropriately depending on its structure and properties.

[0120] (Item 4) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 4 may be the method for analyzing a morpholine ring-containing oligonucleotide according to any one of Items 1 to 3, further comprising: dripping a thin film solution prepared by dissolving THAP in a solvent onto a sample plate of the matrix-assisted laser desorption / ionization mass spectrometer and drying to form a thin film of THAP; then dripping a matrix solution containing the mixed matrix and a sample solution containing the sample onto the thin film; or dripping a sample / matrix mixed solution prepared by premixing the matrix solution and the sample solution onto the thin film and drying to form a sample / matrix mixed crystal on the thin film; and then analyzing the sample / matrix mixed crystal by the matrix-assisted laser desorption / ionization mass spectrometer.

[0121] According to the analytical method for morpholine ring-containing oligonucleotides according to the fourth aspect, sample / matrix mixed crystals in which the sample and the mixed matrix are mixed relatively uniformly are formed relatively uniformly over the entire well of the sample plate, so that the morpholine ring-containing oligonucleotides contained in the sample can be more appropriately analyzed. Specifically, the [M+H] of the morpholine ring-containing oligonucleotides can be determined from the mass spectrum obtained by measurement using MALDI-MS. + or [MH] - The peak can be detected more reproducibly and with higher peak intensity.

[0122] (Item 5) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 5 may be the method for analyzing a morpholine ring-containing oligonucleotide according to Item 4, wherein the concentration of THAP contained in the thin film solution is 4 mg / mL or more and a saturation concentration or less.

[0123] According to the method for analyzing morpholine ring-containing oligonucleotides relating to paragraph 5, sample / matrix mixed crystals in which the sample and mixed matrix are more uniformly mixed can be formed over the entire well of the sample plate, thereby enabling more appropriate analysis of the morpholine ring-containing oligonucleotides contained in the sample.

[0124] (Item 6) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 6 is the method for analyzing a morpholine ring-containing oligonucleotide according to Item 4 or 5, wherein the solvent contained in the thin film solution is acetonitrile.

[0125] According to the method for analyzing morpholine ring-containing oligonucleotides according to item 6, a thin film of THAP can be easily and efficiently formed.

[0126] (Item 7) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 7 is the method for analyzing a morpholine ring-containing oligonucleotide according to any one of Items 1 to 6, wherein the matrix-assisted laser desorption / ionization mass spectrometer is a digital ion trap type matrix-assisted laser desorption / ionization mass spectrometer.

[0127] Digital ion trap MALDI-MS is prone to fragmentation due to its instrument characteristics. The method according to the present invention has the effect of suppressing fragmentation, and therefore can effectively suppress peaks resulting from fragment ions appearing in mass spectra when a digital ion trap MALDI-MS is used. Furthermore, since digital ion trap MALDI-MS can be easily miniaturized, the method for analyzing morpholine ring-containing oligonucleotides according to item 7 allows mass analysis of morpholine ring-containing oligonucleotides contained in a sample to be performed in a small space.

[0128] (Item 8) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 8 is the method for analyzing a morpholine ring-containing oligonucleotide according to any one of Items 1 to 7, wherein the matrix-assisted laser desorption / ionization mass spectrometer is a time-of-flight matrix-assisted laser desorption / ionization mass spectrometer.

[0129] According to the method for analyzing a morpholine ring-containing oligonucleotide according to the eighth aspect, the [M+H] of the morpholine ring-containing oligonucleotide can be determined from the mass spectrum. + or [MH] - The peak can be detected with higher sensitivity.

[0130] (Item 9) The method for analyzing a morpholine ring-containing oligonucleotide according to Item 9 is the method for analyzing a morpholine ring-containing oligonucleotide according to any one of Items 1 to 8, wherein the morpholine ring-containing oligonucleotide is a morpholino nucleic acid.

[0131] According to the method for analyzing a morpholino ring-containing oligonucleotide according to the ninth aspect, morpholino nucleic acids used in nucleic acid medicines can be appropriately analyzed.

Claims

1. A method for analyzing morpholine ring-containing oligonucleotides, in which a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2',4',6'-trihydroxyacetophenone (THAP) is used to analyze morpholine ring-containing oligonucleotides contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer.

2. The method for analyzing a morpholine ring-containing oligonucleotide according to claim 1, wherein the morpholine ring-containing oligonucleotide has a hydroxy group bonded to the 5' carbon of the 5'-terminal nucleotide, and the mixed matrix contains 3-HPA and THAP in a mixing ratio of 2:1 to 5:

1.

3. The method for analyzing a morpholine ring-containing oligonucleotide according to claim 1, wherein the morpholine ring-containing oligonucleotide has a structure containing an alkylene glycol group or a polyalkylene glycol group bound to the 5' carbon of the 5'-terminal nucleotide, and the mixed matrix contains 3-HPA and THAP in a mixing ratio of 3:1 to 1:

5.

4. A method for analyzing a morpholine ring-containing oligonucleotide according to any one of claims 1 to 3, comprising: dropping a thin film solution prepared by dissolving THAP in an organic solvent onto a sample plate of the matrix-assisted laser desorption / ionization mass spectrometer; drying the thin film solution on the sample plate to form a thin film of THAP; dropping a matrix solution containing the mixed matrix and a sample solution containing the sample onto the thin film, respectively, and drying to form a sample / matrix mixed crystal on the thin film; or dropping a sample / matrix mixed solution prepared by premixing the matrix solution and the sample solution onto the thin film and drying to form a sample / matrix mixed crystal on the thin film; and analyzing the sample / matrix mixed crystal by the matrix-assisted laser desorption / ionization mass spectrometer.

5. The method for analyzing a morpholine ring-containing oligonucleotide according to claim 4, wherein the concentration of THAP contained in the thin film solution is 4 mg / mL or more and not more than the saturation concentration.

6. The method for analyzing a morpholine ring-containing oligonucleotide according to claim 4, wherein the solvent contained in the thin film solution is acetonitrile.

7. The method for analyzing a morpholine ring-containing oligonucleotide according to any one of claims 1 to 3, wherein the matrix-assisted laser desorption / ionization mass spectrometer is a digital ion trap type matrix-assisted laser desorption / ionization mass spectrometer.

8. The method for analyzing a morpholine ring-containing oligonucleotide according to any one of claims 1 to 3, wherein the matrix-assisted laser desorption / ionization mass spectrometer is a time-of-flight matrix-assisted laser desorption / ionization mass spectrometer.

9. A method for analyzing a morpholine ring-containing oligonucleotide according to any one of claims 1 to 3, wherein the morpholine ring-containing oligonucleotide is a morpholino nucleic acid.

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