Oligonucleotide drug impurity identification method based on central cutting two-dimensional liquid chromatography-mass spectrometry

Abstract

The invention discloses an oligonucleotide drug impurity identification method based on central cutting two-dimensional liquid chromatography-mass spectrometry. Comprising the following steps: acquiring an oligonucleotide sample solution; preparing a mobile phase RA and a mobile phase RB of a one-dimensional liquid chromatographic column and a mobile phase LA and a mobile phase LB of a two-dimensional liquid chromatographic column; separating a target component from a to-be-identified sample solution by utilizing a one-dimensional liquid chromatographic column according to a center cutting method to obtain the target component; further separating the target component through a two-dimensional liquid chromatographic column, and desalting on line; and detecting the molecular weight and the sequence of the target product by using a mass spectrometer to obtain the molecular weight and the sequence of the target product. The method covers a wide range of oligonucleotide sequences, and is suitable for purity detection of oligonucleotides with various sequences and different lengths; the method can be combined with mass spectrometry to quickly analyze the purity, the molecular weight and the sequence of the sample.

Description

Technical Field

[0001] The present invention relates to the field of oligonucleotide biochemical analysis, in particular to an oligonucleotide drug impurity identification method based on heart-cutting two-dimensional liquid chromatography-mass spectrometry. Background Art

[0002] Oligonucleotide drugs are a class of molecules composed of short chains of nucleotides that are used to target specific gene sequences and have broad therapeutic potential. However, due to their complex chemical structure and synthesis process, a variety of impurities may be generated during the production of oligonucleotide drugs, including deletions, insertions, or mutations caused by incomplete reactions or side reactions, degradation products such as chain breaks, deamination, and dephosphorylation caused by physical and chemical conditions during storage or use, and incompletely coupled nucleotide small molecules. The analysis and control of these impurities are crucial to the quality and safety of drugs. At present, the impurity analysis of oligonucleotide drugs faces the following challenges:

[0003] 1) Small molecular weight difference: impurities are usually very similar to the main component in structure, and the molecular weight difference may be only one nucleotide unit;

[0004] 2) Diverse chemical structures: Impurities may include isomers, sequence isomers, and chemical modifications;

[0005] 3) High detection sensitivity requirements: Oligonucleotide impurities have high biological activity and potential biological toxicity. The content of impurities is strictly controlled, requiring the analytical method to have extremely high sensitivity.

[0006] In order to effectively analyze impurities in oligonucleotide drugs, there are currently a variety of analytical techniques, among which high performance liquid chromatography (HPLC) can be widely used for purity detection and impurity analysis of oligonucleotides. Combined with a mass spectrometer (MS) detector, the impurities of oligonucleotides can be qualitatively and sequenced. However, the current liquid chromatography-mass spectrometry method usually first uses hydrophilic interaction chromatography (HILIC) or reverse ion pair chromatography (IR-RP) to separate oligonucleotide drugs and impurities, which has limited separation and a long analysis time, usually more than one hour. The anion exchange chromatography method with better separation is not compatible with mass spectrometry because it contains non-volatile salt solutions. Summary of the invention

[0007] The present invention aims at the lack of existing oligonucleotide drug impurity analysis methods, and provides an oligonucleotide drug impurity identification method based on heart-cutting two-dimensional liquid chromatography-mass spectrometry. The method of the present invention first uses anion exchange chromatography as the first-dimensional chromatography to perform preliminary separation of oligonucleotide drugs and impurities, and has the advantages of good component peak separation effect, high resolution, high analysis accuracy, good reproducibility, and rapid separation; further uses ion pair reverse phase chromatography as the second-dimensional chromatography to further separate the target sample or impurities, and has the advantage of being compatible with mass spectrometry. The molecular weight and sequence of oligonucleotides and drugs are identified by mass spectrometry to achieve the purpose of effectively controlling the quality of oligonucleotides.

[0008] The present invention adopts the following technical solution:

[0009] 1. Oligonucleotide drug impurity identification method based on heart-cutting two-dimensional liquid chromatography-mass spectrometry

[0010] The oligonucleotide drug impurity identification method comprises the following steps:

[0011] 1) Obtain a sample solution to be identified. The sample solution to be identified contains at least one target product, the target product is an oligonucleotide with a length of 2 to 40mer, and the concentration of each target product in the sample solution to be identified is 1 to 100 pmol / μL. The types of the oligonucleotide samples include but are not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA-RNA hybrids. The oligonucleotide sample is an antisense oligonucleotide (ASO), dsRNA, siRNA, nucleic acid aptamer or microRNA. The sample solution to be identified uses DEPC-treated water as a solvent.

[0012] 2) Prepare mobile phase RA and mobile phase RB of the one-dimensional liquid chromatography column, and mobile phase LA and mobile phase LB of the two-dimensional liquid chromatography column.

[0013] The mobile phase RA of the one-dimensional liquid chromatography column adopts tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer or sodium borate (BBS) buffer; the mobile phase RB of the one-dimensional liquid chromatography column adopts a mixed solution of the mobile phase RA of the one-dimensional liquid chromatography column and a sodium salt, the sodium salt is sodium chloride (NaCl) or sodium bromide (NaBr), preferably sodium chloride, and the concentration of the sodium salt is 0.4-1.5 mol / L.

[0014] Preferably, the mobile phase RA of the one-dimensional liquid chromatography column is a sodium borate (BBS) buffer, the concentration of the sodium borate buffer is 1-25 mmol / L, the pH of the sodium borate buffer is 8.0-9.0, preferably pH = 8.0, 8.5 or 9.0. The concentration of sodium salt in the mobile phase RB of the one-dimensional liquid chromatography column is 1.0 mol / L.

[0015] The mobile phase LA of the two-dimensional liquid chromatography column uses methanol. The mobile phase LB of the two-dimensional liquid chromatography column uses a mixed aqueous solution of N,N-diisopropylethylamine and hexafluoroisopropanol, the concentration of N,N-diisopropylethylamine in the mobile phase LB is 20-50 mmol / L, and the concentration of hexafluoroisopropanol in the mobile phase LB is 100-500 mmol / L.

[0016] As a preferred technical solution of the present invention, the mobile phase RA of the one-dimensional liquid chromatography column is a sodium borate (BBS) buffer with a concentration of 20 mmol / L and a pH of 8.0; the concentration of sodium salt in the mobile phase RB of the one-dimensional liquid chromatography column is 1.0 mol / L. The mobile phase LA of the two-dimensional liquid chromatography column is methanol, and the mobile phase LB of the two-dimensional liquid chromatography column contains diisopropylethylamine with a concentration of 30 mmol / L and hexafluoroisopropanol with a concentration of 200 mmol / L.

[0017] 3) Separating the target component from the sample solution to be identified by a one-dimensional liquid chromatography column through the heart cutting method. The separation conditions of the one-dimensional liquid chromatography column are: chromatographic flow rate 0.5-0.8 mL / min, injection volume 1-10 μL, column temperature 20-60°C, injection plate temperature 4-25°C. The elution gradient of the one-dimensional liquid chromatography column is:

[0018] 0 min, 63% mobile phase RA, 37% mobile phase RB;

[0019] 12 min, 35% mobile phase RA, 65% mobile phase RB;

[0020] 12.5 min, 20% mobile phase RA, 80% mobile phase RB;

[0021] 15 min, 20% mobile phase RA, 80% mobile phase RB;

[0022] 15.5 min, 63% mobile phase RA, 37% mobile phase RB.

[0023] 4) Inject the target component into the two-dimensional liquid chromatography column, further separate the target component through the two-dimensional liquid chromatography column and desalt it online; the separation conditions of the two-dimensional liquid chromatography column are: chromatographic flow rate 0.5-0.8mL / min, injection volume 50-200μL, column temperature 20-60℃, injection plate temperature 4-25℃. The elution gradient of the two-dimensional liquid chromatography column is:

[0024] 0 min, 10% mobile phase LA, 90% mobile phase LB;

[0025] 9 min, 10% mobile phase LA, 90% mobile phase LB;

[0026] 12 min, 100% mobile phase LA, 0% mobile phase LB;

[0027] 15.5 min, 100% mobile phase LA, 0% mobile phase LB.

[0028] 5) Using a mass spectrometer to detect the molecular weight and sequence of the target product to obtain the molecular weight and sequence of the target product. In the step 5), the molecular weight of the target product is determined by a primary mass spectrometer, and the target product is fragmented by a secondary mass spectrometer (MS / MS) for sequence detection, and the conditions for the fragmentation treatment are: using high energy collision dissociation (HCD) technology, the normalized collision energy (NCE) is 15% to 35%, preferably 30%, and the extraction window is 2Da.

[0029] As a preferred technical solution of the present invention, the mass spectrometer model used in step 5) is Thermo Fisher Orbitrap Exploris 120, the mass spectrometer scanning range is 200-3000 m / z, the resolution is 60000, the negative ion mode, RFLens 100%, the ion source voltage is 2500 V, Sheath Gas 60 Arb, Aux Gas 15 Arb, Sweep Gas 2 Arb, the ion transfer tube temperature is 350 ° C, and the evaporation temperature is 350 ° C.

[0030] As a preferred technical solution of the present invention, the separation conditions of the one-dimensional and two-dimensional liquid chromatography columns are: chromatographic flow rate 0.6mL / min; injection volume 4μL; column temperature 40°C; injection plate temperature 4°C.

[0031] Furthermore, in the step 3), at least one target component is separated according to the retention time of the one-dimensional liquid chromatography column, and each target component corresponds to at least one target product. After each target component is collected, each target component is sequentially separated by a two-dimensional liquid chromatography column and detected by a mass spectrometer according to the steps 4) to 5), and the molecular weight and sequence of the target product in the target component are obtained. Among them, each target component corresponds to at least one target product means that if the one-dimensional separation is not enough, the target products are not separated at one retention time, that is, the target component at one retention time corresponds to more than one target product, and the two-dimensional separation can further separate the target components and desalt them online.

[0032] Preferably, the one-dimensional liquid chromatography column is an anion exchange chromatography column; and the two-dimensional liquid chromatography column is a C18 chromatography column.

[0033] Furthermore, an ultraviolet detector and a six-way valve are arranged between the one-dimensional liquid chromatography column and the two-dimensional liquid chromatography column. The ultraviolet detector is used to detect the concentration of the target product in the target component, and the detection wavelength of the ultraviolet detector is set to 260nm; the six-way valve is used to perform heart cutting.

[0034] Furthermore, a quantitative loop for collecting target components is provided in the six-way valve, and the volume of the quantitative loop is 50 μL or 200 μL, preferably 50 μL.

[0035] 2. A heart-cutting two-dimensional liquid chromatography-mass spectrometry system for oligonucleotide drug impurity identification

[0036] The heart-cutting two-dimensional liquid chromatography-mass spectrometry system comprises a binary pump, an injector, a one-dimensional liquid chromatography column, an ultraviolet detector, a six-way valve, a two-dimensional liquid chromatography column, a three-way valve and a mass spectrometer.

[0037] The first outlet of the binary pump is connected to the inlet of a one-dimensional liquid chromatography column through an injector, the outlet of the one-dimensional liquid chromatography column is connected to the inlet of an ultraviolet detector, the outlet of the ultraviolet detector is connected to the third valve port of a six-way valve, the second valve port of the six-way valve is connected to the inlet of an external waste liquid collection or treatment device, the first valve port and the fourth valve port of the six-way valve are respectively connected to the inlet and outlet of a quantitative loop, the fifth valve port of the six-way valve is connected to the second outlet of the binary pump, the sixth valve port of the six-way valve is connected to the inlet of a two-dimensional liquid chromatography column, the outlet of the two-dimensional liquid chromatography column is connected to the three-way inlet of a three-way valve, the three-way second outlet of the three-way valve is connected to the inlet of a mass spectrometer, and the three-way first outlet of the three-way valve is connected to the inlet of an external waste liquid collection or treatment device.

[0038] When the third valve port of the six-way valve is connected to the second valve port, and the fifth valve port, the fourth valve port, the first valve port and the sixth valve port are connected in sequence, the six-way valve is used to cut the non-target components of the one-dimensional liquid chromatography column into the waste liquid, and if the target component is stored in the quantitative loop, the six-way valve is also used to inject the target component into the two-dimensional liquid chromatography column under the drive of the binary pump;

[0039] When the third valve port, the fourth valve port and the first valve port of the six-way valve are connected in sequence, and the fifth valve port is connected to the sixth valve port, the six-way valve is used to collect the target components separated by the one-dimensional liquid chromatography column in the quantitative loop.

[0040] As a preferred technical solution of the present invention, the heart-cutting two-dimensional liquid chromatography-mass spectrometry system includes a high performance liquid chromatograph Thermo Fisher Scientific Vanquish Series equipped with a binary pump and an ultraviolet detector, and a mass spectrometer Thermo Fisher Orbitrap Exploris 120.

[0041] As a preferred technical solution of the present invention, the one-dimensional liquid chromatography column is Thermo Fisher Scientific DNAPac TM PA200RS (filler particle size is 4 μm, column diameter × column length is 4.6 × 50 mm). The two-dimensional liquid chromatography column is Thermo Fisher Scientific Hypersil GOLD TM (The filler particle size is 3 μm, and the diameter × column length of the chromatographic column is 2.1 × 100 mm).

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] 1. The analytical method of the present invention covers a wide range of oligonucleotide sequences and is suitable for purity detection of oligonucleotides of various sequences and different lengths.

[0044] 2. The analytical method of the present invention uses two-dimensional chromatography for online desalting, and can then be combined with mass spectrometry to quickly analyze sample purity, molecular weight and sequence.

[0045] 3. The chromatographic method adopted in the analytical method of the present invention has a short separation time and a high degree of separation.

[0046] 4. The analytical method of the present invention can effectively control the quality of oligonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The chromatograms of oligonucleotide separation by one-dimensional ion exchange chromatography in different buffer salt systems in Examples 1 to 9 of the present invention;

[0048] Figure 2 is a chromatographic mass spectrum of oligonucleotide separation by two-dimensional reverse ion pair chromatography in Example 10 of the present invention;

[0049] Figure 3 Schematic diagram of the heart-cutting two-dimensional liquid chromatography-mass spectrometry system of the present invention; wherein (A) is a flow path connection diagram when the third valve port, the fourth valve port, the first valve port and the second valve port of the six-way valve are connected, and the fifth valve port and the sixth valve port are connected; (B) is a flow path connection diagram when the third valve port and the second valve port of the six-way valve are connected, and the fifth valve port, the fourth valve port, the first valve port and the sixth valve port are connected; (C) is a flow path connection diagram when the first valve port and the third valve port of the three-way valve are connected;

[0050] Figure 4 The following are the results of the identification of 16mer oligonucleotides based on heart-cutting two-dimensional liquid chromatography-mass spectrometry in Example 11 of the present invention; wherein (A) is an ultraviolet detection diagram, (B) is an extracted ion current diagram, (C) is a mass spectrometry primary spectrum diagram, and (D) is a mass spectrometry secondary spectrum diagram;

[0051] Figure 5 Schematic diagram of the naming method of oligonucleotide mass spectrometry fragments in Example 11 of the present invention;

[0052] Figure 6 This is a schematic diagram of the sequencing results of the 16mer oligonucleotide drug using mass spectrometry in Example 11 of the present invention. DETAILED DESCRIPTION

[0053] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0054] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0055] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0056] The oligonucleotide drug impurity identification method provided by the present invention comprises the following steps: sample processing, two-dimensional liquid phase oligonucleotide separation, ultraviolet and mass spectrometry detector detection, and mass spectrometry secondary sequence identification. The first-dimensional liquid chromatography column is an anion exchange chromatography. Oligonucleotide drugs and impurities can be separated, and the sample to be identified after central cutting enters the second-dimensional liquid phase reverse ion pair chromatography for further separation, and the sample is desalted at the same time to reduce the contamination of the sample to the mass spectrometry detector.

[0057] The specific embodiments of the present invention are as follows:

[0058] Example 1

[0059] Separation of Oligonucleotides by One-Dimensional Ion Exchange Chromatography

[0060] (1) Prepare an oligonucleotide standard mixture with a total concentration of 20 pmol / μL, the mixture containing oligonucleotides with the following sequences:

[0061] 10T:TTTTTTTTTT;

[0062] 15T:TTTTTTTTTTTTTTTT;

[0063] 20T:TTTTTTTTTTTTTTTTTTTT;

[0064] 25T:TTTTTTTTTTTTTTTTTTTTTTTT;

[0065] 30T:TTTTTTTTTTTTTTTTTTTTTTTTTTTT;

[0066] 35T:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0067] (2) Prepare a Tris-HCl buffer solution with a concentration of 20 mmol / L and pH = 8.0 as the mobile phase RA, and prepare a 1.0 mol / L sodium chloride NaCl + mobile phase RA as the mobile phase RB.

[0068] (3) Flow rate 0.6 mL / min. Elution gradient:

[0069] 0 min, 63% mobile phase RA, 37% mobile phase RB;

[0070] 12 min, 35% mobile phase RA, 65% mobile phase RB;

[0071] 12.5 min, 20% mobile phase RA, 80% mobile phase RB;

[0072] 15 min, 20% mobile phase RA, 80% mobile phase RB;

[0073] 15.5 min, 63% mobile phase RA, 37% mobile phase RB.

[0074] (4) The samples after one-dimensional separation are detected using an ultraviolet detector with an ultraviolet wavelength of 260 nm.

[0075] Embodiment 2 to Embodiment 9

[0076] The present invention also carried out Examples 2 to 9 based on the oligonucleotide standard mixture prepared in Example 1. Except for using different types and pH buffers as the mobile phase RA, the other steps and parameters of Examples 2 to 9 are the same as those of Example 1. The specific differences between Examples 2 to 9 and Example 1 are as follows:

[0077] In Example 2 and Example 3, Tris-HCl buffer with pH=8.8 and pH=9.5 were used as mobile phase RA respectively;

[0078] Examples 4 to 6 respectively used HEPES buffer with pH=7.5, pH=8.0, and pH=8.5 as the mobile phase RA;

[0079] Examples 7 to 9 respectively used BBS buffer with pH=8.0, pH=8.5, and pH=9.0 as the mobile phase RA.

[0080] The chromatograms obtained in Examples 1 to 9 are shown in Figure 1 .Depend on Figure 1 It can be seen that the five oligonucleotides can be completely separated under the above liquid phase conditions.

[0081] The separation degree is shown in the following table:

[0082]

[0083] In the above table, ND means not detected, and * represents half peak width data

[0084] It can be seen from the above table that the six oligonucleotides have the best separation when BBS buffer (Example 7) at pH=8.0 is used as the RA mobile phase.

[0085] The calculation method of separation R is:

[0086]

[0087] or:

[0088]

[0089] Where, 1 / t1 and 1 / t2 are the chromatographic retention times of the two oligonucleotides, w1 and w2 are the chromatographic peak widths of the two oligonucleotides, and w 1,1 / 2 and w 2,1 / 2 is the half-peak width of the chromatographic peaks of the two oligonucleotides.

[0090] Example 10

[0091] Separation of Oligonucleotides by Two-Dimensional Reverse Ion-Pair Chromatography

[0092] (1) Prepare 20 pmol / μL of a 21mer oligonucleotide, the oligonucleotide sequence of which is as shown in SEQ ID No. 1, specifically 5'-UUGUACUACACAAAAGUACUG-3', with a molecular weight of 6661.9237 Da.

[0093] (2) Prepare methanol as the mobile phase LA, and 30 mmol / L diisopropylethylamine and 200 mmol / L hexafluoroisopropanol as the mobile phase LB.

[0094] (3) Flow rate 0.6 mL / min. Elution gradient:

[0095] 0 min, 10% mobile phase LA, 90% mobile phase LB;

[0096] 6 min, 10% mobile phase LA, 90% mobile phase LB;

[0097] 9 min, 100% mobile phase LA, 0% mobile phase LB;

[0098] 15.5 min, 100% mobile phase LA, 0% mobile phase LB.

[0099] (4) A mass spectrometer was used to detect the eluent in the separation process. The mass spectrometer scanning range was 300-3000 m / z, the resolution was 60000, the negative ion mode, the RF Lens was 100%, the ion source voltage was 2500 V, the Sheath Gas was 60 Arb, the AuxGas was 15 Arb, the Sweep Gas was 2 Arb, the ion transfer tube temperature was 350 °C, and the evaporation temperature was 350 °C.

[0100] In Example 10, Figure 2 The extracted ion chromatogram (upper) with a mass range of 2200-2600 m / z and the mass spectrum signal at 8.04 min (lower). The signal peak appears at 8.04 min, indicating that the reverse ion pair chromatography has a certain retention capacity for oligonucleotides. When the mobile phase LB ratio is high, the target compound does not elute, but when the mobile phase LA ratio is high, the target compound elutes. The mass spectrometry signal peak [M-3H] detected at the same time 3- The m / z is 2219.6289, the theoretical m / z is 2219.6339, and the difference between the theoretical mass-to-charge ratio and the actual mass-to-charge ratio is small. It can be seen that mass spectrometry can perform qualitative molecular weight detection on the target oligonucleotide.

[0101] Embodiment 11

[0102] Identification of 16mer oligonucleotides based on heart-cutting two-dimensional liquid chromatography-mass spectrometry

[0103] (1) Prepare 20 pmol / μL of 16mer oligonucleotide, the oligonucleotide sequence is as shown in SEQ ID No. 2, specifically: 5'-CUCAACGACUAUCACU-3', with a molecular weight of 4982.7032 Da.

[0104] (2) Prepare a 20 mmol / L BBS buffer solution with a pH of 8.0 as the mobile phase RA, prepare a 1.0 mol / L NaCl + mobile phase RA as the mobile phase RB. Prepare methanol as the mobile phase LA, and a mixed aqueous solution containing 30 mmol / L diisopropylethylamine and 200 mmol / L hexafluoroisopropanol as the mobile phase LB.

[0105] (3) The first-dimensional elution gradient was: 0 min, 63% mobile phase RA, 37% mobile phase RB; 12 min, 35% mobile phase RA, 65% mobile phase RB; 12.5 min, 20% mobile phase RA, 80% mobile phase RB; 15 min, 20% mobile phase RA, 80% mobile phase RB; 15.5 min, 63% mobile phase RA, 37% mobile phase RB. The flow rate was 0.6 mL / min. The second-dimensional elution gradient was: 0 min, 10% mobile phase LA, 90% mobile phase LB; 9 min, 10% mobile phase LA, 90% mobile phase LB; 12 min, 100% mobile phase LA, 0% mobile phase LB; 15.5 min, 100% mobile phase LA, 0% mobile phase LB. The flow rate was 0.6 mL / min.

[0106] (4) A UV detector with a UV wavelength of 260 nm was used. A mass spectrometer was also used with a mass spectrometer scanning range of 300-3000 m / z, a resolution of 60000, negative ion mode, RF Lens 100%, ion source voltage of 2500 V, Sheath Gas 60 Arb, Aux Gas 15 Arb, Sweep Gas 2 Arb, ion transfer tube temperature of 350 °C, and evaporation temperature of 350 °C.

[0107] (5) The m / z = 1659.8894 in the sample was collided using secondary mass spectrometry (MS / MS) high energy collision dissociation (HCD) with a collision energy (NCE) of 30% and an extraction window of 2 Da.

[0108] In Example 11, the liquid phase flow path and the method of linking with the mass spectrometer are as follows Figure 3 .Depend on Figure 3 It can be seen that this embodiment provides a heart-cutting two-dimensional liquid chromatography-mass spectrometry system. The heart-cutting two-dimensional liquid chromatography-mass spectrometry system includes a binary pump, an injector, a one-dimensional liquid chromatography column, an ultraviolet detector, a six-way valve, a two-dimensional liquid chromatography column, a three-way valve and a mass spectrometer. Figure 3In the figure, the Arabic numerals 1, 2, 3, 4, 5, and 6 in the six-way valve respectively represent the first valve port, the second valve port, the third valve port, the fourth valve port, the fifth valve port, and the sixth valve port of the six-way valve; the Arabic numerals 1, 2, and 3 in the three-way valve respectively represent the three-way first outlet, the three-way inlet, and the three-way second outlet of the three-way valve.

[0109] Among them, the first outlet of the binary pump ( Figure 3 The right outlet in the flowmeter) is connected to the inlet of the one-dimensional liquid chromatography column DNAPac100 through the injector, the outlet of the one-dimensional liquid chromatography column DNA Pac100 is connected to the inlet of the ultraviolet detector, the outlet of the ultraviolet detector is connected to the third valve port of the six-way valve, the second valve port of the six-way valve is connected to the inlet of an external waste liquid collection or treatment device, the first valve port and the fourth valve port of the six-way valve are respectively connected to the inlet and outlet of the quantitative loop, the fifth valve port of the six-way valve is connected to the second outlet of the binary pump, the sixth valve port of the six-way valve is connected to the inlet of the two-dimensional liquid chromatography column, the outlet of the two-dimensional liquid chromatography column is connected to the three-way inlet of the three-way valve, the three-way second outlet of the three-way valve is connected to the inlet of the mass spectrometer, and the three-way first outlet of the three-way valve is connected to the inlet of the external waste liquid collection or treatment device.

[0110] like Figure 3 As shown in (A), when the third valve port, the fourth valve port and the first valve port of the six-way valve are connected in sequence, and the fifth valve port is connected to the sixth valve port, the six-way valve is used to collect the target components separated by the one-dimensional liquid chromatography column in a quantitative ring.

[0111] like Figure 3 As shown in (B), when the third valve port of the six-way valve is connected to the second valve port, and the fifth valve port, the fourth valve port, the first valve port and the sixth valve port are connected in sequence, the six-way valve is used to cut the non-target components of the one-dimensional liquid chromatography column into the waste liquid. If the target component is stored in the quantitative loop, the six-way valve is also used to inject the target component into the two-dimensional liquid chromatography column under the drive of the left pump of the binary pump.

[0112] When the three-way inlet of the three-way valve is connected to the three-way first outlet, the three-way valve is used to cut the eluate separated by the two-dimensional liquid chromatography column and not containing the target product into waste liquid.

[0113] like Figure 3 As shown in (C), when the three-way inlet of the three-way valve is connected to the three-way second outlet, the three-way valve is used to inject the eluate containing the target product separated by the two-dimensional liquid chromatography column into the mass spectrometer.

[0114] First, the oligonucleotide drug and impurities to be analyzed enter the quantitative loop after passing through the right pump and the first-dimensional liquid phase separation. At this time, the gear position of the six-way valve where the quantitative loop is located is switched ( Figure 3(B) , use the left pump to pump the liquid in the quantitative loop into the second-dimensional liquid phase. After the second-dimensional liquid phase separation, the three-way valve in front of the mass spectrometer is switched to select whether to enter the mass spectrometer analysis to achieve the purpose of desalting.

[0115] Figure 4 This is the result of the 16mer liquid phase analysis by center-cutting two-dimensional liquid chromatography-mass spectrometry. Figure 4 (A) shows that the oligonucleotide peak time in the first dimension chromatogram is 7.57 min. Figure 4 (B) shows that after the central cutting, the peak time of the target analyte in the second dimension chromatogram is 11.49min. Figure 4 (C) shows the mass spectrum at 11.49 min, [M-3H] 3- The m / z is 1659.8894, and the theoretical mass-to-charge ratio is m / z 1659.8938. It can be seen that the method of the present invention can perform qualitative molecular weight detection on oligonucleotide impurities.

[0116] Figure 4 (D) shows that 1659.8894 is used as the parent ion for mass spectrometry fragmentation sequencing analysis, and the obtained fragment information is as follows:

[0117]

[0118]

[0119] The fragment naming method is shown in Figure 5 , the sequence coverage reached 64% ( Figure 6 ).

[0120] It can be seen that the method of the present invention can further identify the sequence of the target product.

[0121] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept, which should be regarded as falling within the protection scope of the present invention.

[0122] The nucleotide sequence involved in the present invention is as follows:

[0123] SEQ ID No.1

[0124] Name: Oligonucleotide sample sequence in Example 10

[0125] DNA type: RNA (other RNA)

[0126] Organism source: synthetic construct

[0127] 5'-UUGUACUACACAAAAGUACUG-3'

[0128] SEQ ID No.2

[0129] Name: Oligonucleotide sample sequence in Example 11

[0130] DNA type: RNA (other RNA)

[0131] Organism source: synthetic construct

[0132] 5′-CUCAACGACUAUCACU-3′.

Claims

1. A method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: 1) Obtaining a sample solution to be identified; 2) preparing mobile phase RA and mobile phase RB of a one-dimensional liquid chromatography column, and mobile phase LA and mobile phase LB of a two-dimensional liquid chromatography column; 3) Separating the target component from the sample solution to be identified using a one-dimensional liquid chromatography column by a heart-cutting method; 4) injecting the target component into a two-dimensional liquid chromatography column, further separating the target component through the two-dimensional liquid chromatography column and performing online desalting; 5) Using a mass spectrometer to detect the molecular weight and sequence of the target product to obtain the molecular weight and sequence of the target product.

2. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step 1), the sample solution to be identified contains at least one target product, the target product is an oligonucleotide with a length of 2 to 40 mer, and the concentration of each target product in the sample solution to be identified is 1 to 100 pmol / μL.

3. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step 3), at least one target component is separated according to the retention time of the one-dimensional liquid chromatography column. After each target component is collected, each target component is sequentially separated by a two-dimensional liquid chromatography column and detected by a mass spectrometer according to the steps 4) to 5) to obtain the molecular weight and sequence of the target product in the target component.

4. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step 2), the mobile phase RA of the one-dimensional liquid chromatography column adopts tris(hydroxymethyl)aminomethane hydrochloride buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer or sodium borate buffer; the mobile phase RB of the one-dimensional liquid chromatography column adopts a mixed solution of the mobile phase RA of the one-dimensional liquid chromatography column and a sodium salt, wherein the sodium salt is sodium chloride or sodium bromide, and the concentration of the sodium salt is 0.4 to 1.5 mol / L; In the step 2), the mobile phase LA of the two-dimensional liquid chromatography column is methanol; the mobile phase LB of the two-dimensional liquid chromatography column is a mixed aqueous solution of N,N-diisopropylethylamine and hexafluoroisopropanol, the concentration of the N,N-diisopropylethylamine is 20-50 mmol / L, and the concentration of the hexafluoroisopropanol is 100-500 mmol / L.

5. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 4, characterized in that: In the step 2), the mobile phase RA of the one-dimensional liquid chromatography column is a sodium borate buffer, the concentration of the sodium borate buffer is 1-25 mmol / L, and the pH of the sodium borate buffer is 8.0-9.0; the concentration of the sodium salt in the mobile phase RB of the one-dimensional liquid chromatography column is 1.0 mol / L.

6. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 4, characterized in that: In the step 3), the separation conditions of the one-dimensional liquid chromatography column are: chromatographic flow rate 0.5-0.8 mL / min, injection volume 1-10 μL, column temperature 20-60° C., injection plate temperature 4-25° C.; the elution gradient of the one-dimensional liquid chromatography column is: 0 min, 63% mobile phase RA, 37% mobile phase RB; 12 min, 35% mobile phase RA, 65% mobile phase RB; 12.5 min, 20% mobile phase RA, 80% mobile phase RB; 15 min, 20% mobile phase RA, 80% mobile phase RB; 15.5 min, 63% mobile phase RA, 37% mobile phase RB; In the step 4), the separation conditions of the two-dimensional liquid chromatography column are: chromatographic flow rate 0.5-0.8 mL / min, injection volume 50-200 μL, column temperature 20-60° C., injection plate temperature 4-25° C.; the elution gradient of the two-dimensional liquid chromatography column is: 0 min, 10% mobile phase LA, 90% mobile phase LB; 9 min, 10% mobile phase LA, 90% mobile phase LB; 12 min, 100% mobile phase LA, 0% mobile phase LB; 15.5 min, 100% mobile phase LA, 0% mobile phase LB.

7. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: An ultraviolet detector and a six-way valve are arranged between the one-dimensional liquid chromatography column and the two-dimensional liquid chromatography column; the ultraviolet detector is used to detect the concentration of the target product in the target component. A quantitative ring for collecting the target component is arranged in the six-way valve, and the volume of the quantitative ring is 50μL or 200μL.

8. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step 5), the molecular weight of the target product is determined using a primary mass spectrometer, and the target product is fragmented using a secondary mass spectrometer for sequence detection. The conditions for the fragmentation treatment are: using high-energy collision dissociation technology, the normalized collision energy is 15% to 35%, and the extraction window is 2Da.

9. The method for identifying impurities in oligonucleotide drugs based on heart-cutting two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that: The one-dimensional liquid chromatography column adopts an anion exchange chromatography column; the two-dimensional liquid chromatography column adopts a C18 chromatography column.

10. A heart-cutting two-dimensional liquid chromatography-mass spectrometry system for implementing the method for identifying impurities in oligonucleotide drugs according to any one of claims 1 to 9, characterized in that: The heart-cutting two-dimensional liquid chromatography-mass spectrometry system comprises a binary pump, an injector, a one-dimensional liquid chromatography column, an ultraviolet detector, a six-way valve, a two-dimensional liquid chromatography column, a three-way valve and a mass spectrometer; The first outlet of the binary pump is connected to the inlet of the one-dimensional liquid chromatography column through the injector, the outlet of the one-dimensional liquid chromatography column is connected to the inlet of the ultraviolet detector, the outlet of the ultraviolet detector is connected to the third valve port of the six-way valve, the second valve port of the six-way valve is connected to the inlet of an external waste liquid collection or treatment device, the first valve port and the fourth valve port of the six-way valve are respectively connected to the inlet and the outlet of the quantitative loop, the fifth valve port of the six-way valve is connected to the second outlet of the binary pump, the sixth valve port of the six-way valve is connected to the inlet of the two-dimensional liquid chromatography column, the outlet of the two-dimensional liquid chromatography column is connected to the three-way inlet of the three-way valve, the three-way second outlet of the three-way valve is connected to the inlet of the mass spectrometer, and the three-way first outlet of the three-way valve is connected to the inlet of the external waste liquid collection or treatment device; When the third valve port of the six-way valve is connected to the second valve port, and the fifth valve port, the fourth valve port, the first valve port and the sixth valve port are connected, the six-way valve is used to cut the non-target components of the one-dimensional liquid chromatography column into the waste liquid, and if the target component is stored in the quantitative loop, the six-way valve is also used to inject the target component into the two-dimensional liquid chromatography column under the drive of the binary pump; When the third valve port, the fourth valve port and the first valve port of the six-way valve are connected, and the fifth valve port is connected to the sixth valve port, the target components separated by the one-dimensional liquid chromatography column are collected in the quantitative loop.

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