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CN-121978238-A - Method for confirming oligonucleotide sequence by liquid chromatography-mass spectrometry

CN121978238ACN 121978238 ACN121978238 ACN 121978238ACN-121978238-A

Abstract

The invention provides a method for confirming an oligonucleotide sequence by liquid chromatography-mass spectrometry, and relates to the technical field of instrument analysis. The confirmation method comprises the steps of detecting a sample solution containing oligonucleotides by adopting an IPRP-UPLC-MS/MS method to obtain fragment ion data, and processing the fragment ion data by adopting a CONFIRM Sequence to obtain a Sequence matching rate. The invention adopts IPRP-LC-MS/MS to detect the sample solution and adopts CONFIRM Sequence to process data so as to obtain the Sequence matching rate. The invention adopts the IPRP high performance liquid chromatography and QTOF MS combined method to analyze, has the advantages of short analysis time, strong substantivity, high analysis precision, high resolution, high accuracy, simple operation and the like, and achieves the aim of identifying the oligonucleotide sequences.

Inventors

  • SHEN LIHUI
  • LIU TING
  • HOU LIQIANG

Assignees

  • 杭州诺泰诺和生物医药科技有限公司

Dates

Publication Date
20260505
Application Date
20260206

Claims (10)

  1. 1. A method for confirming an oligonucleotide sequence by liquid chromatography-mass spectrometry, comprising: Detecting a sample solution containing the oligonucleotides by adopting an IPRP-UPLC-MS/MS method to obtain fragment ion data; And processing the fragment ion data by adopting CONFIRM Sequence to obtain a Sequence matching rate.
  2. 2. The method for confirming oligonucleotide sequences for liquid chromatography-mass spectrometry according to claim 1, wherein the sample solution containing the oligonucleotide drug is prepared by the steps of: Dissolving the oligonucleotide to be detected in water to obtain a sample solution containing the oligonucleotide; Preferably, the concentration of the sample solution containing the oligonucleotide is 0.1-1.0 mg/mL.
  3. 3. The method for confirming a sequence of an oligonucleotide used in combination with liquid chromatography-mass spectrometry according to claim 1, wherein a WATERS PREMIER ultra high performance liquid chromatograph is used for the liquid chromatography in the detection and a Waters Xevo G3 high performance time-of-flight tandem mass spectrometer is used for the mass spectrum in the detection.
  4. 4. The method for confirming a sequence of an oligonucleotide used in combination with liquid chromatography-mass spectrometry according to claim 1, wherein the chromatographic column used for the detection is an ostbhe C18 chromatographic column; Preferably, the specification parameters of the chromatographic column used for detection comprise that the length of the chromatographic column is 50-150 mm, the inner diameter of the chromatographic column is 1.5-4.6 mm, and the particle size of the filler is 1.5-5.0 mu m; Preferably, the chromatographic column used for the detection is Waters Acquity Premier OST BEH C chromatographic column with specification of length 100 mm, inner diameter 2.1 mm and filler particle size 3.5 μm.
  5. 5. The method for confirming a sequence of an oligonucleotide used in combination with liquid chromatography-mass spectrometry according to claim 1, wherein mobile phase a used in the detection liquid chromatography comprises dibutylamine acetate, acetonitrile and water, and mobile phase B comprises dibutylamine acetate, acetonitrile and water; Preferably, in the mobile phase A, the concentration of the dibutylamine acetate is 5-15 mM, the volume percentage of the acetonitrile is 5-15%, and the balance is water; Preferably, in the mobile phase B, the concentration of the dibutylamine acetate is 5-15 mM, the volume percentage of the acetonitrile is 75-90%, and the balance is water; Preferably, the pH of the mobile phase a and the mobile phase B are each independently 7.0 to 8.0.
  6. 6. The method for confirming a sequence of an oligonucleotide used in combination with liquid chromatography-mass spectrometry according to claim 1, wherein the gradient elution conditions used in the detection liquid chromatography are as follows: 0min, 85-95% of mobile phase A and 5-15% of mobile phase B; 2 min, 85-95% of mobile phase A and 5-15% of mobile phase B; 12 min, 5-15% of mobile phase A and 85-95% of mobile phase B; 12.1 min, 85-95% of mobile phase A and 5-15% of mobile phase B; 15 min, 85-95% of mobile phase A and 5-15% of mobile phase B; Preferably, the gradient elution conditions employed in the liquid chromatography in the detection are as follows: 0min, 90% of mobile phase A and 10% of mobile phase B; 2 min, 90% of mobile phase A and 10% of mobile phase B; 12 min, 10% of mobile phase A and 90% of mobile phase B; 12.1 min, 90% of mobile phase A and 10% of mobile phase B; 15 min, 90% of mobile phase A and 10% of mobile phase B.
  7. 7. The method for confirming the oligonucleotide sequence used in the liquid chromatography-mass spectrometry according to claim 1, wherein the conditions adopted in the detection comprise a wavelength of 250-270 nm, a flow rate of 0.1-1.0 mL/min, a sample injection volume of 3-10 mu L, a column temperature of 35-70 ℃ and a temperature of 2-10 ℃ of a sample injection disk.
  8. 8. The method for confirming a liquid chromatography-mass spectrometry combined oligonucleotide sequence according to claim 1, wherein the conditions adopted in the detection of mass spectrometry comprise ionization mode of ESI, scanning mode of negative polarity, analysis mode of sensitivity mode, parent ion of 1782.06 m/z, capillary voltage of 1.5-2.5 kV, sample taper hole voltage of 40-100V, taper hole gas flow of 45-55L/h, desolvation gas flow of 700-900L/h, ion source temperature of 100-150 ℃, desolvation temperature of 300-450 ℃, mass range of 500-3000 m/z, collision energy voltage of 10-50V and scanning time of 0.5-2 s.
  9. 9. The method for confirming oligonucleotide sequences for liquid chromatography-mass spectrometry according to claim 1, wherein the mobile phase a and mobile phase B are formulated and then subjected to the following post-treatment steps; Filtering the mobile phase A and the mobile phase B through microporous filter membranes respectively, and then carrying out ultrasonic degassing.
  10. 10. The method for confirming a sequence of an oligonucleotide used in combination with liquid chromatography-mass spectrometry according to claim 9, wherein the pore size of the microporous filter membrane is 0.20-0.25 μm; Preferably, the power of the ultrasonic deaeration is 400-600W, the frequency is 40-60 kHz, and the time of the ultrasonic deaeration is 5-30 min.

Description

Method for confirming oligonucleotide sequence by liquid chromatography-mass spectrometry Technical Field The invention relates to the technical field of instrument analysis, in particular to a method for confirming an oligonucleotide sequence by liquid chromatography-mass spectrometry. Background Oligonucleotide drugs (e.g., ASO, siRNA, aptamer, etc.) are a class of short-chain nucleic acid molecules consisting of tens to tens of nucleotides. The correctness of the sequence is the fundamental guarantee of the pharmacological activity, the specificity and the safety. Thus, accurate sequence validation (Sequence Confirmation) of the post-synthesis peptide is a critical component in drug development and quality control. Sequencing of oligonucleotide drugs is primarily to confirm whether the synthesized product is consistent with the designed theoretical sequence. This includes detecting the presence of sequence impurities such as deletions, insertions, base-pairing errors, and the like. Because oligonucleotides have the characteristics of high polarity, high charge, easy modification, complex structure and the like, the sequence analysis of the oligonucleotides is required to depend on high-resolution and high-selectivity analysis technology. The oligonucleotide sequencing method based on mass spectrum has high sensitivity, high resolution and direct structural analysis capability, can realize sequence confirmation through fragment ions, and can accurately identify base deletion, modification and degradation impurities, thereby being the most reliable sequence confirmation means at present. Compared with the traditional indirect methods such as enzymolysis and electrophoresis, the MS-based sequencing can obtain the molecular weight, structure and purity information simultaneously in one-time analysis, and has the remarkable advantages of high flux, strong quantification, visual result and the like. However, the technology also has the challenges of complex data analysis, huge fragment patterns and the like. Despite the complexity of nucleotide mass spectrometry fragmentation processes, the popularity of LC-MS/MS technology in the field of oligonucleotide sequencing has benefited from the continued development of bioinformatics tools that can be used to facilitate data analysis and improve throughput. The present invention uses UPLC-MS/MS (QTOF MS) technology and CONFIRM Sequence application by Waters corporation to automatically Sequence synthetic oligonucleotides and their impurities by automatically processing the MS/MS spectra of oligonucleotides obtained from targeted MS/MS or non-targeted MS E (DIA). In view of this, the present invention has been made. Disclosure of Invention The invention provides a method for confirming an oligonucleotide sequence by combining liquid chromatography-mass spectrometry, which combines a high-resolution mass spectrometry technology with intelligent software analysis, and solves the problems of lack of research on the oligonucleotide sequence confirming and analyzing method in the prior art, complex data processing, low confirming efficiency and the like in the traditional method. In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: In a first aspect, the present invention provides a method for validating a sequence of an oligonucleotide for liquid chromatography-mass spectrometry, the method comprising: Detecting a sample solution containing the oligonucleotides by adopting an IPRP-UPLC-MS/MS method to obtain fragment ion data; And processing the fragment ion data by adopting CONFIRM Sequence to obtain a Sequence matching rate. Further, the sample solution containing the oligonucleotide drug is prepared by the following steps: dissolving the oligonucleotide to be detected in water to obtain the sample solution containing the oligonucleotide. Further, the concentration of the sample solution containing the oligonucleotide is 0.1-1.0 mg/mL, preferably 0.2-1.0 mg/mL. Further, WATERS PREMIER ultra-high performance liquid chromatography is used for the in-detection liquid chromatography, and a Waters Xevo G3 high performance time-of-flight tandem mass spectrometer is used for the in-detection mass spectrum. Further, the chromatographic column used in the detection is an OST BHE C18 chromatographic column. Further, the chromatographic column used in the detection may be another C18 chromatographic column equivalent to the OST BHE C18 chromatographic column, for example, may be Waters ACQUITY Premier, ACQUITY UPLC column. Further, the specification parameters of the chromatographic column used for detection comprise that the length of the chromatographic column is 50-150 mm, the inner diameter of the chromatographic column is 1.5-4.6 mm, and the particle size of the filler is 1.5-5.0 mu m. Further, the chromatographic column used in the detection is Waters Acquity Premier OST BEH C chromatographic column, its