A non-ion pair LC-MS analytical method for the identification of oligonucleotide drug metabolites

Abstract

The application provides a non-ion pair LC-MS analysis method for identifying metabolites of an oligonucleotide drug. The hydrophilic interaction chromatography technology does not require ion pair reagents, has good mass spectrometry compatibility, and avoids mass spectrometry pollution caused by ion pair reagents. Preferably, a mixed solvent of hexafluoroisopropanol-ammonium hydroxide is used as a mobile phase additive, which significantly improves the mass spectrometry response of the oligonucleotide, and rich and comprehensive secondary mass spectrometry fragment information is obtained under the optimized collision energy, which is helpful for rapid analysis of metabolites. The method has the advantages of high efficiency, accuracy and rapidness, and meets the requirements of rapid metabolite identification and analysis of chemically modified oligonucleotide drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a non-ion-pair liquid chromatography-mass spectrometry method for the identification of chemically modified oligonucleotide drug metabolites. Background Technology

[0002] With continuous improvements in oligonucleotide chemical modification and its widespread application to various targets, the development of oligonucleotide therapy has grown exponentially, with a rapid increase in oligonucleotide drugs approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) in recent years. To improve their affinity for binding to targets, metabolic stability, and pharmacokinetics, various chemical modification methods for oligonucleotides have been developed, including bioconjugation (e.g., N-acetylgalactosamine conjugation, GalNAc3), phosphodiester linkage (e.g., phosphate thio), and ribose modification (e.g., 2'-O-methyl, 2'-fluorine). Understanding the metabolic pathways of chemically modified oligonucleotides is crucial for drug development. Unlike small molecule drugs, which are primarily metabolized by cytochrome P450 enzymes, oligonucleotides are mainly metabolized by exonucleases and endonucleases, producing truncated oligonucleotides. Furthermore, due to the numerous modifications involved, oligonucleotide drugs exhibit greater heterogeneity, and the impact of chemical modifications on metabolic stability and biotransformation pathways remains unclear. Therefore, there is an urgent need to develop analytical techniques capable of fully analyzing the metabolites of these oligonucleotide drugs.

[0003] Hybridization enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qPCR) are highly sensitive and widely used in the analysis of oligonucleotides. However, these techniques cannot distinguish between truncated metabolites and full-length oligonucleotides. Ion-pair reversed-phase liquid chromatography-mass spectrometry (IPC-MS) has been widely used in the bioanalysis of oligonucleotides. This technique typically uses alkylamines and hexafluoroisopropanol as ion-pairing reagents. While this improves the chromatographic retention and mass spectrometric response of oligonucleotides, the high proton affinity of these reagents and their tendency to adhere to the surface of the mass spectrometer easily lead to contamination, resulting in problems such as analyte ion suppression, poor detection reproducibility, mobile phase aging, and shortened column life. Hydrophilic interaction chromatography is a preferred method for analyzing polar and hydrophilic compounds, but its application in the identification of oligonucleotide drug metabolites has not yet been explored. Summary of the Invention

[0004] To overcome the problems of mass spectrometry contamination, poor reproducibility, mobile phase aging, and short column life inherent in traditional ion-pair reversed-phase liquid chromatography-mass spectrometry, this invention develops a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method and applies it to the identification of chemically modified oligonucleotide drug metabolites, which includes the following steps: In the above method, preferably, the solution preparation includes: 100 μM N11-GalNAc solution and 100 μM N9 solution; nuclease buffer (pH 8.0) containing 100 mM Tris-HCl and 1 mM magnesium acetate; 50 mM ammonium acetate solution (pH 5.5) as SPE equilibration buffer; mixing the SPE equilibration buffer with acetonitrile at a 1:1 ratio to prepare SPE washing buffer; preparing 200 mM ammonium bicarbonate solution (pH 9.5) and mixing it with acetonitrile at a 1:1 ratio to prepare elution buffer.

[0005] In the above method, preferably, the liver homogenate is prepared from SD rats, and the specific method includes: Rat livers were perfused with ice-cold phosphate-buffered saline (PBS) free of calcium or magnesium. The livers were removed and placed in ice-cold nuclease buffer for further chopping. Cold nuclease buffer was added to each sample and homogenized.

[0006] In the above method, preferably, the sample incubation and pretreatment include: Take 20 μL of N11-GalNAc or N9 solution (final concentration 10 μM), add 120 μL of liver homogenate (333.3 mg / mL protein) and 60 μL of nuclease buffer, mix, and incubate at 37 °C. After incubation for the specified time (0, 1, 6 and 24 hours), add 450 μL of ice-cold lysis buffer to terminate the reaction. Use 20 μL of ultrapure water instead of oligonucleotide solution. Metabolic studies for each group are prepared in parallel.

[0007] Adjust the SPE column using 1 mL methanol and 1 mL equilibration buffer. Load the sample, already mixed with lysis buffer, onto the SPE column. Wash the SPE column with 3 mL wash buffer and elute the analyte with 0.5 mL elution buffer. Evaporate the eluent to near dryness under nitrogen and reconstitute with the initial mobile phase solvent (a mixture of 45 mM ammonium hydroxide and 100 mM hexafluoroisopropanol aqueous solution: acetonitrile, 2:8). Vortex dissolve and transfer to a 1.5 mL EP tube. In the above method, preferably, the chromatographic column used for liquid chromatography separation is a NanoChromChromCore HILIC-Amide column, 3.0 μm, 2.1 × 100 mm. The mobile phase is: Phase A is an aqueous solution containing 45 mM ammonium hydroxide and 100 mM hexafluoroisopropanol, and Phase B is acetonitrile.

[0008] In the above method, preferably, the liquid chromatography separation and elution conditions are as follows: Gradient elution: 0.0 ~ 5.0 min, 80%-50% B phase; 20%-50% A phase; 5.0 ~ 12.0 min, 50% B phase; 50% A phase; 12.0 ~ 12.5 min, 50%-80% B phase; 50%-20% A phase; 12.5 ~ 25.0 min, 80% B phase; 20% A phase; Wash-off time: 25.0 min; Flow rate: 0.100 mL / min; Injection volume: 10.00 μL; Autosampler temperature: 6℃; Column temperature: 40℃.

[0009] In the above method, preferably, the mass spectrometry conditions are: Ion source: Thermo-spray ionization source (HESI); Detection method: Negative ion mode; Scan range: m / z 400-2000 Full scan resolution: 70000; dd-MS 2 Resolution: 17500; Spray voltage: -3200 V; Capillary temperature: 325℃ HESI probe heater temperature: 300℃; Step normalized collision energy (NCE): 25, 35, and 45.

[0010] The beneficial effects of this invention are: (1) This invention utilizes a HILIC-HRMS method for oligonucleotide metabolites using HFIP-NH4OH as the mobile phase additive. This method provides sufficient retention and resolution for the metabolic analysis of chemically modified oligonucleotides in rat liver homogenate. Traditional HILIC mobile phase acid modifiers use ammonium formate, which causes significant mass spectrometry signal suppression. HFIP, however, is a volatile mobile phase additive that can adjust the solution pH and is more easily volatilized in the gas phase under electrospray ionization conditions. Therefore, it promotes the ionization efficiency of oligonucleotides and enhances the mass spectrometry signal intensity of oligonucleotides. Furthermore, HFIP has a low boiling point, making it more compatible with electrospray mass spectrometry and enabling efficient negative ion mode electrospray ionization. Normalized collision energy (NCE) provides information fragment ions with near 100% coverage of the oligonucleotide sequence, and metabolites can be identified by comparing characteristic fragments. Twenty-seven N11-GalNAc (modified with thiophosphate, 2'-fluorine, 2'-O-methyl and 3'-N-acetylgalactosamine) and 29 N9 (modified with thiophosphate and 2'-fluorine) metabolites were identified.

[0011] (2) The HILIC-HRMS method used in this invention covers a wide range of mass numbers and chromatographic elution intensities, and can capture truncated metabolites down to one nucleotide, thus supporting metabolic studies of various chemically modified oligonucleotides.

[0012] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1HILIC-MS extraction ion chromatograms of N11-GalNAc and N9 prototypes under different mobile phase A conditions were obtained, where: (A) 5 mM ammonium formate + 15 mM ammonium hydroxide (pH 10); (B) 5 mM ammonium formate + 15 mM ammonium hydroxide + 50 mM hexafluoroisopropanol (pH 9.01); (C) 5 mM ammonium formate + 15 mM ammonium hydroxide + 100 mM hexafluoroisopropanol (pH 8.33); (D) 5 mM ammonium formate + 15 mM ammonium hydroxide + 200 mM hexafluoroisopropanol (pH 7.96); (E) 2 mM ammonium formate + 15 mM ammonium hydroxide + 50 mM hexafluoroisopropanol (pH 8.91); (F) 45 mM ammonium hydroxide + 100 mM hexafluoroisopropanol (pH 9.16).

[0015] Figure 2 Extraction ion chromatogram of metabolites of N11-GalNac.

[0016] Figure 3 The extracted ion chromatogram of N9 metabolites.

[0017] Figure 4 This is a first-order full-scan mass spectrum of 3' n-11, a representative metabolite of N11-GalNAc.

[0018] Figure 5 Full scan mass spectrum of fragment ions of 3' n-11, a representative metabolite of N11-GalNAc (including full spectrum and magnified views of three mass number ranges).

[0019] Figure 6 This is a first-order full-scan mass spectrum of 5' n-6, a representative metabolite of N9.

[0020] Figure 7 Full scan mass spectra of fragment ions of 5' n-6, a representative metabolite of N9 (including full spectrum and magnified views of three mass number ranges). Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except for those specifically mentioned, are all common knowledge and general knowledge in the art, and the present invention does not have any special limitations.

[0022] This invention addresses the shortcomings of existing technologies by establishing analytical methods based on liquid chromatography and mass spectrometry.

[0023] I. Chromatographic Analysis: A ChromCore HILIC-Amide column was used for gradient elution [0.0 ~ 5.0 min, 80%-50% phase B; 20%-50% phase A; 5.0 ~ 12.0 min, 50% phase B; 50% phase A; 12.0 ~ 12.5 min, 50%-80% phase B; 50%-20% phase A; 12.5 ~ 25.0 min, 80% phase B; 20% phase A]. Mobile phase A was an aqueous solution containing 45 mM NH4OH and 100 mM HFIP, and mobile phase B was acetonitrile. The ChromCore HILIC-Amide column used in this invention provides good retention of both the analyte and the internal standard. Elution time: 25.0 min; flow rate: 0.100 mL / min; injection volume: 10.00 μL; autosampler temperature: 6℃; column temperature: 40℃.

[0024] II. Mass Spectrometry Analysis: Mass spectrometry analysis was performed on a Q-Exactive Plus Orbitrap mass spectrometer using a thermoelectric spray ionization (HESI) source, with a scan range of [missing information]. m / z Operating in negative ion mode, the frequency range is 400-2000. Full scan data and dd-MS were acquired at resolutions of 70000 and 17500, respectively. 2 Data: The spray voltage used was -3200 V, the capillary temperature was 325℃, and the HESI probe heater temperature was 300℃; the step normalized collision energy (NCE) was set to 25, 35, and 45.

[0025] The present invention will now be described in detail through specific embodiments: 1. Materials 1.1 Instruments Chromatograph: Thermo Scientific SII liquid chromatography system, including VF-D11-A UV detector, VF-P10-A infusion pump, VF-A10-A autosampler, and VH-C10-A column oven.

[0026] Mass spectrometer: Thermo Scientific Q Exactive UHMR combined quadrupole Orbitrap mass spectrometer, equipped with a thermoelectric spray ionization source (HESI source) and a UHPLC liquid chromatography system.

[0027] Data software: Xcalibur and Freestyle, Thermo Scientific, USA.

[0028] Centrifuge: Sorvall Legend Micro 21 microcentrifuge, Thermo Scientific, USA.

[0029] Analytical balance: MS105DU Mettler Toledo electronic balance, Mettler Toledo Instruments GmbH, Switzerland.

[0030] 1.2 Reference Standards and Reagents Modified RNA (N11-GalNAc and N9, purity >90%) was synthesized by Chengdu Pioneer Biotechnology Co., Ltd. Hexafluoroisopropanol (chromatographic grade, 99.8%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Ammonium hydroxide (chromatographic grade, 25% in water) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Ammonium bicarbonate was purchased from Shanghai Haohong Biochemical Technology Co., Ltd. Sodium chloride (analytical grade, 99.5%), potassium dihydrogen phosphate (analytical grade, 99.5%), disodium hydrogen phosphate dodecahydrate (analytical grade, 99.0%), and potassium chloride (analytical grade, 99.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Magnesium acetate (chromatographic grade) was purchased from Beijing MREDA Technology Co., Ltd. Tris-HCl was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Clarity OTX SPE columns and lysis buffer (containing 56%-58% guanidine hydrochloride and 1%-3% Triton X-100) were purchased from Finnemax Inc. (USA). Acetonitrile (HPLC grade) was purchased from Sigma-Aldrich (USA).

[0031] 2 Methods 2.1 Solution Preparation Prepare 100 μM N11-GalNAc solution and 100 μM N9 solution; prepare nuclease buffer (pH 8.0) containing 100 mM Tris-HCl and 1 mM magnesium acetate; prepare 50 mM ammonium acetate solution (pH 5.5) as SPE equilibration buffer; mix SPE equilibration buffer with acetonitrile at a 1:1 ratio to prepare SPE washing buffer; prepare 200 mM ammonium bicarbonate solution (pH 9.5) and mix with acetonitrile at a 1:1 ratio to prepare elution buffer.

[0032] 2.2 Preparation of rat liver homogenate Liver homogenates were prepared from SD rats (170-180 g) purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China). Briefly, rats were anesthetized, and the livers were perfused via the portal vein at a flow rate of 35 mL / min with ice-cold phosphate-buffered saline (PBS) free of calcium or magnesium. The livers were then removed and further chopped in ice-cold nuclease buffer (a mixture of 1 mM magnesium acetate and 100 mM Tris-HCl aqueous solution, pH 8.0). Cold nuclease buffer was added to each sample before homogenization. Protein concentrations in the liver homogenates were determined using a dicendamide assay kit (Pierce, Rockford, IL, USA), and the liver homogenates were stored at -70°C.

[0033] 2.3 Incubation and processing of liver homogenate samples

[0034] 2.4 Chromatographic and Mass Spectrometric Conditions Chromatographic conditions:

[0035] Mass spectrometry conditions:

[0036] 3.0 Results and Discussion This invention develops a non-ion-pair liquid chromatography-mass spectrometry (LC-MS / MS) method for the identification of chemically modified oligonucleotide metabolites. The method was developed and optimized by considering both LC separation and mass spectrometry detection conditions. The effects of different mobile phase compositions on chromatographic retention and mass spectrometric response were investigated, and the results are shown in the appendix. Figure 1 As shown, reducing the concentration of ammonium formate (AF) in the mobile phase and introducing HFIP into the mobile phase both improve the LC-MS / MS intensity. However, at higher HFIP concentrations, the ion responses of N9 and N11-GalNAc differ under different charge states. These results indicate that HFIP has different effects on the multi-charge ion distribution of oligonucleotides under HILIC-MS conditions. Further optimization of the mobile phase showed that using a mixture of 45 mM NH4OH and 100 mM HFIP (pH=9.16) as an aqueous mobile phase additive significantly improves the LC-MS / MS intensity (see attached diagram). Figure 1 F). The above results indicate that the traditional HILIC mobile phase acid modifier, ammonium formate, causes significant mass spectrometry signal suppression. In contrast, HFIP, a volatile mobile phase additive, can adjust the solution pH and is more easily volatilized in the gas phase of electrospray ionization. Therefore, it has the characteristics of promoting the ionization efficiency of oligonucleotides and enhancing the mass spectrometry signal intensity of oligonucleotides.

[0037] For the establishment of the mass spectrometry method, this invention employed the NCE / HRMS method to study the nucleotide fragmentation behavior of N11-GalNAc and N9. NCE provides informative fragment ions with near 100% coverage of oligonucleotide sequences, and metabolites can be identified by comparing characteristic fragments. Using this method, this invention identified 27 related metabolites of N11-GalNAc and 29 related metabolites of N9, and their extracted ion chromatograms are shown below. Figure 2 , 3 As shown in the figure. The first-order full-scan mass spectrum of 3'n-11, a representative metabolite of N11-GalNAc, and the first-order full-scan mass spectrum of 5'n-6, a representative metabolite of N9, are shown in the figure. Figure 4 , 6 As shown. Characteristic fragments of nucleotides, dinucleotides, and trinucleotides resulting from c / y-, d / y-, and b / y-type fragmentation were detected in the fragment ion full-scan mass spectra of these two metabolites, and they were assigned (…). Figure 5 , Figure 7 This further confirmed the nucleotide sequences of N11-GalNAc metabolites and N9 metabolites, verifying that the detection method developed in this invention can provide highly complete oligonucleotide sequence coverage.

Claims

1. A non-ionic pair hydrophilic interaction liquid chromatography-high-resolution mass spectrometry method for identifying metabolites of chemically modified oligonucleotide drugs in rat liver homogenate, characterized in that, Includes the following steps: (1) The oligonucleotide drug solution to be tested was mixed with rat liver homogenate and nuclease buffer to make the final concentration of the oligonucleotide drug 10 μM and the protein concentration of the rat liver homogenate 333.3 mg / mL. The mixture was incubated at 37 °C and the reaction was terminated by adding ice-cold lysis buffer at 0, 1, 6 or 24 hours. (2) The incubated sample after termination was subjected to solid phase extraction. The eluent was evaporated to near dryness under nitrogen flow and then reconstituted with a mixture of the initial mobile phase A and the volume ratio of mobile phase B of 2:

8. (3) Separation was performed using a NanoChromChromCore HILIC-Amide column with a column size of 3.0 μm and 2.1 × 100 mm. Mobile phase A was an aqueous solution of 45 mM ammonium hydroxide and 100 mM hexafluoroisopropanol with a pH of 9.

16. Mobile phase B was acetonitrile. The gradient elution program was: 0.0~5.0 min, with phase B decreasing from 80% to 50%; 5.0~12.0 min. min, phase B remains at 50%; 12.0~12.5 min, phase B increases from 50% to 80%; 12.5~25.0 min, phase B remains at 80%; (4) Q-Exactive Orbitrap high-resolution mass spectrometer and thermoelectric spray ionization source are used for detection in negative ion mode, with a scanning range of m / z 400~2000, a data-dependent secondary scan resolution of 17500, and step normalization collision energies set to 25, 35 and 45. The nucleotide sequences of chemically modified oligonucleotide metabolites are confirmed by using c / y type, d / y type and b / y type characteristic fragment ions obtained from the step normalization collision energies.

2. The method according to claim 1, characterized in that, The chemically modified oligonucleotide drug is any one of the following: (i) an oligonucleotide containing thiophosphate and 2'-fluorine modification; (ii) an oligonucleotide simultaneously containing thiophosphate, 2'-fluorine, 2'-O-methyl and 3'-N-acetylgalactosamine modification.

3. The method according to claim 1, characterized in that, The solid-phase extraction process includes: adjusting the SPE column with 1 mL methanol and 1 mL equilibration buffer, loading the terminated incubation sample, washing the SPE column with 3 mL washing buffer, and eluting the analyte with 0.5 mL elution buffer; wherein the equilibration buffer is a 50 mM ammonium acetate solution with a pH of 5.5; the washing buffer is a mixture of the equilibration buffer and acetonitrile at a volume ratio of 1:1; the elution buffer is a 200 mM ammonium bicarbonate solution with a pH of 9.5, mixed with acetonitrile at a volume ratio of 1:1; and retaining approximately 20 μL of solvent during nitrogen drying.

4. The method according to claim 1, characterized in that, The preparation of the rat liver homogenate includes: perfusing the rat liver with ice-cold phosphate buffer that does not contain calcium or magnesium, removing the liver and then placing it in ice-cold nuclease buffer to chop and homogenize; the nuclease buffer contains 100 mM Tris-HCl and 1 mM magnesium acetate, and the pH is 8.

0.

5. The method according to claim 1, characterized in that, The liquid chromatography flow rate was 0.100 mL / min, the injection volume was 10.00 μL, the autosampler temperature was 6 ℃, and the column temperature was 40 ℃. The mass spectrometry spray voltage was -3200 V, and full scan data and data-dependent secondary scan (dd-MS2) data were acquired at resolutions of 70000 and 17500, respectively. The capillary temperature was 325 ℃, and the thermoelectric spray ionization probe heater temperature was 300 ℃.

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