CN117665147B - Method for detecting purity of oligonucleotide compound by LC-MS and related substances in purity - Google Patents

Abstract

The invention discloses a method for detecting purity of an oligonucleotide compound by LC-MS and related substances in the oligonucleotide compound. The method provided by the invention comprises the following steps: dissolving or diluting a sample to be tested in water to prepare a solution to be tested; firstly, carrying out liquid chromatography detection on the solution to be detected, then entering a mass spectrum detector, and analyzing the part which does not reach baseline separation in the liquid phase spectrum through mass spectrum; and quantitatively analyzing the purity of the oligonucleotide compound in the sample and related substances in the oligonucleotide compound according to the detection results of the liquid chromatography and the mass spectrum. The method provided by the invention solves the problem that the CpG ODN target sequence is difficult to distinguish from N+, N-, P=O impurities, and realizes the accurate detection of the purity of the CpG ODN target sequence and related substances.

Description

Method for detecting purity of oligonucleotide compound by LC-MS and related substances in purity

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the purity of an oligonucleotide compound and related substances in the oligonucleotide compound by utilizing LC-MS.

Background

Oligonucleotide drugs (oligonucleotides, ONs) are a class of drugs consisting of 12-30 ribose oligonucleotides single-stranded or double-stranded synthesized by artificial chemistry, which act on target messenger RNAs (mrnas) through Watson-Crick base pairing rules to disable the encoding abnormal genes and thereby prevent expression of "false" proteins, thereby exerting a unique mechanism for regulating the transcriptional translation process of disease genes at the gene level. The ONs are mainly of the following types: antisense oligonucleotides (ASOs), small interfering RNAs (sirnas), micrornas (mirnas), aptamer (Aptamer), decoy oligonucleotides (Decoy ODNs), and the like. Since oligonucleotide drugs exhibit strong pharmaceutical advantages, it is highly necessary to develop accurate analytical methods.

The molecular weight of the oligonucleotide drug is relatively large and almost anionic in water, resulting in the formation of a negatively charged aggregated molecular entity. Based on this structural feature, the analytical methods currently in common use are: reversed phase ion pair chromatography, hydrophilic chromatography, ion exchange chromatography, capillary electrophoresis, and PCR.

The principle of Liquid Chromatography (LC) is that the physical and chemical properties of the components of the mixture to be measured differ, they being distributed to different extents in two mutually immiscible phases; when the two phases are in relative motion, the components are redistributed in the two phases repeatedly a number of times, allowing the mixture to separate. One of the two phases, stationary, is called the stationary phase and the other phase, moving, is called the mobile phase.

Liquid chromatography has stronger separation capability, but like other chromatographic techniques, the liquid chromatography cannot directly obtain chemical structure information of separated components, but rather must rely on comparison with a standard sample to estimate the structure of a separated unknown substance; and for complex mixed unknowns, the structure is difficult to analyze by simply using chromatographic techniques. Common liquid chromatographs employ differential, conductivity, fluorescence, and uv-vis, etc. detectors, and use standard samples to perform qualitative or quantitative analysis of the separated components. However, the detectors such as differential and conductance are susceptible to external environments such as temperature and flow rate, have low sensitivity, and cannot perform gradient elution. Uv-vis and fluorescence detectors, etc., are primarily characterized by the retention time of the material, either as peak intensity (peak height) or peak area, but relying solely on such detectors to draw false conclusions in the case of simultaneous analysis of multiple components and nearly simultaneous washing out of the column due to too close a structure.

Mass Spectrometry (MS) is an instrument analysis technology with a strong structure analysis function, and with the realization of miniaturization of the instrument structure, the MS is currently applied to a plurality of modern instrument combination analysis technologies. The mass spectrometry is carried out by ionizing the molecules of the compound to be detected (M- & gt M ⁺), and separating the obtained ions with different mass-to-charge ratios (including molecular ions and fragment ions) under the action of an electric field and a magnetic field, thereby obtaining a group of characteristic mass spectrograms. Because specific molecules have characteristic fragmentation and ionization rules under the determined mass spectrometry conditions and have good reproducibility, mass spectrometry can provide abundant structural information for analysis of unknown components, and is one of the most effective qualitative and quantitative analysis means.

LC-MS is a device in which LC and MS are combined. On the one hand, LC can effectively separate mixed components and provide information such as retention time associated with qualitative analysis; alternatively, the MS may ionize the different LC eluting components and obtain the mass spectral information that characterizes them, and then infer their molecular weight and chemical structure information. Meanwhile, LC-MS combination techniques are powerful tools for quantitative analysis of complex mixture analytes. The detected molecular weight adds another layer of specificity to the analysis, as the co-eluting analyte can be further distinguished by its mass spectral signal. In quantitative analysis, the intensity of the MS signal is related to the amount of compound in the sample. The normalized ion chromatogram integration gives the percentage of each compound in the sample in a manner similar to the percentage of area of each compound obtained based on the UV chromatogram. This relative quantification is performed assuming the same ionization efficiency for all quantitatively analyzed compounds. Based on this assumption, compounds at the same concentration will produce the same ionic strength in mass spectra, but this assumption is limited. The ionization efficiency depends to a large extent on the hydrophobicity of the compound and the presence of the ionizable groups. MS quantification is generally less preferred than UV, but is necessary if adequate chromatographic separation is not available.

CpG ODN (CpG oligodeoxynucleotide ) is an artificially synthesized Oligodeoxynucleotide (ODN) containing unmethylated cytosine guanine dinucleotides (CpG) that mimics bacterial DNA to stimulate immune cells in a variety of mammals, including humans. CpG ODN is often used in a thio-modified form as a vaccine adjuvant or an antitumor agent, and thus quantification of CpG ODN is an indispensable step and product quality control index for ensuring drug safety and efficacy.

The existing synthetic method of CpG ODN mainly comprises liquid phase synthesis and solid phase synthesis, wherein the solid phase synthesis is to directionally connect a first nucleotide at the 3 'end of a DNA chain to be synthesized with a solid phase carrier of a polymer material (the 3' end is connected to the solid phase carrier); the protecting group at the 5' end of the nucleotide is removed by a deprotection agent, after the second nucleotide to be connected is activated by tetrazole, the 3' phosphate group of the second nucleotide is coupled with the 5' hydroxyl group of the first nucleotide, and a phosphoric acid (or phosphorous acid) triester bond is formed between the two connected nucleotides, so that the method has the advantages of high coupling efficiency, easy separation and purification of a synthesized product, high final yield, very simplified and easy grasp of the whole operation process, and is a main synthesis method (CN 113150041B) at present. However, in the solid phase synthesis of CpG ODN, impurities such as n+, N-, p=o and the like are easily generated, and the impurities are very similar to the structure of CpG ODN, and the separation difficulty is very high, so some solutions are proposed in the prior art, for example:

by adopting a mode of combining two analysis methods, P=O impurities and other impurities are analyzed by anion exchange chromatography, and then N-1 impurities are analyzed by capillary electrophoresis, but the whole analysis process of the method is long in time consumption, more in steps and larger in error.

Chinese patent application CN104195235A discloses a method for detecting CpG ODN sequence purity, and "establishment of method for detecting CpG ODN purity by reverse phase chromatography" (Wang Lili, pharmaceutical biotechnology, 2015, 22 (4): 335-339) discloses a method for detecting CpG ODN by reverse phase chromatography, which has good detection results on N-impurity in CpG ODN synthetic product, but is difficult to distinguish n+ and p=o impurities, thus the purity of target sequence cannot be accurately determined.

Disclosure of Invention

The invention aims to provide a method for detecting the purity of an oligonucleotide compound and related substances in the oligonucleotide compound by using an LC-MS, which solves the problem that a CpG ODN target sequence is indistinguishable from N+, N-, P=O impurities, and realizes the accurate detection of the purity of the CpG ODN target sequence and related substances.

In order to achieve the above purpose, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a method for detecting the purity of an oligonucleotide compound and related substances therein, comprising the steps of:

S1, adding water into a sample to be tested for dissolution or dilution to prepare a solution to be tested;

s2, carrying out liquid chromatography detection on the solution to be detected, entering a mass spectrum detector, and analyzing the part which does not reach baseline separation in the liquid spectrum through mass spectrum;

S3, quantitatively analyzing the purity of the oligonucleotide compound in the sample and related substances in the oligonucleotide compound according to the detection results of the liquid chromatograph and the mass spectrum.

Based on the detection principle of LC-MS and similar structures and constituent units of oligonucleotide compounds, one skilled in the art can envision that the above method is equally applicable to different types of oligonucleotide compounds. For example:

From the viewpoint of the chain structure, the oligonucleotide compounds are single-stranded oligonucleotide compounds and double-stranded oligonucleotide compounds.

From the point of view of the modified structure, the oligonucleotide compound is a modified oligonucleotide compound. In particular, the oligonucleotide compound is a thio-modified oligonucleotide compound.

Preferably, the oligonucleotide compound is a CpG ODN, an antisense oligonucleotide, a small interfering ribonucleic acid (siRNA), a micro ribonucleic acid (miRNA).

In step S1, the water is ultrapure water.

In the step S1, the concentration of the solution to be detected is 2 mug/mL-0.4 mg/mL, preferably 0.02-0.2mg/mL.

In step S2, the conditions for the chromatographic detection are as follows:

Chromatographic column: octadecylsilane chemically bonded silica column;

Column temperature: 60-70deg.C, preferably 65deg.C;

flow rate: 0.2mL/min;

Ultraviolet detector wavelength: 260nm;

Sample injection amount: 5. Mu.L.

In step S2, the mobile phase used for the chromatographic detection is as follows:

The mobile phase A is an aqueous solution containing 90-120mM hexafluoroisopropanol, 7-10mM triethylamine and 143-239mM acetonitrile, preferably the concentration of hexafluoroisopropanol is 100mM, the concentration of triethylamine is 7mM and the concentration of acetonitrile is 191mM.

The mobile phase B consists of 60-80% of mobile phase A by volume fraction and 20-40% of acetonitrile by volume fraction, preferably 70% of mobile phase A by volume fraction and 30% of acetonitrile by volume fraction.

The elution gradient was as follows:

At 0min, mobile phase A was 90% and mobile phase B was 10%;

at 5min, mobile phase A was 81% and mobile phase B was 19%;

at 8min, mobile phase A was 79% and mobile phase B was 21%;

At 13min, mobile phase A was 70% and mobile phase B was 30%;

at 14min, mobile phase A was 90% and mobile phase B was 10%;

at 20min, mobile phase A was 90% and mobile phase B was 10%.

In step S2, the failure to achieve baseline separation means that the degree of separation is less than 1.5.

In step S2, the conditions for mass spectrometry detection are:

Ion source: electrospray ion source ESI;

Ion polarization mode: a negative ion mode;

Capillary voltage: 2.5kV;

taper hole voltage: 5-10V, preferably 5V;

Ion source temperature: 75-100deg.C, preferably 80deg.C;

desolventizing temperature: 300-400 ℃, preferably 400 ℃;

taper hole air flow rate: 50L/h;

desolventizing gas flow rate: 600L/h.

In step S3, the steps of analyzing and detecting the related substances in the oligonucleotide compounds are as follows:

For related substances which can be separated by liquid chromatography, calculating the related substances according to an area normalization method in a liquid chromatogram;

For related substances which cannot be separated by liquid chromatography, the calculation is carried out according to the respective mass spectrum intensity and the ratio after entering the mass spectrum, and the calculation formula is as follows:

related substance mass fraction% = P _UV％×P_{MS Related substances} %

Wherein:

P _UV% = main peak area/total peak area x 100%

P _{MS Related substances} % = related species mass spectrum intensity in main peak/total mass spectrum intensity in main peak x 100%.

In step S3, the relevant substances are impurities generated during the synthesis, storage and transportation of the oligonucleotide compounds. Specifically, the present invention relates to a method for manufacturing a semiconductor device.

The related substances are as follows: n+ impurity, N-impurity, p=o impurity.

The N+ impurities are as follows: n+1.

The N-impurity is as follows: n-1, N-2, N-3, N-4, N-5, N-6, N-7, N-8, N-9, N-10.

The p=o impurity is: p=o generated when the thio reaction in the solid phase synthesis method is not complete, or p=s on the phosphate backbone becomes p=o by slow oxidation during storage.

In step S3, the purity of the oligonucleotide compound is calculated according to the detection results of the liquid chromatography detection and the mass spectrometry detection;

The calculation formula is as follows:

purity% = P _UV％×P_{MS Main component} %

Wherein:

P _UV% = main peak area/total peak area x 100%

P _{MS Main component} % = principal component mass spectrum intensity in principal peak/total mass spectrum intensity in principal peak x 100%.

In a second aspect, the invention further provides the use of the two methods described above for detecting a drug substance or formulation comprising the oligonucleotide compound.

The beneficial effects obtained by the invention are as follows:

1. the invention applies the LC-MS detection technology to the detection of the purity of the oligonucleotide compound and related impurities for the first time, and provides a novel analysis method for the quantification of oligonucleotide drugs.

2. According to the invention, by fumbling the concentration of the solution to be detected and the detection conditions, the more matched detection conditions are determined, the high-efficiency and accurate detection of the purity of the oligonucleotide compound and related substances is realized, and the defect that the oligonucleotide compound is difficult to distinguish from N+, N-, P=O and other impurities is overcome.

Drawings

FIG. 1 is a plot of batch liquid phase integration of WRS007 samples 201908002.

FIG. 2 is a TIC plot of the main peaks (retention time 7.47 min) of a batch of samples from WRS007 201908002.

FIG. 3 is a deconvolution plot of the main peak (retention time 7.47 min) of a batch of samples from WRS007 201908002.

Fig. 4 is a liquid phase integral spectrum of 5 consecutive batches of WRS007 freeze-dried product.

FIG. 5 is a plot of the MS peak of the primary peaks of 5 consecutive batches of WRS007 freeze-dried products.

Fig. 6 is a WRS007 control UV linear (first bar).

Fig. 7 is a WRS007 control MS linearity (first bar).

FIG. 8 shows UV linearity of N-1 (T) impurity control.

FIG. 9 shows linearity of the N-1 (T) impurity control MS.

Fig. 10 is a WRS007 control UV linear (second bar).

Fig. 11 is a WRS007 control MS linearity (second bar).

FIG. 12 shows UV linearity of the N+1 (G) impurity control.

Fig. 13 is a graph showing linearity of the n+1 (G) impurity control MS.

Fig. 14 is a UV linearity of p=o impurity control.

Fig. 15 is a graph of p=o impurity control MS linearity.

FIG. 16 is a chart showing liquid phase integration of CpG-684 202202003 batches of samples; the upper plot is the liquid chromatography integration plot and the lower plot is the TIC peak.

FIG. 17 shows the main component and impurity calculations in the CpG-684 202202003 lot of TIC with a retention time of 8.79 min.

FIG. 18 is a graph showing the purity of CpG-684, an oligonucleotide compound, using method one.

FIG. 19 is a liquid phase UV spectrum of a sample of CpG-684, a compound of a second detector oligonucleotide, using method II.

Fig. 20 is a plot of the localization of the principal component and p=o impurity and n+ impurity in TIC after entry of the principal peak (retention time 7.08 min) in fig. 19 into mass spectrum; the upper graph is the main component, and the lower graph is the impurity.

Fig. 21 is a plot of p=o impurity and n+1 (C) impurity controls at different concentrations in CpG-684 samples.

Fig. 22 is a graph of liquid phase integration of p=o impurity controls and n+1 (C) impurity controls at different concentrations in CpG-684 samples.

Fig. 23 is a TIC integration graph of p=o impurity and n+1 (C) impurity controls added at different concentrations to CpG-684 samples.

Detailed Description

The invention will be further illustrated with reference to the following specific examples, but the invention is not limited to the following examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Reagents, materials, instruments and the like used in the examples described below are commercially available unless otherwise specified.

The compound WRS007 used in the examples below was CpG 6 of Table 1 of CN108728444A, a sequence 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3' (SEQ ID NO: 6), a full thio CpG ODN sequence from Jiangsu Taipren Biotechnology Co.

Example 1 purity of representative Compound WRS007 crude drug and detection of related substances

1. Experimental materials

1.1 Laboratory apparatus

High-resolution quadrupole rod flight time tandem liquid chromatograph-mass spectrometer: model ACGUITY UPLC I-CIASS PIUSXEO G2-XS-QTOF, manufacturer: volter technology (Shanghai) Inc.

An electronic balance: model XS205DU, manufacturer: mertrer.

1.2 Preparation of Experimental solutions

1) Preparation of mobile phase a: 1mL of triethylamine, 10.5mL of hexafluoroisopropanol and 10mL of acetonitrile were placed in a reagent bottle, 975mL of ultrapure water was added, and the mixture was thoroughly and uniformly mixed, and subjected to ultrasonic deaeration to obtain mobile phase A (7 mM triethylamine, 100mM hexafluoroisopropanol).

2) Preparation of mobile phase B: and respectively taking 210mL of mobile phase A and 90mL of acetonitrile, placing the mobile phase A and the acetonitrile into a reagent bottle, uniformly mixing, and carrying out ultrasonic degassing to obtain a mobile phase B (70% of mobile phase A and 30% of acetonitrile).

3) Blank solvent and diluent: ultrapure water was used for 24 hours, and the storage condition was room temperature.

4) Preparation of a System applicability solution: the reference compound WRS007 is weighed into a volumetric flask, dissolved in ultrapure water and scaled to a certain volume, and shaken well to prepare a solution with a concentration of about 0.2 mg/mL.

5) Preparation of a sensitivity solution: and diluting the solution with proper system applicability by 100 times to prepare a solution with the concentration of 0.002 mg/mL.

6) Preparation of test solution: taking a proper amount of each test compound WRS007 bulk drug, respectively placing the bulk drugs into a volumetric flask, adding ultrapure water for dissolution, fixing the volume to a scale, shaking uniformly to prepare a solution with the concentration of about 0.2mg/mL, and preparing 2 parts of test solution in parallel for each sample.

2. LC-MS conditions

2.1 Liquid phase parameters

Chromatographic column: ACQUITY UPLC BEH C18,1.7 μm, 2.1X10 mm;

Mobile phase a:7mM triethylamine, 100mM hexafluoroisopropanol;

mobile phase B: mobile phase a-acetonitrile (70:30);

Ultraviolet detector wavelength: 260nm;

Sample injection amount: 5. Mu.L;

Column temperature: 65 ℃;

flow rate: 0.2mL/min;

Elution gradient:

TABLE 1 elution gradient

Time(min)	Mobile phase A%	Mobile phase B%
			0	90	10
5	81	19
			8	79	21
13	70	30
			14	90	10
20	90	10

2.2 Mass Spectrometry parameters

Table 2 mass spectral parameters

Ion Polarity	negative	Source temperature(℃)	80
				Capillary voltage(kV)	2.5	Desolvation temperature(℃)	400
Sample Cone(V)	5	Cone Gas(L/h)	50
				Ion Source	ESI	Desolvation Gas(L/h)	600

3. Sample injection measurement

Taking blank solvent and each solution according to the liquid chromatography parameters under the 2.1 item and the mass spectrum parameters under the 2.2 item, respectively injecting the blank solvent and each solution into an ultra-high performance liquid chromatograph and a mass spectrometer, operating according to a sample injection sequence shown in the following table, and recording a chromatogram. RSD of main peak area in the 5-needle system applicability solution is less than or equal to 2.0 percent, and RSD of main peak area of all the system applicability solutions after needle return is less than or equal to 2.0 percent.

TABLE 3 sample injection sequence listing

4. Experimental results

The results are detailed in FIGS. 1-5 and Table 4.

As is clear from FIG. 1 (liquid chromatography integration chart), the main peak P _UV% of liquid chromatography was 93.65%. As is clear from fig. 3 (mass spectrum deconvolution chart of the main peak of liquid chromatograph), the MS ratio of the main component in the main peak was 89.6%, the MS ratio of the n+1 (G) impurity was 7.7%, and the MS ratio of the n+1 (C) impurity was 2.6%.

And (3) calculating results:

1) For components that cannot be separated in the liquid phase, after entering the mass spectrum, the calculation is performed according to the respective mass spectrum intensity and the ratio:

main component purity in main peak% = P _UV％×P_{MS Main component} % = 93% = 65% ×89.6% = 83.9%

N+1 (G) impurity in the main peak% = P _UV％×P_{MS Related substances} % = 93.65% ×7.7% = 7.21%

N+1 (C) impurity in the main peak% = P _UV％×P_{MS Related substances} % = 93.65% ×2.6% = 2.43%

2) The purity of the relevant substances which can be separated by liquid chromatography was calculated by an area normalization method in a liquid chromatogram.

3) The peaks in the liquid phase integral plot were assigned as detailed in table 4 below.

TABLE 4 purity of WRS007 crude drug and related substances

From the above results, it can be seen that:

(1) As can be seen from FIGS. 1 and 2, liquid chromatography is capable of analyzing and separating out N-1 impurities. The N+1 impurity is not separated from the main component by baseline, is wrapped in the main peak, and has the separation degree of less than 1.5.

(2) As can be seen from fig. 3, the main peak (retention time 7.47 min) in fig. 1 was identified and analyzed by a mass spectrum detector, and the n+1 (G) impurity, the n+1 (C) impurity and the main component were deconvoluted to obtain a mass-to-charge ratio and a molecular weight, and the identification of the impurity and the main component was realized according to the difference of the molecular weights.

(3) As can be seen from table 4, regarding the substances that can be separated by liquid chromatography, the substances were quantified by the area normalization method in the liquid chromatography; the substances which cannot be separated from the liquid phase enter the mass spectrum, and are quantified according to the respective mass spectrum intensities and the respective duty ratios. Thus, the amount of the main component and the impurity in the compound can be accurately calculated.

(4) As can be seen from FIG. 4 and FIG. 5, the overlapping patterns of the MS patterns of the liquid chromatographic peak and the main peak of 5 batches of samples produced continuously show that the production process is consistent and the impurity spectra are consistent.

Example 2 repeatability

1. Purpose of experiment

The reproducibility of LC-MS analysis of representative compound WRS007 was verified.

2. Experimental materials

2.1 Laboratory apparatus

As in example 1.

2.2 Experimental reagents

Blank solvent and diluent: ultrapure water was used for 24 hours, and the storage condition was room temperature.

WRS007 sample solution preparation: 201907001 and 201909001 batches of WRS007 samples 12.5mg were weighed, placed in a 50mL volumetric flask, dissolved in ultrapure water and diluted to the scale, and 6 samples of solution were prepared in parallel for each batch.

Preparation of mobile phase a: as in example 1.

Preparation of mobile phase B: as in example 1.

3. LC-MS conditions

As in example 1.

4. Sample injection

And respectively injecting the blank solvent and the WRS007 sample solution into an ultra-high performance liquid chromatograph and a mass spectrometer.

5. Calculation of WRS007 purity and related substances

The purity of WRS007 and related substances were calculated as follows:

(1) UV purity calculation: the peak areas of the main peak and each impurity peak are calculated according to an area normalization method, and the UV purity can be obtained through software automatic integration. The main peak UV purity, labeled P _UV%.

P _UV% = main peak area/total peak area x 100%

(2) For the main component and impurities which cannot be separated from the main peak, a mass spectrometer was connected in series after the ultra high performance liquid phase (UPLC), and the region corresponding to the main peak in the liquid phase was analyzed by the mass spectrometer, and the mass spectrum purity was calculated and labeled as P _MS%.

P _MS% = principal component in main peak or individual impurity mass spectrum intensity/total mass spectrum intensity in main peak x 100%

(3) Calculation of the purity of the main component or of the individual impurities which cannot be separated from the main peak;

Purity of main component or respective impurity% = P _UV％×P_MS%

6. Experimental results

See tables 5 and 6.

Table 5 201907001 batch of repeatability verification results

As can be seen from Table 5, 201907001 batches of 100% strength samples of WRS007 were repeatedly assayed 6 times, and the RSD of the total purity of the main component was 0.78%; RSD of impurity n+1 (G) percent is 8.48%; RSD of impurity n+1 (C) percent is 4.46%; the purity of the main component is not more than 2.0% relative standard deviation, and the impurity percentage is not more than 10% relative standard deviation, which indicates that the repeatability verification meets the requirements.

Table 6 201909001 batch of repeatability verification results

As can be seen from Table 6, 201909001 batches of 100% strength samples of WRS007 were repeatedly assayed 6 times, and the RSD of the total purity of the main component was 0.64%; the RSD of the impurity N+1 (G) percentage content higher than the quantitative limit is 6.78%, the RSD of the impurity N+1 (C) percentage content is 7.34%, the requirements of the purity relative standard deviation of the main component of not more than 2.0% and the impurity percentage relative standard deviation of not more than 10% are met, and the repeatability verification meets the requirements.

Example 3, linear

1. Purpose of experiment

By linear measurement of the WRS007 reference substance and the N-1 (T) and P= O, N +1 (G) 3 impurity reference substances, slope correlation coefficients of linear regression of the WRS007 reference substance and the N-1 (T) and P= O, N +1 (G) 3 impurity reference substances are established, the correlation coefficients are 0.9-1.1, and the method can be directly used for purity detection by the percentage ratio of mass spectrum intensity.

2. Experimental materials

2.1 Laboratory apparatus

As in example 1.

2.2 Experimental reagents

Blank solvent and diluent: ultrapure water was used for 24 hours, and the storage condition was room temperature.

A WRS007 control containing 1％(2μg/mL)、1.5％(3μg/mL)、2％(4μg/mL)、5％(10μg/mL)、10％(20μg/mL)、20％(40μg/mL)、50％(100μg/mL)、80％(160μg/mL)、100％(200μg/mL)、120％(240μg/mL)、150％(300μg/mL)、200％(400μg/mL), and N-1 (T), p= O, N +1 (G) 3 impurity control lines of linear solutions were prepared, respectively.

3. LC-MS conditions

As in example 1.

4. Sample injection

5. The blank solvent is advanced, and then a series of linear solutions are added, and the series of linear solutions are sampled according to the order of the concentration from low to high. The receiving requirements are as follows:

1) Linear acceptance criterion

A. And drawing a linear equation by taking the UV peak area of each reference substance component peak as an ordinate and the corresponding concentration as an abscissa, and calculating a linear correlation coefficient R ²,R² to be not less than 0.990.

B. And drawing a linear equation by taking the MS mass spectrum intensity of each reference substance component peak as an ordinate and the corresponding concentration as an abscissa, and calculating a linear correlation coefficient R ²,R² to be not less than 0.990.

The Y-axis intercept of the uv and MS linear equations should be less than 25% of the 100% response value.

2) Acceptance criterion of correlation coefficient

And calculating a correlation coefficient according to the ratio of the slope of the impurity regression line to the slope of the regression line of the WRS007 main component, wherein the correlation coefficient is 0.9-1.1.

6. Experimental results

1) The linear results are shown in FIGS. 6-15.

As can be seen from fig. 6, the first curve 201911001 shows that the concentration of WRS007 control is 0.002187mg/mL to 0.4375mg/mL (1% -200%), the UV peak area of each concentration is linear with the corresponding concentration, y=562305.909x+1540.570, r ² is 0.998, and the Y-axis intercept is less than 25% of the 100% response value.

As can be seen from fig. 7, the MS intensity of each concentration is linearly related to the corresponding concentration, the MS linear equation is y=462878.310x+206386.131, r ² is 0.998, and the Y-axis intercept is less than 25% of the 100% response value.

As can be seen from FIG. 8, the N-1 (T) impurity control was in the concentration range of 0.002152 mg/mL-0.4305 mg/mL (1% -200%), the UV peak area of each concentration was in a linear relationship with the corresponding concentration, Y=598065.395X+2312.053, R ² was 0.997, and the Y-axis intercept was less than 25% of the 100% response value.

As can be seen from fig. 9, the MS intensity at each concentration is in a linear relationship with the corresponding concentration, y=43055971.880x+372264.421, r ² is 0.991, and Y-axis intercept is less than 25% of the 100% response value; the N-1 (T) impurity reference substance has good linear relation in the concentration range of 0.002 mg/mL-0.4 mg/mL (1% -200%).

As can be seen from fig. 10, the second curve 201911001 of WRS007 controls was in the concentration range of 0.002187mg/mL to 0.4375mg/mL (1% -200%), the UV peak area of each concentration was in a linear relationship with the corresponding concentration, y=555829.707x+1427.630, r ² was 0.998, and the Y-axis intercept was less than 25% of the 100% response.

As can be seen from fig. 11, the MS intensities at the respective concentrations are in a linear relationship with the corresponding concentrations, the MS linear equation is y=67006742.260x+290604.939, r ² is 0.996, and the Y-axis intercept is less than 25% of the 100% response value.

As can be seen from fig. 12, the n+1 (G) impurity control was in a concentration range of 0.002110mg/mL to 0.4220mg/mL (1% -200%), the UV peak area of each concentration was in a linear relationship with the corresponding concentration, y=521115.167x+2915.468, r ² was 0.996, and the Y-axis intercept was less than 25% of the 100% response value.

As can be seen from fig. 13, the MS intensity at each concentration is in a linear relationship with the corresponding concentration, y= 66838153.270X-142614.215, r ² is 0.996, and the Y-axis intercept is less than 25% of the 100% response; the N+1 (G) impurity reference substance has good linear relation in the concentration range of 0.002 mg/mL-0.4 mg/mL (1% -200%).

As can be seen from fig. 14, the p=o impurity control was in a concentration range of 0.002186mg/mL to 0.4372mg/mL (1% -200%), the UV peak area of each concentration was in a linear relationship with the corresponding concentration, y=551334.501x+2154.058, r ² was 0.998, and the Y-axis intercept was less than 25% of the 100% response value.

As can be seen from fig. 15, the MS intensity at each concentration is in a linear relationship with the corresponding concentration, y=6713355x+306849.312, r ² is 0.996, and Y-axis intercept is less than 25% of the 100% response value; the P=O impurity reference substance has good linear relation in the concentration range of 0.002 mg/mL-0.4 mg/mL (1% -200%).

2) Correlation coefficient results

The results are detailed in Table 7.

TABLE 7 correlation coefficient table of impurities and principal components

As can be seen from the results of Table 7, the correlation coefficient between the slope of the regression line of the N-1 (T) impurity and the main component is: the UV correction factor was 1.06 and the ms correction factor was 0.93. The slope of the regression line of the n+1 (G) impurity and the slope of the regression line of the main component were 0.94 for the UV correction factor and 1.00 for the ms correction factor. The slope of the regression line for p=o impurity and the slope of the regression line for the principal component are: the UV correction factor was 0.99 and the ms correction factor was 1.00. Therefore, the correlation coefficients of the N-1 (T), the N+1 (G) and the P=O impurities and the main component are all in the range of 0.9-1.1, and meet the requirements, so that the mass spectrum intensity percentage is directly adopted for purity detection.

3) Conclusion:

The WRS007 reference substance, the N-1 (T) reference substance, the P=O impurity reference substance and the N+1 (G) impurity reference substance all have good linear relationship in the concentration range of 0.002 mg/mL-0.4 mg/mL (1% -200%).

Example 4 detection of the purity of oligonucleotide Compounds of different sequences

1. Purpose of experiment

Verification of the LC-MS analysis method is suitable for detecting the purity of the compound CpG-684 and related substances

2. Experimental materials

2.1 Laboratory apparatus

As in example 1.

2.2 Experimental reagents

Blank solvent and diluent: ultrapure water was used for 24 hours, and the storage condition was room temperature.

CpG-684 sample solution preparation: a202202003 batch of CpG-684 (also known as CpG PV 001) samples (12.5 mg) was weighed, placed in a 50mL volumetric flask, dissolved in ultrapure water to dilute to scale, and 2 sample solutions were prepared in parallel.

Preparation of mobile phase a: as in example 1.

Preparation of mobile phase B: as in example 1.

3. LC-MS conditions

As in example 1.

4. Sample injection

And respectively injecting the blank solvent and the CpG-684 sample solution into an ultra-high performance liquid chromatograph and a mass spectrometer.

5. Calculation of CpG-684 purity and related substances

The purity of CpG-684 and the related substances are calculated as follows:

(1) UV purity calculation: the peak areas of the main peak and each impurity peak are calculated according to an area normalization method, and the UV purity can be obtained through software automatic integration. The main peak UV purity, labeled P _UV%.

P _UV% = main peak area/total peak area x 100%

(2) For the main component and impurities which cannot be separated from the main peak, a mass spectrometer was connected in series after the ultra high performance liquid phase (UPLC), and the region corresponding to the main peak in the liquid phase was analyzed by the mass spectrometer, and the mass spectrum purity was calculated and labeled as P _MS%.

P _MS% = principal component in main peak or individual impurity mass spectrum intensity/total mass spectrum intensity in main peak x 100%

(3) Calculation of the purity of the main component or of the individual impurities which cannot be separated from the main peak;

Purity of main component or respective impurity% = P _UV％×P_MS%

6. Experimental results

The results are shown in detail in FIGS. 16 to 17.

And (3) calculating results:

1) The liquid chromatography cannot be separated, analysis is carried out through mass spectrometry, deconvolution software is used for deconvolution of the liquid phase, and the mass spectrum intensity and the duty ratio of the corresponding main component and impurities are calculated. The deconvolution results are shown in FIG. 17, and after analysis according to deconvolution software, the MS ratio of the main component is 81.82%, the MS ratio of the main component +K is 6.19%, the MS ratio of the main component +Na is 6.64%, and the MS ratio of the depurination A impurity is 5.35%. Therefore, the actual ratio of the main component to each impurity in the liquid chromatograph can be calculated.

Main component purity in main peak%p _UV％×P_{MS Main component} %

=93.44% × (81.82% (Main component) +6.64% (+na) +6.19% (+k)

＝88.44％

Apurinic a impurity in the main peak% = P _UV％×P_{MS Related substances} % = 93% = 44% ×5.35% = 5.00%

2) The related substances which can be separated by liquid chromatography are subjected to purity calculation according to an area normalization method in a liquid chromatogram.

3) The peaks in the liquid phase integral plot were assigned as detailed in table 8 below.

TABLE 8 CpG-684 202202003 batch liquid phase diagrams for impurity assignment

Comparative example 1 determination of oligonucleotide Compound purity Using different liquid phase methods

1. Purpose of experiment

The purity of the oligonucleotide compound CpG-684 and the detection effect of the related substances are compared by different analysis methods.

The method comprises the following steps: reference is made to the method described in "establishment of method for detecting CpG ODN purity by reverse phase chromatography" (Wang Lili, biotechnology of medicine, 2015, 22 (4): 335-339)

The second method is as follows: the method of the invention

2. Experimental materials

2.1 Laboratory apparatus: the details are given in the following table.

2.2 Method one Experimental reagent

2.2.1 Method one experimental reagent:

1) Blank solvent and diluent: mobile phase

2) Sample solution preparation: 16.5mg of the sample was weighed, placed in a 50mL volumetric flask, and dissolved and diluted to a scale with ultrapure water to prepare a sample solution having a concentration of 0.33mg/mL.

2.2.2 Method two experimental reagents:

1) Blank solvent and diluent: ultrapure water was used for 24 hours, and the storage condition was room temperature.

2) CpG-684 sample solution preparation: 12.5mg of CpG-684 sample is weighed and placed in a 50mL volumetric flask, dissolved and diluted to a scale by ultrapure water, and the concentration of the prepared sample solution is 0.2mg/mL.

3) P=o impurity control solution preparation: 12.5mg of P=O impurity reference substance is weighed, placed in a 50mL volumetric flask, dissolved and diluted to a scale with ultrapure water, and the concentration of the P=O impurity reference substance solution is prepared to be 0.2mg/mL.

4) Preparation of N+1 (C) impurity reference substance solution: 12.5mg of the N+1 (C) impurity reference substance is weighed, placed in a 50mL volumetric flask, dissolved and diluted to a scale by ultrapure water, and the concentration of the N+1 (C) impurity reference substance solution is 0.2mg/mL.

5) P=o impurity and n+1 (C) impurity plus standard solution preparation: 1ml of 0.2mg/ml CpG-684 sample is removed to a 10ml volumetric flask, 25 μl, 50 μl, 75 μl and 100 μl of the P=O impurity reference solution and the N+1 (C) impurity reference solution of 0.2mg/ml are respectively added, and the volumes are respectively fixed to scale marks, so as to obtain standard adding solutions with P=O impurity concentration and N+1 (C) impurity concentration of 2.5%, 5%, 7.5% and 10%.

3. The test method comprises the following steps: see Table 9 for details.

Table 9 parameters of detection for both methods

4. Detection result

4.1 Purity was determined by the method I, by referring to the method for detecting the purity of CpG ODN sequences, by the method for detecting CpG ODN by reverse phase chromatography disclosed in "establishment of method for detecting CpG ODN purity by reverse phase chromatography" (Wang Lili, biotechnology of medicine, 2015, 22 (4): 335-339). The Xbridge OST C18 column in its patent is shut down and Xbridge BEH C18 is used instead. The detection results are shown in FIG. 18.

As can be seen from FIG. 18, the purity of the oligonucleotide compounds can be detected by the method, and only N-1 and N-2 impurities can be separated, and impurities which are very close to the molecular weight and the charge number of the target substance in the nucleic acid sample can not be effectively separated and detected, for example, the following steps: p=o impurity and n+ impurity.

4.2 Adopting a second method, namely the method disclosed by the invention is characterized in that the detection spectrum is as follows:

Fig. 19 is a liquid phase UV spectrum of CpG-684 sample, and fig. 20 is a localization map of the main component and p=o impurity and n+ impurity in TIC after the main peak (retention time 7.08 min) in fig. 19 enters mass spectrum.

Fig. 21 is a plot of p=o impurity and n+1 (C) impurity controls at different concentrations in CpG-684 samples. Fig. 22 is a graph showing the liquid phase integration of the control with different concentrations of p=o impurity and n+1 impurity added to CpG-684 samples to observe the effect of liquid phase separation. Fig. 23 is a TIC integral graph of a control with different concentrations of p=o impurities and n+1 impurities added to CpG-684 samples.

5. Conclusion(s)

As can be seen from FIG. 19, the liquid phase detection method of the present invention can separate not only N-1 and N-2 impurities but also N-3, N-4, N-5 and other impurities. But the liquid phase process is also difficult to separate for p=o impurity and n+1 impurity. Therefore, the concentrations of the p=o impurity reference substance and the n+1 (C) impurity reference substance added to the sample are respectively 2.5%, 5%, 7.5% and 10% as verified by the respective labeling experiments.

As can be seen from fig. 21 to 22, as the concentration of p=o impurity and the concentration of n+1 (C) impurity increases, the liquid phase with the concentration of n+1 impurity being 2.5% can be identified by automatic integration, but when the concentration of p=o impurity reaches 7.5%, a significantly small peak is observed, and the automatic integration of the liquid phase cannot be identified until the concentration of p=o reaches 10%, but the requirement of the separation degree of 1.5 cannot be met.

As can be seen from fig. 23, mass spectrometry was performed on the substance corresponding to the main peak period of 7.08min in fig. 21 by a mass spectrometer, the mass-to-charge ratio of the p=o impurity was 664.8m/z, the mass-to-charge ratio of the main component was 666.3m/z, and the mass-to-charge ratio of the n+1 (C) impurity was 694.1m/z, and the mass spectrum was quantified according to the percentage of the mass spectrum intensity of the impurity and the main component.

Based on the detection principle of LC-MS and similar structure and constituent units of oligonucleotide compounds, one skilled in the art would envision that the above method is equally applicable to the detection of the purity and related substances of oligonucleotide compound CpG-684、CpG-685、D-SL01、CpG-1018、CpG-1862、CpG-2006、CpG-2007、CpG-2216、CpG-2336、CpG-2395、CpG-M362、D-SL03、G10 CpG ODN、SD-101 shown in table 10.

TABLE 10 oligonucleotide compound sequences

Numbering device	Code number	Sequence(s)
			1	CpG-684	5’-tcg acg ttc gtc gtt cgt cgt tc-3’(23mer)
2	CpG-685	5’-tcgtcgacgtcgttcgttct c-3’(21mer)
			3	D-SL01	5’-tcg cga cgt tcg ccc gac gtt cgg ta-3’(26mer)
4	CpG-1018	5’-tgactgtgaa cgttcgagat ga-3’(22mer)
			5	CpG-1826	5’-tccatgacgttcctgacgtt-3’(20mer)
6	CpG-2006	5’-tcgtcgttttgtcgttttgtcgtt-3’(24mer)
			7	CpG-2007	5’-tcg tcg ttg tcg ttt tgt cgt t-3’(22mer)
8	CpG-2216	5’-ggGGGACGA:TCGTCgggggg-3’(20mer)
			9	CpG-2336	5’-gggGACGAC:GTCGTGgggggg-3’(21mer)
10	CpG-2395	5’-tcgtcgttttcggcgc:gcgccg-3’(22mer)
			11	CpG-M362	5’-tcgtcgtcgttc:gaacgacgttgat-3’(25mer)
12	D-SL03	5’-tcg cga acg ttc gcc gcg ttc gaa cgc gg-3’(29mer)
			13	G10 CpG ODN	5'-GGGGGGGGGGGACGATCGTCGGGGGGGGGG-3'
14	SD-101	5'-tcgaacgttc gaacgttcga acgttcgaat-3’(30-mer)

All of the above compounds are available from Jiangsu Tiapril Biotechnology Co.

The embodiment proves that a collaborative detection method combining liquid chromatography and mass spectrometry detection is established, namely, the liquid chromatography technology is utilized to carry out chromatographic detection on the oligonucleotide compound, then the mass spectrum detector is utilized to carry out mass spectrometry analysis on substances corresponding to a time period which does not reach baseline separation, and the purity of the main component and related substances in the oligonucleotide compound are accurately determined according to the chromatographic detection result and the mass spectrum detection result.

The method for liquid chromatography-mass spectrometry has high repeatability and accuracy, and through methodological verification, linearity and repeatability are in a verification acceptable range, the method can identify impurities which cannot realize baseline separation in a liquid phase, and the method has high sensitivity and can accurately quantify related impurities and target objects in the compound. The method is suitable for detecting the purity of pure products of oligonucleotide compounds with different sequences and related substances.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (7)

1. A method for detecting the purity of an oligonucleotide compound and related substances therein, comprising the steps of:

S1, adding water into a sample to be tested for dissolution or dilution to prepare a solution to be tested;

s2, carrying out liquid chromatography detection on the solution to be detected, entering a mass spectrum detector, and analyzing the part which does not reach baseline separation in the liquid spectrum through mass spectrum;

S3, quantitatively analyzing the purity of the oligonucleotide compound in the sample and related substances in the oligonucleotide compound according to detection results of the liquid chromatograph and the mass spectrum;

in step S2, the conditions for the chromatographic detection are as follows:

Chromatographic column: octadecylsilane chemically bonded silica column;

column temperature: 60-70 ℃;

flow rate: 0.2mL/min;

Ultraviolet detector wavelength: 260nm;

Sample injection amount: 5. Mu.L;

the mobile phase used for the chromatographic detection is as follows:

mobile phase A is aqueous solution containing 90-120mM hexafluoroisopropanol, 7-10mM triethylamine and 143-239mM acetonitrile;

the mobile phase B consists of 60-80% of mobile phase A by volume fraction and 20-40% of acetonitrile by volume fraction;

The elution gradient was as follows:

At 0min, mobile phase A was 90% and mobile phase B was 10%;

at 5 min, mobile phase A was 81% and mobile phase B was 19%;

At 8min, mobile phase A was 79% and mobile phase B was 21%;

At 13 min, mobile phase A was 70% and mobile phase B was 30%;

at 14 min, mobile phase A was 90% and mobile phase B was 10%;

At 20 min, mobile phase A was 90% and mobile phase B was 10%;

In step S2, the conditions for mass spectrometry detection are:

Ion source: electrospray ion source ESI;

Ion polarization mode: a negative ion mode;

Capillary voltage: 2.5kV;

Taper hole voltage: 5-10V;

Ion source temperature: 75-100 ℃;

Desolventizing temperature: 300-400 ℃;

taper hole air flow rate: 50L/h;

desolventizing gas flow rate: 600L/h;

In step S3, the steps of analyzing and detecting the related substances in the oligonucleotide compounds are as follows:

For related substances which can be separated by liquid chromatography, calculating the related substances according to an area normalization method in a liquid chromatogram;

For related substances which cannot be separated by liquid chromatography, calculating according to the respective mass spectrum intensity and the ratio after entering a mass spectrum;

The calculation formula is as follows:

Related substance mass fraction% = P _UV%×P_{MS Related substances} %

Wherein:

P _UV% = main peak area/total peak area x 100%

P _{MS Related substances} % = related species mass spectrum intensity in main peak/total mass spectrum intensity in main peak x 100%;

in step S3, the purity of the oligonucleotide compound is calculated according to the detection results of the liquid chromatography detection and the mass spectrometry detection;

The calculation formula is as follows:

Purity% = P _UV%×P_{MS Main component} %

Wherein:

P _UV% = main peak area/total peak area x 100%

P _{MS Main component} % = principal component mass spectrum intensity in principal peak/total mass spectrum intensity in principal peak x 100%.

2. The method according to claim 1, characterized in that: in the step S1, the concentration of the solution to be detected is 2 mug/mL-0.4 mg/mL.

3. The method according to claim 1, characterized in that: the oligonucleotide compound is as follows: single-stranded oligonucleotide compounds and double-stranded oligonucleotide compounds.

4. The method according to claim 1, characterized in that: the oligonucleotide compound is a modified oligonucleotide compound.

5. The method according to claim 1, characterized in that: the oligonucleotide compound is a thio-modified oligonucleotide compound.

6. The method according to claim 1, characterized in that: the oligonucleotide compound is CpG ODN, antisense oligonucleotide, small interference ribonucleic acid and micro ribonucleic acid.

7. Use of the method of any one of claims 1-6 for detecting a drug substance or formulation comprising said oligonucleotide compound.