Method for detecting nucleotide, cap analogue and nucleic acid in in-vitro transcription product

Abstract

The invention provides a method for analyzing NTP, cap analogues and mRNA components in in-vitro transcription product components.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese Patent Application No. 2023108891018 filed on July 19, 2023, which is incorporated herein by reference in its entirety. 1. Technical Field

[0003] The invention relates to the technical fields of mRNA drug process development, NTPs analysis and mRNA analysis, and in particular to a high-resolution and high-sensitivity ion-pair reverse phase chromatography method for simultaneous rapid separation and detection of NTP, cap analogs and mRNA. 2. Background technology

[0004] Naturally occurring RNA is synthesized from four basic ribonucleotides: ATP, CTP, UTP, and GTP. A variety of different nucleoside modifications have been applied to prepare RNA by in vitro transcription (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). Through in vitro transcription (IVT), natural ribonucleotides and modified ribonucleotides can be used to synthesize mRNA, screen RNA polymerases (RNAPs), analyze promoter strength, screen drugs that inhibit transcription, etc. The quality of in vitro transcription directly affects the quality of mRNA stock solution, thereby affecting the quality and yield of mRNA drugs. Therefore, analyzing the components of IVT products and optimizing the IVT system are of great significance to the pharmaceutical industry. 3. Summary of the invention

[0005] The present invention provides a method for analyzing nucleoside triphosphates (NTPs), cap analogs and polynucleotides (e.g., RNA polynucleotides, mRNA polynucleotides) in in vitro transcription product components based on ultra-high performance liquid chromatography (UPLC), comprising the following steps: (1) preparing a sample of in vitro transcription product for UPLC detection and (2) detecting using the UPLC method.

[0006] In certain embodiments, the present invention provides a method for analyzing NTP, cap analog and mRNA components in an in vitro transcription product, comprising the following steps: (1) preparing a sample of the in vitro transcription product for UPLC detection and (2) detecting using the UPLC method.

[0007] In certain embodiments, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) the separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 11 minutes: 84% mobile phase A + 16% mobile phase B; 22 minutes: 0% mobile phase A + 100% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and (c) Chromatogram of NTPs, cap analogs, and mRNA collected from in vitro transcription product components.

[0008] In certain embodiments, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) the separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 11+T minutes: 84% mobile phase A + 16% mobile phase B; 22+T+U minutes: 0% mobile phase A + 100% mobile phase B; 25+T+U minutes: 0% mobile phase A + 100% mobile phase B; 25.1+T+U minutes: 95% mobile phase A + 5% mobile phase B; 30+T+U minutes: 95% mobile phase A + 5% mobile phase B; and

[0009] (c) collecting a chromatogram of NTP, cap analog and mRNA in the in vitro transcription product components; wherein the range of T value is: 0≤T≤25; the range of U value is: -5≤U≤10. In certain embodiments, the detection by UPLC method in step (2) comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) The separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: (100%-P%) mobile phase A + P% mobile phase B; 11 minutes: (100%-Q%) mobile phase A + Q% mobile phase B; 22 minutes: (100%-R%) mobile phase A + R% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and (c) Collecting chromatograms of NTP, cap analogs and mRNA in the components of in vitro transcription products; wherein the range of P value is: 5≤P≤10, the range of Q value is: 10≤Q≤16, and the range of R value is: 16≤R≤100.

[0010] In certain embodiments, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) The separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: (100% - P%) mobile phase A + P% mobile phase B; 11 + T minutes: (100% - Q%) mobile phase A + Q% mobile phase B; 22 + T + U minutes: (100% - R%) mobile phase A + R% mobile phase B; 25 + T + U minutes: 0% mobile phase A + 100% mobile phase B; 25.1 + T + U minutes: 9 5% mobile phase A + 5% mobile phase B; 30+T+U minutes: 95% mobile phase A + 5% mobile phase B; and (c) collecting chromatograms of NTPs, cap analogs and mRNA in the in vitro transcription product components; wherein the range of T values ​​is: 0≤T≤25; the range of U values ​​is: -5≤U≤10; the range of P values ​​is: 5≤P≤10, the range of Q values ​​is: 10≤Q≤16, and the range of R values ​​is: 16≤R≤100.

[0011] In one embodiment, the analytical method further comprises the following quantitative step: d) Collect chromatograms of NTP, cap analog and mRNA in the mixed standard solution at no less than three concentration levels e) After taking the log of the concentrations and peak areas of NTP, cap analog and mRNA in the mixed standard solution, a standard curve is drawn to obtain a linear fitting equation; and f) Calculate the concentration of each NTP and cap analog based on the linear fitting equation of the standard.

[0012] In one embodiment, the NTP is native CTP, UTP, GTP, or ATP.

[0013] In another embodiment, the NTP comprises two or more natural NTPs, wherein the natural NTPs include: CTP, UTP, GTP, and ATP.

[0014] In another embodiment, the NTP is a modified NTP.

[0015] In another embodiment, the modified NTP is modified UTP: N1-methyl-pseudouridine triphosphate.

[0016] In another embodiment, the NTP comprises two or more NTPs, wherein the NTP is selected from: CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0017] In another embodiment, the NTP is CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0018] In another embodiment, the NTP is CTP, UTP, GTP, and ATP.

[0019] In one embodiment, the cap analog is a trinucleotide cap analog. In certain embodiments, the cap analog is: 7 (3'OMeG)(5')ppp(5')G(ARCA),m 7 G(5')ppp(5')(2'OMeA)pG、m 7 (3'OMeG)(5')ppp(5')(2'OMeA)pG,m 7 G(5')ppp(5')(2'OMeA)pU、m 7 G(5')ppp(5')(2'OMeG)pG, or m 7 (3'OMeG)(5')ppp(5')(2'OMeG)pG.

[0020] In another embodiment, the cap analog is: 7 G(5')ppp(5')(2'OMeA)pG

[0021] In another embodiment, the cap analog is a compound of formula (1):

[0022] In certain embodiments, the cap analog is a dinucleotide cap analog.

[0023] In certain embodiments, the cap analog is a trinucleotide cap analog. In certain embodiments, the cap analog is: m7(3'OMeG)(5')ppp(5')G(ARCA), m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, m7G(5') ppp(5')(2'OMeG)pG, or m7(3'OMeG)(5')ppp(5')(2'OMeG)pG.

[0024] In certain embodiments, the cap analog is a tetranucleotide cap analog.

[0025] In one embodiment, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) the separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 11 minutes: 84% mobile phase A + 16% mobile phase B; 22 minutes: 0% mobile phase A + 100% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and

[0026] (c) Collecting chromatograms of NTP, cap analogs and mRNA in the components of the in vitro transcription product. In one embodiment, the mobile phase A in (a) contains 200mM triethylamine-acetic acid and 100mM butanediamine-acetic acid. In one embodiment, the mobile phase B in (a) is an acetonitrile aqueous solution containing 200mM triethylamine-acetic acid and 100mM butanediamine-acetic acid with a volume concentration of 30%-60%. In one embodiment, the C18 column in (b) is a hybrid particle bonded C18 column with an inner diameter of 2.1mm and a particle size of 1.7μm. In certain embodiments, the total flow rate of mobile phase A and phase B in (b) is: 0.2-0.6mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.2mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.3mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.4 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.5 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.6 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.2 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.3 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.4 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.5 mL / min. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.6 mL / min. In certain embodiments, the column temperature of the C18 column in (b) is 50-70°C. In one embodiment, the column temperature of the C18 column in (b) is 50°C. In one embodiment, the column temperature of the C18 column in (b) is 55°C. In one embodiment, the column temperature of the C18 column in (b) is 60°C. In one embodiment, the column temperature of the C18 column in (b) is 65°C. In one embodiment, the column temperature of the C18 column in (b) is 70°C. In one embodiment, the column temperature of the C18 column in (b) is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C. In one embodiment, the column temperature of the C18 column in (b) is 60°C. In certain embodiments, the detection wavelength of the target color peak in (c) is 250-270nm. In one embodiment, the detection wavelength of the target color peak in (c) is 250 nm. In one embodiment, the detection wavelength of the target color peak in (c) is 260 nm.In one embodiment, the detection wavelength of the target color peak in (c) is 265 nm. In one embodiment, the detection wavelength of the target color peak in (c) is 270 nm. In certain embodiments, step (1) adopts a method comprising the following steps: (i) the in vitro transcription product is terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; the termination mixture in (i) is precipitated by 2-4 M LiCl, washed with 70% ethanol, and the precipitate is re-dissolved in enzyme-free water to obtain an in vitro transcribed mRNA stock solution sample for UPLC detection.

[0027] In certain embodiments, step (1) adopts a method comprising the following steps: (i) terminating the in vitro transcription product with 20-100 mM EDTA (final concentration) to obtain a termination mixture; purifying the termination mixture in (i) by chromatography or other means to obtain a sample of the in vitro transcription product for UPLC detection.

[0028] In certain embodiments, step (1) adopts a method comprising the following steps: (i) terminating the in vitro transcription product with 20-100 mM EDTA (final concentration) to obtain a termination mixture; and diluting the termination mixture in (i) 20-100 times with enzyme-free water to obtain a sample of the in vitro transcription product for UPLC detection.

[0029] In certain embodiments, the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes to 150 minutes.

[0030] In certain embodiments, the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes, 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 130 minutes, or 150 minutes.

[0031] In certain embodiments, the in vitro transcription product in step (1) is any mRNA in vitro transcription product.

[0032] In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 4173nt. In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 500-1000nt. In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 2000-3000nt. In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 3000-4000nt. In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 4000-10000nt. In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, 1500nt, 2000nt, 2500nt, 3000nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, or 10000nt.

[0033] In certain embodiments, step (d) comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mixed standard solutions of different concentrations.

[0034] In certain embodiments, step (d) comprises six concentration levels of NTP in the mixed standard solution.

[0035] In certain embodiments, the six concentration levels of the mixed standard solutions are: Mixed standard solution 1: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 1 μM concentrations, and mRNA concentration of 0.015625 mg / mL; Mixed standard solution 2: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 4 μM concentrations, and mRNA concentration of 0.03125 mg / mL; Mixed standard solution 3: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 16 μM, and mRNA concentration of 0.0625 mg / mL; Mixed standard solution 4: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 64 μM, and mRNA concentration of 0.125 mg / mL; Mixed standard solution 5: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 256 μM, and mRNA at 0.25 mg / mL; Mixed standard solution 6: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 500 μM, respectively, and mRNA at a concentration of 0.5 mg / mL;

[0036] In certain embodiments, the method has a limit of quantitation, LOQ, of 1 μM for NTPs and cap analogs.

[0037] In certain embodiments, the method has a limit of quantitation (LOQ) concentration of mRNA of 0.004 mg / mL.

[0038] In certain embodiments, the standard curve concentration range is: NTP and cap analog concentration range is 1-500 μM

[0039] In certain embodiments, the standard curve concentration range is: mRNA concentration range is 0.004-0.5 mg / mL.

[0040] The method according to embodiment 2 is characterized in that: the standard curve fitting method is: after taking the Log value of the concentration and the peak area, a linear fit is performed. In certain embodiments, the relationship obtained by the linear fit is: CTP: y = 1.1213x - 1.4971; N1-methyl-pseudouridine triphosphate: y = 1.098x-1.4653; UTP: y = 1.118x - 1.3636; GTP: y = 1.2619x - 1.5964; ATP: y = 1.1325x - 1.2056; m 7 G(5')ppp(5')(2'OMeA)pG:y=1.0172x-0.6385; mRNA:y=1.2323x+2.4969 Where x is the Log(concentration) value and y is the Log(peak area) value.

[0041] In certain embodiments, the R2 of the fitted linear equation is > 0.99.

[0042] In certain embodiments, the quantitative accuracy of the method within the concentration range of claim 10 is 90%-110%. 3.1. Implementation plan

[0043] 1. A method for analyzing NTP, cap analog and mRNA components in in vitro transcription product components based on UPLC, comprising the following steps: (1) preparing a sample of in vitro transcription product for UPLC detection and (2) detecting using the UPLC method; Wherein step (2) adopts UPLC method to carry out detection and comprises: a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), pH 6.90-7.10, and mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, pH 6.90-7.10; b) using a C18 column for chromatographic separation: the separation gradient program is as follows: initial 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 2 minutes: 90% mobile phase A + 10% mobile phase B; 11 minutes: 84% mobile phase A + 16% mobile phase B; 22 minutes: 0% mobile phase A + 100% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and c) Collect chromatograms of NTPs, cap analogs and mRNA in the in vitro transcription product components.

[0044] 2. The method according to embodiment 1, wherein the analytical method further comprises the following quantitative step: d) Collect chromatograms of NTP, cap analog and mRNA in the mixed standard solution at no less than three concentration levels e) After taking the log of the concentrations and peak areas of NTP, cap analog and mRNA in the mixed standard solution, a standard curve is drawn to obtain a linear fitting equation; and f) Calculate the concentration of each NTP and cap analog based on the linear fitting equation of the standard.

[0045] 3. The method according to embodiment 1 or 2 is characterized in that the NTP is natural CTP, UTP, GTP, or ATP.

[0046] 4. The method according to embodiment 1 or 2 is characterized in that: the NTP comprises two or more natural NTPs, wherein the natural NTPs include: CTP, UTP, GTP, and ATP.

[0047] 5. The method according to embodiment 1 or 2, characterized in that the NTP is a modified NTP.

[0048] 6. The method according to embodiment 1 or 2 is characterized in that the modified NTP is modified UTP: N1-methyl-pseudouridine triphosphate.

[0049] 7. The method according to embodiment 1 or 2 is characterized in that the modified NTP is N6-methyl-adenosine triphosphate, N1-methyl-adenosine triphosphate, 2-amino-adenosine triphosphate, 2-oxymethyl-adenosine triphosphate, 5-methyl-cytidine triphosphate, 5-hydroxymethyl-cytidine triphosphate, N4-acetyl-cytidine triphosphate, 2-oxymethyl-cytidine triphosphate, N7-methyl-guanosine triphosphate, 2-oxymethyl-guanosine triphosphate, N1-methyl-pseudouridine triphosphate, pseudouridine triphosphate, 2-thio-uridine triphosphate, 5-methoxy-uridine triphosphate, or 2-oxymethyl-uridine triphosphate.

[0050] 8. The method according to embodiment 1 or 2 is characterized in that: the NTP comprises two or more NTPs, wherein the NTP is selected from: CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0051] 9. The method according to embodiment 1 or 2 is characterized in that the NTP is CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0052] 10. The method according to embodiment 1 or 2, characterized in that the NTP is CTP, UTP, GTP, and ATP.

[0053] 11. The method according to embodiment 1 or 2 is characterized in that the NTP is CTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0054] 12. The method according to embodiment 1 or 2 is characterized in that the cap analog is a trinucleotide cap analog.

[0055] 13. The method according to embodiment 1 or 2, characterized in that: the hat analog is m 7 (3'OMeG)(5')ppp(5')G(ARCA),m 7 G(5')ppp(5')(2'OMeA)pG、m 7 (3'OMeG)(5')ppp(5')(2'OMeA)pG,m 7 G(5')ppp(5')(2'OMeA)pU、m 7 G(5')ppp(5')(2'OMeG)pG, or m 7(3'OMeG)(5')ppp(5')(2'OMeG)pG.

[0056] 14. The method according to embodiment 1 or 2, characterized in that: the hat analog is: m 7 G(5')ppp(5')(2'OMeA)pG

[0057] 15. The method according to embodiment 1 or 2 is characterized in that the cap analog is a tetranucleotide cap analog.

[0058] 16. The method according to embodiment 1 or 2 is characterized in that the cap analog is a dinucleotide cap analog.

[0059] 17. The method according to embodiment 1 or 2 is characterized in that the mobile phase A in a) contains 200 mM triethylamine-acetic acid and 100 mM butanediamine-acetic acid.

[0060] 18. The method according to embodiment 1 or 2 is characterized in that the mobile phase B in a) is an acetonitrile aqueous solution containing 200 mM triethylamine-acetic acid and 100 mM butanediamine-acetic acid with a volume concentration of 30%-60%.

[0061] 19. The method according to embodiment 1 or 2 is characterized in that the C18 column in b) is a hybrid particle bonded C18 column with an inner diameter of 2.1 mm and a particle size of 1.7 μm.

[0062] 20. The method according to embodiment 1 or 2 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.2 to 0.6 mL / min.

[0063] 21. The method according to embodiment 1 or 2 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.2 mL / min.

[0064] 22. The method according to embodiment 1 or 2 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.3 mL / min.

[0065] 23. The method according to embodiment 1 or 2 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.4 mL / min.

[0066] 24. The method according to embodiment 1 or 2 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.5 mL / min.

[0067] 25. The method according to embodiment 1 or 2 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.6 mL / min.

[0068] 26. The method according to embodiment 1 or 2, characterized in that the column temperature of the C18 column in b) is 50-70°C.

[0069] 27. The method according to embodiment 1 or 2, characterized in that the column temperature of the C18 column in b) is 50°C.

[0070] 28. The method according to embodiment 1 or 2, characterized in that the column temperature of the C18 column in b) is 60°C.

[0071] 29. The method according to embodiment 1 or 2, characterized in that the column temperature of the C18 column in b) is 70°C.

[0072] 30. The method according to embodiment 1 or 2 is characterized in that the detection wavelength of the target color peak in c) is 250-270nm.

[0073] 31. The method according to embodiment 1 or 2 is characterized in that the detection wavelength of the target color peak in c) is 260 nm.

[0074] 32. The method according to embodiment 1 or 2, characterized in that step (1) adopts a method comprising the following steps: i. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; ii. The termination mixture described in i was precipitated with 2-4M LiCl, washed with 70% ethanol, and the precipitate was redissolved in enzyme-free water to obtain an in vitro transcribed mRNA stock solution sample for UPLC detection.

[0075] The method according to embodiment 1 or 2 is characterized in that step (1) adopts a method comprising the following steps: i. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; ii. Purify the termination mixture described in i by chromatography or other means to obtain a sample of the in vitro transcription product for UPLC detection.

[0076] 34. The method according to embodiment 1 or 2, characterized in that step (1) adopts a method comprising the following steps: i. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; and ii. Dilute the termination mixture described in i by 20-100 times with enzyme-free water to obtain a sample of the in vitro transcription product for UPLC detection.

[0077] 35. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes to 150 minutes.

[0078] 36. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes, 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 130 minutes, or 150 minutes.

[0079] 37. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is any mRNA in vitro transcription product.

[0080] 38. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an in vitro mRNA transcription product with a length of 500-1000 nt.

[0081] 39. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of mRNA with a length of 1000-2000nt.

[0082] 40. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of mRNA with a length of 2000-3000 nt.

[0083] 41. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 3000-4000nt.

[0084] 42. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of mRNA with a length of 4000-10000 nt.

[0085] 43. The method according to embodiment 1 or 2 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 4173 nt.

[0086] 44. The method of embodiment 2, wherein step (d) comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mixed standard solutions of different concentrations.

[0087] 45. The method of embodiment 2, wherein step (d) comprises NTP in a mixed standard solution at 6 concentration levels.

[0088] 46. ​​The method according to embodiment 25, characterized in that the mixed standard solutions at 6 concentration levels are: Mixed standard solution 1: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 1 μM concentrations, and mRNA concentration of 0.015625 mg / mL; Mixed standard solution 2: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 4 μM concentrations, and mRNA concentration of 0.03125 mg / mL; Mixed standard solution 3: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 16 μM, and mRNA concentration of 0.0625 mg / mL; Mixed standard solution 4: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 64 μM, and mRNA concentration of 0.125 mg / mL; Mixed standard solution 5: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 256 μM, and mRNA at 0.25 mg / mL; Mixed standard solution 6: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 500 μM, respectively, and mRNA at a concentration of 0.5 mg / mL;

[0089] 47. The method according to embodiment 2 is characterized in that the limit of quantification LOQ of the method is: 1 μM for NTP and cap analogs.

[0090] 48. The method according to Embodiment 2 is characterized in that the LOQ concentration of the method is: mRNA concentration 0.004 mg / mL.

[0091] 49. The method according to embodiment 2, characterized in that the concentration range of the standard curve is: the concentration range of NTP and cap analog is 1-500 μM

[0092] 50. The method according to Embodiment 2 is characterized in that the concentration range of the standard curve is: the mRNA concentration range is 0.004-0.5 mg / mL.

[0093] 51. The method according to Embodiment 2 is characterized in that: the standard curve fitting method is: after taking Log values ​​of both concentration and peak area, linear fitting is performed.

[0094] 52. The method according to embodiment 36, characterized in that the relationship obtained by the linear fitting is: CTP: y = 1.1213x - 1.4971; N1-methyl-pseudouridine triphosphate: y = 1.098x-1.4653; UTP: y = 1.118x - 1.3636; GTP: y = 1.2619x - 1.5964; ATP: y = 1.1325x - 1.2056; m 7 G(5')ppp(5')(2'OMeA)pG:y=1.0172x-0.6385; mRNA:y=1.2323x+2.4969 Where x is the Log(concentration) value and y is the Log(peak area) value.

[0095] 53. The method according to implementation scheme 2 is characterized in that: R2 of the fitted linear equation is greater than 0.99.

[0096] 54. The method according to embodiment 2 is characterized in that the quantitative accuracy of the method within the concentration range of claim 10 is 90%-110%.

[0097] 55. A method for analyzing NTP, cap analog and mRNA components in in vitro transcription product components based on UPLC, comprising the following steps: (1) preparing a sample of in vitro transcription product for UPLC detection and (2) detecting using the UPLC method;

[0098] Wherein step (2) adopts UPLC method to carry out detection and comprises: a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), pH 6.90-7.10, and mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, pH 6.90-7.10; b) using a C18 column for chromatographic separation: the separation gradient program is as follows: initial 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 2 minutes: 90% mobile phase A + 10% mobile phase B; 11+T minutes: 84% mobile phase A + 16% mobile phase B; 22+T+U minutes: 0% mobile phase A + 100% mobile phase B; 25+T+U minutes: 0% mobile phase A + 100% mobile phase B; 25.1+T+U minutes: 95% mobile phase A + 5% mobile phase B; 30+T+U minutes: 95% mobile phase A + 5% mobile phase B; and c) collecting chromatograms of NTP, cap analogs and mRNA in the in vitro transcription product components; The range of T value is: 0≤T≤25; the range of U value is: -5≤U≤10.

[0099] 56. The method according to embodiment 55, wherein the analytical method further comprises the following quantitative step: d) Collect chromatograms of NTP, cap analog and mRNA in the mixed standard solution at no less than three concentration levels e) After taking the log of the concentrations and peak areas of NTP, cap analog and mRNA in the mixed standard solution, a standard curve is drawn to obtain a linear fitting equation; and f) Calculate the concentration of each NTP and cap analog based on the linear fitting equation of the standard.

[0100] 57. The method according to embodiment 55 or 56 is characterized in that the NTP is natural CTP, UTP, GTP, or ATP.

[0101] 58. The method according to embodiment 55 or 56 is characterized in that the NTP comprises two or more natural NTPs, wherein the natural NTPs include: CTP, UTP, GTP, and ATP.

[0102] 59. The method according to embodiment 55 or 56, characterized in that the NTP is a modified NTP.

[0103] 60. The method according to embodiment 55 or 56 is characterized in that the modified NTP is modified UTP: N1-methyl-pseudouridine triphosphate.

[0104] 61. The method according to embodiment 55 or 56 is characterized in that the modified NTP is N6-methyl-adenosine triphosphate, N1-methyl-adenosine triphosphate, 2-amino-adenosine triphosphate, 2-oxymethyl-adenosine triphosphate, 5-methyl-cytidine triphosphate, 5-hydroxymethyl-cytidine triphosphate, N4-acetyl-cytidine triphosphate, 2-oxymethyl-cytidine triphosphate, N7-methyl-guanosine triphosphate, 2-oxymethyl-guanosine triphosphate, N1-methyl-pseudouridine triphosphate, pseudouridine triphosphate, 2-thio-uridine triphosphate, 5-methoxy-uridine triphosphate, or 2-oxymethyl-uridine triphosphate.

[0105] 62. The method according to embodiment 55 or 56 is characterized in that the NTP comprises two or more NTPs, wherein the NTP is selected from: CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0106] 63. The method according to embodiment 55 or 56 is characterized in that the NTP is CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0107] 64. The method according to embodiment 55 or 56 is characterized in that the NTP is CTP, UTP, GTP, and ATP.

[0108] 65. The method according to embodiment 55 or 56 is characterized in that the NTP is CTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0109] 66. The method of embodiment 55 or 56, wherein the cap analog is a trinucleotide cap analog.

[0110] 67. A method according to embodiment 55 or 56, characterized in that the cap analog is m7(3'OMeG)(5')ppp(5')G(ARCA), m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, m7G(5')ppp(5')(2'OMeG)pG, or m7(3'OMeG)(5')ppp(5')(2'OMeG)pG.

[0111] 68. The method according to embodiment 55 or 56, characterized in that: the hat analog is: m7G(5')ppp(5')(2'OMeA)pG

[0112] 69. The method according to embodiment 55 or 56 is characterized in that the cap analog is a tetranucleotide cap analog.

[0113] 70. The method of embodiment 55 or 56, wherein the cap analog is a dinucleotide cap analog.

[0114] 71. The method according to embodiment 55 or 56 is characterized in that the mobile phase A in a) contains 200 mM triethylamine-acetic acid and 100 mM butanediamine-acetic acid.

[0115] 72. The method according to embodiment 55 or 56 is characterized in that the mobile phase B in a) is an acetonitrile aqueous solution containing 200 mM triethylamine-acetic acid and 100 mM butanediamine-acetic acid with a volume concentration of 30%-60%.

[0116] 73. The method according to embodiment 55 or 56 is characterized in that the C18 column in b) is a hybrid particle bonded C18 column with an inner diameter of 2.1 mm and a particle size of 1.7 μm.

[0117] 74. The method according to embodiment 55 or 56 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.2 to 0.6 mL / min.

[0118] 75. The method according to embodiment 55 or 56 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.2 mL / min.

[0119] 76. The method according to embodiment 55 or 56 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.3 mL / min.

[0120] 77. The method according to embodiment 55 or 56 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.4 mL / min.

[0121] 78. The method according to embodiment 55 or 56 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.5 mL / min.

[0122] 79. The method according to embodiment 55 or 56 is characterized in that the total flow rate of mobile phase A and phase B in b) is: 0.6 mL / min.

[0123] 80. The method according to embodiment 55 or 56, characterized in that the column temperature of the C18 column in b) is 50-70°C.

[0124] 81. The method according to embodiment 55 or 56, characterized in that the column temperature of the C18 column in b) is 50°C.

[0125] 82. The method according to embodiment 55 or 56, characterized in that the column temperature of the C18 column in b) is 60°C.

[0126] 83. The method according to embodiment 55 or 56, characterized in that the column temperature of the C18 column in b) is 70°C.

[0127] 84. The method according to embodiment 55 or 56 is characterized in that the detection wavelength of the target color peak in c) is 250-270nm.

[0128] 85. The method according to embodiment 55 or 56 is characterized in that the detection wavelength of the target color peak in c) is 260 nm.

[0129] 86. The method according to embodiment 55 or 56, characterized in that step (1) adopts a method comprising the following steps: iii. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; iv. The termination mixture described in i was precipitated with 2-4M LiCl, washed with 70% ethanol, and the precipitate was redissolved in enzyme-free water to obtain an in vitro transcribed mRNA stock solution sample for UPLC detection.

[0130] 87. The method according to embodiment 55 or 56, characterized in that step (1) adopts a method comprising the following steps: iii. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; iv. Purify the termination mixture described in i by chromatography or other means to obtain a sample of the in vitro transcription product for UPLC detection.

[0131] 88. The method according to embodiment 55 or 56, characterized in that step (1) adopts a method comprising the following steps: iii. The in vitro transcription product is terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; and iv. Dilute the termination mixture described in i 20-100 times with enzyme-free water to obtain a sample of the in vitro transcription product for UPLC detection.

[0132] 89. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes to 150 minutes.

[0133] 90. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes, 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 130 minutes, or 150 minutes.

[0134] 91. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is any mRNA in vitro transcription product.

[0135] 92. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 500-1000 nt.

[0136] 93. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 1000-2000 nt.

[0137] 94. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 2000-3000nt.

[0138] 95. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an in vitro mRNA transcription product with a length of 3000-4000 nt.

[0139] 96. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 4000-10000 nt.

[0140] 97. The method according to embodiment 55 or 56 is characterized in that the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 4173 nt.

[0141] 98. The method of embodiment 56, wherein step (d) comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mixed standard solutions of different concentrations.

[0142] 99. The method of embodiment 56, wherein step (d) comprises NTP in a mixed standard solution at 6 concentration levels.

[0143] 100. The method according to embodiment 99, characterized in that the mixed standard solutions at 6 concentration levels are: Mixed standard solution 1: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 1 μM concentrations, and mRNA concentration of 0.015625 mg / mL; Mixed standard solution 2: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 4 μM concentrations, and mRNA concentration of 0.03125 mg / mL; Mixed standard solution 3: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 16 μM, and mRNA concentration of 0.0625 mg / mL; Mixed standard solution 4: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 64 μM, and mRNA concentration of 0.125 mg / mL; Mixed standard solution 5: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 256 μM, and mRNA at 0.25 mg / mL; Mixed standard solution 6: contains CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 500 μM, respectively, and mRNA concentration of 0.5 mg / mL.

[0144] 101. The method according to embodiment 56 is characterized in that the quantification limit LOQ of the method is: 1 μM for NTP and cap analogs.

[0145] 102. The method according to Embodiment 56 is characterized in that the LOQ concentration of the method is: mRNA concentration 0.004 mg / mL.

[0146] 103. The method according to embodiment 56, characterized in that the concentration range of the standard curve is: the concentration range of NTP and cap analog is 1-500 μM

[0147] 104. The method according to Embodiment 56 is characterized in that the concentration range of the standard curve is: the mRNA concentration range is 0.004-0.5 mg / mL.

[0148] 105. The method according to embodiment 56 is characterized in that: the standard curve fitting method is: after taking the Log value of the concentration and the peak area, a linear fitting is performed.

[0149] 106. The method according to embodiment 105, characterized in that the relationship obtained by the linear fitting is: CTP: y = 1.1213x - 1.4971; N1-methyl-pseudouridine triphosphate: y = 1.098x-1.4653; UTP: y = 1.118x - 1.3636; GTP: y = 1.2619x - 1.5964; ATP: y = 1.1325x - 1.2056; m7G(5')ppp(5')(2'OMeA)pG:y=1.0172x-0.6385; mRNA:y=1.2323x+2.4969 Where x is the Log(concentration) value and y is the Log(peak area) value.

[0150] 107. The method according to embodiment 56, characterized in that: the R2 of the fitted linear equation is greater than 0.99

[0151] 108. The method according to embodiment 56 is characterized in that the quantitative accuracy of the method within the concentration range of claim 10 is 90%-110%. 4. Description of the drawings

[0152] Figure 1 The effect of different ion pair reagents in the mobile phase on retention and separation. In the figure, 1-7 are CTP, N1-methyl-pseudouridine triphosphate, UTP, cap analog, GTP, ATP, and mRNA. The ion pair reagents represented by the curves from top to bottom are: TEAA+DBAA (optimal), TBAP (the ion pair effect is too strong, NTP and cap analog can be eluted and separated, but mRNA cannot be eluted), TEAA (the ion pair effect is too weak, NTP and cap analog are weakly retained and poorly separated, and mRNA can be eluted).

[0153] Figure 2 This is the limit of quantification (LOQ) detection spectrum of the method. 1-7 in the figure are CTP, N1-methyl-pseudouridine triphosphate, UTP, cap analog, GTP, ATP, and mRNA. The LOQ of mRNA is 0.004 mg / mL, and the LOQ of the other components is 1 μM.

[0154] Figure 3 is the method accuracy. Within the relevant range, the quantitative accuracy of each component is 90%-110%.

[0155] Figure 4 This is the detection spectrum of NTP and cap analogs in crude mRNA in Example 1, in which 1-5 are CTP, N1-methyl-pseudouridine triphosphate, UTP, cap analog, GTP, and ATP, respectively.

[0156] Figure 5This is the standard curve fitting diagram of the method.

[0157] Figure 6 This is a dynamic detection graph of NTP, cap analog and mRNA during IVT in Example 2. In the figure, 1-6 are CTP, N1-methyl-pseudouridine triphosphate, cap analog, GTP, ATP and mRNA respectively.

[0158] Figure 7 This is the dynamic monitoring curve of NTP, cap analog and mRNA during the IVT process in Example 2. 4.1. Definitions

[0159] Unless otherwise defined herein, technical and scientific terms used in the specification have the meanings that are commonly understood by one of ordinary skill in the art.

[0160] As used in the present invention, "cap analogue" refers to a structure at the 5' end of mature mRNA formed by post-transcriptional modification in eukaryotic organisms, namely, m7GPPPN structure, also known as methylguanosine cap. This structure can prevent mRNA from being degraded at the 5' end, help RNA transcription products pass through the selective pores of the nuclear membrane and enter the cytoplasm, enhance translation, and help complete the entire shearing process.

[0161] As used in the present invention, "base" and "natural base" can be used interchangeably, and both can be called nucleobases, nitrogenous bases, which are nitrogen-containing compounds that form nucleosides, which are components of nucleotides; they can be adenine (A), guanine (G), cytosine (C), uracil (U), or thymine (T).

[0162] A "modified base" refers to a substance in which one or more hydrogen atoms of a natural base are replaced, such as, but not limited to, N1-methyl-pseudouracil (U). 5. Specific implementation methods

[0163] The present invention provides a method for analyzing NTP, cap analogs and mRNA components in in vitro transcription product components based on UPLC, comprising the following steps: (1) preparing a sample of in vitro transcription product for UPLC detection and (2) detecting using the UPLC method.

[0164] In certain embodiments, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) the separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 11 minutes: 84% mobile phase A + 16% mobile phase B; 22 minutes: 0% mobile phase A + 100% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and (c) Chromatogram of NTPs, cap analogs, and mRNA collected from in vitro transcription product components.

[0165] In certain embodiments, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) the separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 11+T minutes: 84% mobile phase A + 16% mobile phase B; 22+T+U minutes: 0% mobile phase A + 100% mobile phase B; 25+T+U minutes: 0% mobile phase A + 100% mobile phase B; 25.1+T+U minutes: 95% mobile phase A + 5% mobile phase B; 30+T+U minutes: 95% mobile phase A + 5% mobile phase B; and

[0166] (c) collecting a chromatogram of NTP, cap analog and mRNA in the in vitro transcription product components; wherein the range of T value is: 0≤T≤25; the range of U value is: -5≤U≤10. In certain embodiments, the detection by UPLC method in step (2) comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) The separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: (100% - P%) mobile phase A + P% mobile phase B; 11 minutes: (100% - Q%) mobile phase A + Q% mobile phase B; 22 minutes: (100% - R%) mobile phase A + R% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and (c) Collecting chromatograms of NTP, cap analogs and mRNA in the components of in vitro transcription products; wherein the range of P value is: 5≤P≤10, the range of Q value is: 10≤Q≤16, and the range of R value is: 16≤R≤100.

[0167] In certain embodiments, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10;

[0168] (b) The separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 1 minute: (100% - P%) mobile phase A + P% mobile phase B; 11 + T minutes: (100% - Q%) mobile phase A + Q% mobile phase B; 22 + T + U minutes: (100% - R%) mobile phase A + R% mobile phase B; 25 + T + U minutes: 0% mobile phase A + 100% mobile phase B; 25.1 + T + U minutes: 9 5% mobile phase A + 5% mobile phase B; 30 + T + U minutes: 95% mobile phase A + 5% mobile phase B; and (c) collecting chromatograms of NTP, cap analogs and mRNA in the components of in vitro transcription products; wherein the range of T values ​​is: 0 ≤ T ≤ 25; the range of U values ​​is: -5 ≤ U ≤ 10; the range of P values ​​is: 5 ≤ P ≤ 10, the range of Q values ​​is: 10 ≤ Q ≤ 16, and the range of R values ​​is: 16 ≤ R ≤ 100. In one embodiment, the analytical method further comprises the following quantitative steps: d) Collect chromatograms of NTP, cap analog and mRNA in the mixed standard solution at no less than three concentration levels e) After taking the log of the concentrations and peak areas of NTP, cap analog and mRNA in the mixed standard solution, a standard curve is drawn to obtain a linear fitting equation; and f) Calculate the concentration of each NTP and cap analog based on the linear fitting equation of the standard.

[0169] In one embodiment, the NTP is native CTP, UTP, GTP, or ATP.

[0170] In another embodiment, the NTP comprises two or more natural NTPs, wherein the natural NTPs include: CTP, UTP, GTP, and ATP.

[0171] In another embodiment, the NTP is a modified NTP.

[0172] In another embodiment, the modified NTP is modified UTP: N1-methyl-pseudouridine triphosphate.

[0173] In another embodiment, the NTP comprises two or more NTPs, wherein the NTP is selected from: CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0174] In another embodiment, the NTP is CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

[0175] In another embodiment, the NTP is CTP, UTP, GTP, and ATP.

[0176] In one embodiment, the cap analog is a trinucleotide cap analog.

[0177] In another embodiment, the cap analog is: 7 G(5')ppp(5')(2'OMeA)pG

[0178] In another embodiment, the cap analog is a compound of formula (1):

[0179] In certain embodiments, the cap analog is a dinucleotide cap analog.

[0180] In certain embodiments, the cap analog is a trinucleotide cap analog.

[0181] In certain embodiments, the cap analog is a tetranucleotide cap analog.

[0182] In one embodiment, the step (2) of detecting by UPLC method comprises: (a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), with a pH of 6.90-7.10; Mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, with a pH of 6.90-7.10; (b) the separation gradient program is as follows: start 95% mobile phase A + 5% mobile phase B; 2 minutes: 90% mobile phase A + 10% mobile phase B; 11 minutes: 84% mobile phase A + 16% mobile phase B; 22 minutes: 0% mobile phase A + 100% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and (c) Collecting chromatograms of NTP, cap analogs and mRNA in the components of the in vitro transcription product. In one embodiment, the mobile phase A in (a) contains 200mM triethylamine-acetic acid and 100mM butanediamine-acetic acid. In one embodiment, the mobile phase B in (a) is an acetonitrile aqueous solution containing 200mM triethylamine-acetic acid and 100mM butanediamine-acetic acid with a volume concentration of 30%-60%. In one embodiment, the C18 column in (b) is a hybrid particle bonded C18 column with an inner diameter of 2.1mm and a particle size of 1.7μm. In one embodiment, the total flow rate of mobile phase A and phase B in (b) is: 0.2-0.6mL / min. In certain embodiments, the column temperature of the C18 column in (b) is 50-70°C. In one embodiment, the column temperature of the C18 column in (b) is 60°C. In certain embodiments, the detection wavelength of the target color peak in (c) is 250-270 nm. In one embodiment, the detection wavelength of the target color peak in (c) is 260 nm.

[0183] In certain embodiments, step (1) adopts a method comprising the following steps: (i) terminating the in vitro transcription product with 20-100 mM EDTA (final concentration) to obtain a termination mixture; and precipitating the termination mixture in (i) with 2-4 M LiCl, washing with 70% ethanol, and re-dissolving the precipitate in enzyme-free water to obtain an in vitro transcribed mRNA stock solution sample for UPLC detection.

[0184] In certain embodiments, step (1) adopts a method comprising the following steps: (i) terminating the in vitro transcription product with 20-100 mM EDTA (final concentration) to obtain a termination mixture; purifying the termination mixture in (i) by chromatography or other means to obtain a sample of the in vitro transcription product for UPLC detection.

[0185] In certain embodiments, step (1) adopts a method comprising the following steps: (i) terminating the in vitro transcription product with 20-100 mM EDTA (final concentration) to obtain a termination mixture; and diluting the termination mixture in (i) 20-100 times with enzyme-free water to obtain a sample of the in vitro transcription product for UPLC detection.

[0186] In certain embodiments, the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes to 150 minutes.

[0187] In certain embodiments, the in vitro transcription product in step (1) is an in vitro transcription product of 0 minutes, 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 130 minutes, or 150 minutes.

[0188] In certain embodiments, the in vitro transcription product in step (1) is any mRNA in vitro transcription product.

[0189] In certain embodiments, the in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 4173 nt.

[0190] In certain embodiments, step (d) comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mixed standard solutions of different concentrations.

[0191] In certain embodiments, step (d) comprises six concentration levels of NTP in the mixed standard solution.

[0192] In certain embodiments, the six concentration levels of the mixed standard solutions are: Mixed standard solution 1: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 1 μM concentrations, and mRNA concentration of 0.015625 mg / mL; Mixed standard solution 2: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 4 μM concentrations, and mRNA concentration of 0.03125 mg / mL; Mixed standard solution 3: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 16 μM, and mRNA concentration of 0.0625 mg / mL; Mixed standard solution 4: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 64 μM, and mRNA concentration of 0.125 mg / mL; Mixed standard solution 5: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at 256 μM, and mRNA at 0.25 mg / mL; Mixed standard solution 6: containing CTP, UTP, N1-methyl-pseudouridine triphosphate, GTP, and ATP at concentrations of 500 μM, respectively, and mRNA at a concentration of 0.5 mg / mL;

[0193] In certain embodiments, the method has a limit of quantitation, LOQ, of 1 μM for NTPs and cap analogs.

[0194] In certain embodiments, the method has a limit of quantitation (LOQ) concentration of mRNA of 0.004 mg / mL.

[0195] In certain embodiments, the standard curve concentration range is: NTP and cap analog concentration range is 1-500 μM. In certain embodiments, the standard curve concentration range is: NTP and cap analog concentration range is 1-100 μM. In certain embodiments, the standard curve concentration range is: NTP and cap analog concentration range is 1-200 μM. In certain embodiments, the standard curve concentration range is: NTP and cap analog concentration range is 1-300 μM. In certain embodiments, the standard curve concentration range is: NTP and cap analog concentration range is 1-400 μM.

[0196] In certain embodiments, the standard curve concentration range is: mRNA concentration range is 0.004-0.5 mg / mL. In certain embodiments, the standard curve concentration range is: mRNA concentration range is 0.004-0.1 mg / mL. In certain embodiments, the standard curve concentration range is: mRNA concentration range is 0.004-0.2 mg / mL. In certain embodiments, the standard curve concentration range is: mRNA concentration range is 0.004-0.3 mg / mL. In certain embodiments, the standard curve concentration range is: mRNA concentration range is 0.004-0.4 mg / mL.

[0197] The method according to embodiment 2 is characterized in that: the standard curve fitting method is: after taking the Log value of the concentration and the peak area, a linear fit is performed. In certain embodiments, the relationship obtained by the linear fit is: CTP: y = 1.1213x - 1.4971; N1-methyl-pseudouridine triphosphate: y = 1.098x-1.4653; UTP: y = 1.118x - 1.3636; GTP: y = 1.2619x - 1.5964; ATP: y = 1.1325x - 1.2056; m 7 G(5')ppp(5')(2'OMeA)pG:y=1.0172x-0.6385; mRNA:y=1.2323x+2.4969 Where x is the Log(concentration) value and y is the Log(peak area) value.

[0198] In certain embodiments, the R2 of the fitted linear equation is > 0.99.

[0199] In certain embodiments, the quantitative accuracy of the method within the concentration range of claim 10 is 90%-110%.

[0200] It should be understood that modifications that do not substantially affect the activity of the various embodiments of the invention may also be encompassed within the definition of the invention provided herein. Therefore, the following examples are intended to illustrate the invention but not to limit the invention.

[0201] In certain embodiments, modifications of the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) include, but are not limited to, the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2′ -O-methyladenosine; 2′-O-ribosyladenosine (phosphate); isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine; N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2(aminopropyl)adenine adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-ATP; 2'-azido-2'-deoxy-ATP; 2'-deoxy-2'-a-aminoadenosine TP; 2'-deoxy-2'-a-azidoadenosine TP; 6(alkyl)adenine; 6(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7(deaza)adenine; 8(alkenyl)adenine; 8(alkynyl)adenine; 8(amino)adenine; 8(thioalkyl)adenine; 8-(alkenyl)adenine; 8 -(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; azaadenine; deazaadenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-deazaadenosine TP; 2'fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine TP; 2-amino-ATP; 2'-a-trifluoromethyladenosine TP;2-azidoadenosine TP; 2′-b-ethynyladenosine TP; 2-bromoadenosine TP; 2′-b-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2′-deoxy-2′, 2′-difluoroadenosine TP; 2′-deoxy-2′-a-mercaptoadenosine TP; 2′-deoxy-2′-a-thiomethoxyadenosine TP; 2′-deoxy-2′-b-aminoadenosine TP; 2′-deoxy-2′-b-azidoadenosine TP; 2′-deoxy-2′-b-bromoadenosine TP; 2′-deoxy-2′-b-chloroadenosine TP; 2′-deoxy-2′-b-fluoroadenosine TP; 2′-deoxy-2′-b-iodoadenosine TP; 2′-deoxy-2′-b-mercaptoadenosine TP; 2′-deoxy-2′-b-thiomethoxy adenosine TP; 2-fluoroadenosine TP; 2-iodoadenosine TP; 2-thioadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-trifluoromethyladenosine TP; 3-deaza-3-bromoadenosine TP; 3-deaza-3-chloroadenosine TP; 3-deaza-3-fluoroadenosine TP; 3-deaza-3-iodoadenosine TP; 3-deazaadenosine TP; 4′-azidoadenosine TP; 4′-carbocyclic adenosine TP; 4′-ethynyladenosine TP; 5′-homo-adenosine TP; 8-aza-ATP; 8-bromoadenosine TP; 8-trifluoromethyladenosine TP; 9-deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza -8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine; 5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine; N4-acetyl-2′-O-methylcytidine; N4-methylcytidine; N4,N4-dimethyl-2′-OMe-cytidine TP; 4-methylcytidine; 5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-a-aminocytidine TP; 2'-deoxy-2'-a-azidocytidine TP; 3(deaza)5(aza)cytosine; 3(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza)5(aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5(halo)cytosine; 5(methyl)cytosine; 5(propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynylcytosine; 6-(azo)cytosine; 6-aza-cytidine; azacytosine; deazacytosine; N4(acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine EN; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'fluoro-N4-Bz-cytidine TP; 2'fluoro-N4-acetyl-cytidine TP; 2'-O-methyl-N4-acetyl-cytidine TP; 2'O-methyl-N4-Bz-cytidine TP; 2'-a-ethynylcytidine TP; 2'-a-trifluoromethylcytidine TP; 2'-b-ethynylcytidine TP; 2'-b-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP; 2'-deoxy-2'-a-thiocytidine TP; 2'-deoxy-2'-a-thiomethoxycytidine TP; 2'-deoxy-2'-b-aminocytidine TP; 2'-deoxy-2'-b -azidocytidine TP; 2′-deoxy-2′-b-bromocytidine TP; 2′-deoxy-2′-b-chlorocytidine TP; 2′-deoxy-2′-b-fluorocytidine TP; 2′-deoxy-2′-b-iodocytidine TP; 2′-deoxy-2′-b-thiolcytidine TP; 2′-deoxy-2′-b-thiomethoxycytidine TP; 2′-O-methyl-5-(1-propynyl)cytidine TP; 3′-ethynylcytidine TP; 4′-azidocytidine TP; 4′-carbocyclic cytidine TP; 4′-ethynylcytidine TP; 5-(1-propynyl)arabino-cytidine TP; 5-(2-chloro-phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl- CTP; 5-cyanocytidine TP; 5-ethynylarabino-cytidine TP; 5-ethynylcytidine TP; 5′-homo-cytidine TP; 5-methoxycytidine TP; 5-trifluoromethyl-cytidine TP; N4-amino-cytidine TP; N4-benzoyl-cytidine TP; pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine; N2-methylguanosine; gamma-gamma-dinaline; 1,2′-O-dimethylguanosine; 1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; archaeocyanine; methyl gamma-gamma-dinaline; N2,7-dimethylguanosine;N2, N2, 2′-O-trimethylguanosine; N2, N2, 7-trimethylguanosine; N2, N2-dimethylguanosine; N2, 7, 2′-O-trimethylguanosine; 6-thioguanosine; 7-deazaguanosine; 8-oxoguanosine; N1-methylguanosine; α-thioguanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-amino-2′-deoxy-GTP; 2′-azido-2′-deoxy-GTP; 2′-deoxy-2′-a-aminoguanosine TP; 2′-deoxy-2′-a-azidoguanosine TP; 6(methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methylguanosine; 7(alkyl)guanine; 7(deaza)guanine; 7(methyl )guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8(alkyl)guanine; 8(alkynyl)guanine; 8(halo)guanine; 8(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thio)guanine; azaguanine; deazaguanine; N(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl- Guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2, N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a-ethynylguanosine TP; 2'-a-trifluoromethylguanosine TP; 2'-b-ethynylguanosine TP; 2'-b-trifluoromethylguanosine TP; 2'-deoxy-2', 2'-difluoroguanosine TP; 2'-deoxy-2'-a-thioguanosine TP; 2'-deoxy-2'-a-thiomethoxyguanosine TP; 2'-deoxy-2'-b-aminoguanosine TP; 2'-deoxy-2'-b-azidoguanosine Guanosine TP; 2′-deoxy-2′-b-bromoguanosine TP; 2′-deoxy-2′-b-chloroguanosine TP; 2′-deoxy-2′-b-fluoroguanosine TP; 2′-deoxy-2′-b-iodoguanosine TP; 2′-deoxy-2′-b-thioguanosine TP; 2′-deoxy-2′-b-thiomethoxyguanosine TP; 4′-azidoguanosine TP; 4′-carbocyclic guanosine TP; 4′-ethynylguanosine TP; 5′-homo-guanosine TP; 8-bromo-guanosine TP; 9-deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; inosine; 1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine; 2′-O-methylinosine; epoxy Q nucleosides; galactosyl-Q nucleosides;Mannosyl Q nucleoside; Q nucleoside; allylamino-thymidine; azathymidine; deazathymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinemethyl-2-thiouridine; 5-taurinemethyluridine; dihydrouridine; pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseudouridine; 1-methyl-pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O- dimethyluridine; 3-methyl-pseudo-uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2′-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine TP; 5-methoxycarbonyl methyl-2′-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine, 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-oxyacetic acid-uridine TP; 5-oxyacetic acid-methyl ester-uridine TP; N1-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2′-O-methyluridine TP; 5-(isopentenylaminomethyl)- 1-substituted 2-(thio)-pseudouracil; 1-substituted 2-(thio)-pseudouracil; 1-substituted 2-(dithio)-pseudouracil; 1-substituted 2-(dithio)-pseudouracil; 1-substituted 2-(thio)-pseudouracil;1-substituted 4(thio)pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine-UTP; 1-methyl-pseudouridine-UTP; 2(thio)pseudouridine; 2′deoxyuridine; 2′fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouridine; 2′methyl, 2′amino, 2′azido, 2′fluoro-guanosine; 2′-amino-2′-deoxy-UTP; 2′-azido-2′-deoxy-UTP; 2′-azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxyuridine; 2′-fluorouridine; 2′-deoxy-2′-a-aminouridine TP; 2′-deoxy-2′-a-azidouridine TP; 2-methylpseudouridine; 3(3-amino-3-carboxypropyl)uracil; 4(thio)pseudouridine; 4-(thio)pseudouridine; 4-(thio)uracil; 4-thiouracil; 5(1,3-oxadiazol-1-alkyl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidinyl)uracil; 5(methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5(methyl)2(thio)uracil; 5(methyl)2,4(dithio)uracil; 5(methyl uracil; 5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4-(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4-(dithio)pseudouracil; 5-(alkyl)-4-(thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(Guanidinyl)uracil; 5-(halo)uracil; 5-(1,3-oxadiazol-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2(thio)uracil; 5-(methyl)2,4(dithio)uracil; 5-(methyl)4(thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4(dithio)pseudouracil; 5-(methyl)-4(thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil;5-(Methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylamino-uracil; aza-uracil; deaza-uracil; N3(methyl)uracil; pseudo-UTP-1-2-acetic acid; pseudouracil; 4-thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-methoxy-4- Thiouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; dihydropseudouridine; (±) 1-(2-hydroxypropyl)pseudouridine TP; (2R)-1-(2-hydroxypropyl)pseudouridine TP; (2S)-1-(2-hydroxypropyl)pseudouridine TP; (E)-5-(2-bromo-vinyl)arabinouridine TP; (E)-5-(2-bromo-vinyl)uridine TP; (Z)-5-(2-bromo-vinyl)arabino-uridine TP; (Z)-5-(2-bromo-vinyl)uridine TP; 1-(2,2,2-trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP; 1-(2,2-diethoxyethyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-trimethyl-phenyl)pseudo-UTP; 1-(2-amino-2-carboxyethyl)pseudo-UTP; 1-(2-amino-ethyl)pseudo-UTP; 1-(2-hydroxyethyl)pseudouridine TP; 1-(2-methoxyethyl)pseudouridine TP; 1-(3 , 4-bis-trifluoromethoxybenzyl) pseudouridine TP; 1-(3,4-di-methoxybenzyl) pseudouridine TP; 1-(3-amino-3-carboxypropyl) pseudo-UTP; 1-(3-amino-propyl) pseudo-UTP; 1-(3-cyclopropyl-prop-2-ynyl) pseudouridine TP; 1-(4-amino-4-carboxybutyl) pseudo-UTP; 1-(4-amino-benzyl) pseudo-UTP; 1-(4-amino-butyl) pseudo-UTP; 1-(4-amino-phenyl) pseudo-UTP; 1-(4-azidobenzyl) pseudouridine TP; 1-(4-bromobenzyl) pseudouridine TP; 1-(4-chlorobenzyl) pseudouridine TP; 1-(4-fluorobenzyl) pseudouridine TP; 1-(4-iodobenzyl) pseudouridine TP;1-(4-methanesulfonylbenzyl)pseudouridine TP; 1-(4-methoxybenzyl)pseudouridine TP; 1-(4-methoxy-benzyl)pseudouridine TP; 1-(4-methoxy-phenyl)pseudouridine TP; 1-(4-methylbenzyl)pseudouridine TP; 1-(4-methyl-benzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitro-benzyl)pseudouridine TP; 1(4-nitro-phenyl)pseudouridine TP; 1-(4-thiomethoxybenzyl)pseudouridine TP; 1-(4-trifluoromethoxybenzyl)pseudouridine TP; 1-(4-trifluoromethylbenzyl)pseudouridine TP; 1-(5-amino-pentyl)pseudouridine TP; 1-(6-amino-hexyl)pseudouridine TP; 1,6 -dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl] pseudouridine TP; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-acetyl pseudouridine TP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-UTP; 1-alkyl-6-allyl-pseudo-UTP; 1-alkyl-6-ethynyl-pseudo-UTP; 1-alkyl-6-homoallyl-pseudo-UTP; 1-alkyl-6-vinyl-pseudo-UTP; 1-allyl pseudouridine TP; 1-aminomethyl-pseudo-UTP; 1-benzoyl pseudouridine uridine TP; 1-benzyloxymethyl pseudouridine TP; 1-benzyl-pseudo-UTP; 1-biotinyl-PEG2-pseudouridine TP; 1-biotinyl-pseudouridine TP; 1-butyl-pseudo-UTP; 1-cyanomethyl pseudouridine TP; 1-cyclobutylmethyl-pseudo-UTP; 1-cyclobutyl-pseudo-UTP; 1-cycloheptylmethyl-pseudo-UTP; 1-cycloheptyl-pseudo-UTP; 1-cyclohexylmethyl-pseudo-UTP; 1-cyclohexyl-pseudo-UTP; 1-cyclooctylmethyl-pseudo-UTP; 1-cyclooctyl-pseudo-UTP; 1-cyclopentylmethyl-pseudo-UTP; 1-cyclopentyl-pseudo-UTP; 1-cyclopropylmethyl-pseudo-UTP; 1-cyclopropyl-pseudo-UTP; 1-ethyl-pseudo-UTP P; 1-hexyl-pseudo-UTP; 1-homoallyl pseudouridine TP; 1-hydroxymethyl pseudouridine TP; 1-isopropyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-α-thio-pseudo-UTP; 1-methanesulfonylmethyl pseudouridine TP; 1-methoxymethyl pseudouridine TP; 1-methyl-6-(2,2,2-trifluoroethyl) pseudo-UTP; 1-methyl-6-(4-morpholino)-pseudo-UTP; 1-methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-methyl-6-(substituted phenyl) pseudo-UTP; 1-methyl-6-amino-pseudo-UTP; 1-methyl-6-azido-pseudo-UTP;1-methyl-6-bromo-pseudo-UTP; 1-methyl-6-butyl-pseudo-UTP; 1-methyl-6-chloro-pseudo-UTP; 1-methyl-6-cyano-pseudo-UTP; 1-methyl-6-dimethylamino-pseudo-UTP; 1-methyl-6-ethoxy-pseudo-UTP; 1-methyl-6-ethylcarboxylate-pseudo-UTP; 1-methyl-6-ethyl-pseudo-UTP; 1-methyl-6-fluoro-pseudo-UTP; 1-methyl-6-formyl-pseudo-UTP; 1-methyl-6-hydroxyamino-pseudo-UTP; 1-methyl-6-hydroxy-pseudo-UTP; 1-methyl-6-iodo-pseudo-UTP; 1-methyl-6-isopropyl-pseudo-UTP; 1-methyl-6-methoxy-pseudo-UTP; 1-methyl-6-methylamino-pseudo-UTP; 1-methyl-6-phenyl-pseudo-UTP; 1-methyl-6-propyl-pseudo-UTP; 1-methyl-6-tert-butyl-pseudo-UTP; 1-methyl-6-trifluoromethoxy-pseudo-UTP; 1-methyl-6-trifluoromethyl-pseudo-UTP; 1-morpholinomethylpseudouridine TP; 1-pentyl-pseudo-UTP; 1-phenyl-pseudo-UTP; 1-pivaloylpseudouridine TP; 1-propargylpseudouridine TP; 1-propyl-pseudouridine; 1-propynyl-pseudouridine; 1-p-tolyl-pseudouridine; 1-tert-butyl-pseudouridine; 1-thiomethoxymethylpseudouridine TP; 1-thiomorpholinomethylpseudouridine TP; 1-trifluoroacetylpseudouridine uridine TP; 1-trifluoromethyl-pseudo-UTP; 1-vinyl pseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-ethynyl uridine TP; 2'-a-trifluoromethyl uridine TP; 2'-b-ethynyl uridine TP; 2'-b-trifluoromethyl uridine TP; 2'-deoxy-2',2'-difluorouridine TP; 2'-deoxy-2'-a-thiouridine TP; 2'-deoxy-2'-a-thiomethoxyuridine TP; 2'-deoxy-2'-b-aminouridine TP; 2'-deoxy-2'-b-azido uridine TP; 2′-deoxy-2′-b-bromouridine TP; 2′-deoxy-2′-b-chlorouridine TP; 2′-deoxy-2′-b-fluorouridine TP; 2′-deoxy-2′-b-iodouridine TP; 2′-deoxy-2′-b-thiouridine TP; 2′-deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2′-O-methyl-5-(1-propynyl)uridine TP; 3-alkyl-pseudo-UTP; 4′-azidouridine TP; 4′-carbocyclic uridine TP; 4′-ethynyl uridine TP; 5-(1-propynyl)arabino-uridine TP; 5-(2-furanyl)uridine TP; 5-cyanouridine TP; 5-dimethylaminouridine TP;5′-homo-uridine TP; 5-iodo-2′-fluoro-deoxyuridine TP; 5-phenylethynyl uridine TP; 5-trideuteromethyl-6-deuterouidine TP; 5-trifluoromethyl-uridine TP; 5-vinylarabinouridine TP; 6-(2,2,2-trifluoroethyl)-pseudo-UTP; 6-(4-morpholino)-pseudo-UTP; 6-(4-thiomorpholino)-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-amino-pseudo-UTP; 6-azido-pseudo-UTP; 6-bromo-pseudo-UTP; 6-butyl-pseudo-UTP; 6-chloro-pseudo-UTP; 6-cyano-pseudo-UTP; 6-dimethylamino-pseudo-UTP; 6-ethoxy-pseudo-UTP; 6-ethylcarboxylic acid ester-pseudo-UTP; 6-ethyl-pseudo-UTP; 6-fluoro-pseudo-UTP; 6-formyl-pseudo-UTP; 6-hydroxyamino-pseudo-UTP; 6-hydroxy-pseudo-UTP; 6-iodo-pseudo-UTP; 6-isopropyl-pseudo-UTP; 6-methoxy-pseudo-UTP; 6-methylamino-pseudo-UTP; 6-methyl-pseudo-UTP; 6-phenyl-pseudo-UTP; 6-phenyl-pseudo-UTP; 6-propyl-pseudo-UTP; 6-tert-butyl-pseudo-UTP; 6-trifluoromethoxy-pseudo-UTP; 6-trifluoromethyl-pseudo-UTP; α-thio-pseudo-UTP; pseudouridine 1-(4-toluenesulfonic acid) TP; pseudouridine 1-(4-methylbenzoic acid) TP; pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-methylphosphonic acid; pseudouridine TP 1-methylphosphonic acid diethyl ester; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric acid; pseudo-UTP-N1-5-pentanoic acid; pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Iso-wybutosine; Peroxywybutosine; Undermodified hydroxywybutosine; 4-demethyl-wybutosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxo)-naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2'methyl, 2'amino, 2'azido, 2'fluoro-cytidine; 2'methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo -pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isoquinolone; 3-(methyl)isoquinolone; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isoquinolone; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(amino) 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1-(aza)- 7-(Guaniniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(Guaniniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl; 7-(Guaniniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl; 7-(substituted)-phenoxazin-1-yl; 7-(propynyl)isoquinolone; 7-(propynyl)isoquinolone, propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;9-(Methyl)-imidazopyridinyl; Aminoindolyl; Anthracenyl; Bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Bis-o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; Difluorotolyl; Hypoxanthine; Imidazopyridinyl; Inosinyl; Isoquinolone; Isoguanisine; N2-substituted purine; N6-methyl-2-amino-purine; N6- Substituted purines; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; Nubularine; O6-substituted purines; O-alkylated derivatives; o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentacene; phenanthrenyl; Phenanthracenyl; Phenyl; Propynyl-7-(aza)indolyl; Pyrenyl; Pyridopyrimidin-3-yl; Pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; Pyrrolo-pyrimidin-2-one-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrazinyl; Stilbenzyl; Substituted 1,2,4-triazole; Tetraphenyl; Tuberculocidin (Tubercidine); xanthine; xanthine-5'-TP; 2-thio-zebulin; 5-aza-2-thio-zebulin; 7-deaza-2-amino-purine; pyridin-4-one, ribonucleoside; 2-amino-ribonucleoside-TP; mesomorphin A TP; mesomorphin B TP; pyrrolosine TP; 2'-OH-arabino-adenosine TP; 2'-OH-arabino-cytidine TP; 2'-OH-arabino-uridine TP; 2'-OH-arabino-guanosine TP; 5-(2-methoxyformylvinyl)uridine TP; and N6-(19-amino-pentaoxanonadecanyl)adenosine TP.

[0202] In some embodiments, a polynucleotide comprises a combination of at least two (eg, 2, 3, 4, or more) of the foregoing modified nucleobases.

[0203] In some embodiments, the modified nucleobase in the polynucleotide is selected from the group consisting of pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0204] In some embodiments, the modified nucleobase in the polynucleotide is selected from the group consisting of: 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, the polynucleotide comprises a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0205] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0206] In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides with modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.

[0207] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having modified uridine include 5-cyanouridine or 4'-thiouridine.

[0208] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A) and 2,6-diaminopurine.

[0209] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanine include inosine (I), 1-methyl-inosine (m1I), imG, methyl imG, 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0210] In some embodiments, the UPLC instrument is a Thermo Vanquish F ultra-high performance liquid chromatograph and a Chromeleon chromatography workstation.

[0211] In some embodiments, the UPLC column is a hybrid particle bonded C18 column of ACQUITY UPLC Oligonucleotide BEH C18 (1.7 μm, 2.1×100 mm). 5.1. Example 1: Selection of ion-pair reagents in the mobile phase (1) Instrument and chromatographic conditions

[0212] Ultra-high performance liquid chromatograph: Thermo Vanquish F ultra-high performance liquid chromatograph and Chromeleon chromatography workstation;

[0213] Chromatographic column: ACQUITY UPLC Oligonucleotide BEH C18 (1.7 μm, 2.1 × 100 mm) hybrid particle bonded C18 column;

[0214] Selection of ion pair reagents in mobile phase: Prepare the following three mobile phase stock solutions: 200mM TEAA (pH 7.0), 100mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.0) + 20mM tetrabutylammonium dihydrogen phosphate solution (TBAP), and 400mM TEAA + 200mM DBAA (pH 7.0). Mix the above stock solutions with ultrapure water in equal volumes as mobile phase A; mix the above stock solutions with acetonitrile in equal volumes as mobile phase B; set the flow rate to 0.4mL / min, and the elution gradient to: 0, 1, 2, 11, 22, 25, 25.1, 30min, the proportion of phase B is 5%, 5%, 10%, 16%, 100%, 100%, 5%, 5% respectively. The detection wavelength is 260nm, and the column temperature is 60℃. (2) Experimental procedures

[0215] Preparation of mixed test solution: Use nuclease-free water as diluent to dilute to obtain a mixed solution containing 64 μM of each NTP and cap analog and 0.125 mg / mL of mRNA.

[0216] Inject 5 μL of the above mixed test solution and record the chromatogram. See attached Figure 1 .

[0217] In Example 1, 200 mM TEAA + 100 mM DBAA was selected as the ion pair reagent to achieve baseline separation of NTP cap analogs and mRNA, with good separation, stable method and simple operation. 5.2. Example 2: Detection of NTP and cap analog residues in mRNA stock solution (1) Instrument and chromatographic conditions

[0218] Ultra-high performance liquid chromatograph: Thermo Vanquish F ultra-high performance liquid chromatograph and Chromeleon chromatography workstation;

[0219] Chromatographic column: ACQUITY UPLC Oligonucleotide BEH C18 (1.7 μm, 2.1 × 100 mm) hybrid particle bonded C18 column;

[0220] Prepare an aqueous solution containing 400mM TEAA and 200mM DBAA, adjust the pH to 7.0 with acetic acid, and use it as a mobile phase stock solution. Mix the stock solution with an equal volume of ultrapure water as mobile phase A; mix the stock solution with an equal volume of pure acetonitrile as mobile phase B. Set the flow rate to 0.4mL / min, and the elution gradient to: 0, 1, 2, 11, 22, 25, 25.1, 30min, with the corresponding proportions of phase B being 5%, 5%, 10%, 16%, 100%, 100%, 5%, 5%. The detection wavelength was 260nm, and the column temperature was 60℃. (2) Experimental procedures

[0221] Preparation of mixed standard solution: Dilute the standard stock solution with nuclease-free water to obtain mixed standard solutions of different concentration levels, where the concentration range of each NTP and cap analog is 1-500 μM, and the concentration range of mRNA is 0.004-0.5 mg / mL.

[0222] Preparation of accuracy and sensitivity test solutions: The standard stock solution was diluted with nuclease-free water to obtain mixed solutions of different concentration levels, in which the concentrations of each NTP and cap analog were 1 μM, 10 μM, 100 μM, and 500 μM, respectively, and the concentrations of mRNA were 0.004 mg / mL, 0.05 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively.

[0223] 5 μL of each standard solution was injected for analysis and the chromatogram was recorded. After the chromatogram was processed, the peak area of ​​each component was obtained, and the concentration and peak area were taken as logarithms, and linear fitting was performed to obtain the fitting equation. The results are shown in the attached Figure 5 .

[0224] 5 μL of each sensitivity solution and accuracy solution were injected for analysis and the chromatogram was recorded. The spectrum of the sensitivity solution and the peak result statistics are shown in the attached Figure 2 After taking the log of the peak area result of the accuracy solution, substitute it into the fitting equation to calculate the test concentration and recovery rate. The results are shown in the attached Figure 3 .

[0225] Sample solution preparation: After the IVT reaction is completed, add a certain volume of 0.5M EDTA (to make the final EDTA concentration 50mM) to terminate it, then precipitate overnight with a final concentration of 2.7M LiCl solution, wash with 70% ethanol, and finally re-dissolve the precipitate in enzyme-free water to obtain the mRNA stock solution. Inject 5μL for detection and record the chromatogram. The results are shown in Figure 4 The peak area was obtained by integration, and then the residual concentrations of NTP and cap analogs were calculated according to the linear fitting equation. The results are shown in Table 1.

[0226] Table 1: Test results of residual concentration of NTP and cap analogs in mRNA stock solution

[0227] In Example 2, NTP and cap analogs in the mRNA stock solution were effectively separated, with good separation and stability. Within the concentration range investigated, the linearity was good, and the residual concentrations of NTP and cap analogs in the mRNA stock solution could be accurately calculated according to the linear fitting equation, providing data support for release testing. 5.3. Example 3: Detection of NTPs, cap analogs and product mRNA during IVT reaction (1) Instrument and chromatographic conditions

[0228] Ultra-high performance liquid chromatograph: Thermo Vanquish F ultra-high performance liquid chromatograph and Chromeleon chromatography workstation;

[0229] Chromatographic column: ACQUITY UPLC Oligonucleotide BEH C18 (1.7 μm, 2.1 × 100 mm) hybrid particle bonded C18 column;

[0230] Prepare an aqueous solution containing 400mM TEAA and 200mM DBAA, adjust the pH to 7.0 with acetic acid, and use it as a mobile phase stock solution. Mix the stock solution with an equal volume of ultrapure water as mobile phase A; mix the stock solution with an equal volume of pure acetonitrile as mobile phase B. Set the flow rate to 0.4mL / min, and the elution gradient to: 0, 1, 2, 11, 22, 25, 25.1, 30min, with the corresponding proportions of phase B being 5%, 5%, 10%, 16%, 100%, 100%, 5%, 5%. The detection wavelength was 260nm, and the column temperature was 60℃. (2) Experimental procedures

[0231] Preparation of mixed standard solution: Dilute the standard stock solution with nuclease-free water to obtain 6 mixed standard solutions with different concentration levels, in which the concentration range of each NTPs and cap analogs is 1-500 μM, and the concentration range of mRNA is 0.015625-0.5 mg / mL.

[0232] 5 μL of each standard solution was injected for analysis and the chromatogram was recorded. The peak area of ​​each component was obtained after integration of the chromatogram, and the concentration and peak area were taken as logarithms, and linear fitting was performed to obtain the fitting equation.

[0233] Preparation of samples during the IVT reaction process: prepare a co-transcription IVT reaction solution. Among them, the starting concentration of the cap analog is 6.4mM, and the starting concentrations of the four NTPs are: N1-methyl-pseudouridine triphosphate is 4mM, CTP, GTP and ATP are 8mM. Divide the prepared IVT reaction solution into 80μL / tube, place it on a constant temperature mixer, and react at 37°C, 200rpm. Every 20-30 minutes, take out a tube and add 8.9μL 0.5M EDTA solution to terminate the reaction. After the terminated solution is diluted 50 times with enzyme-free water, 5μL is injected for detection, and the chromatogram is recorded. The results are shown in Figure 6 The peak area was obtained by integration, and the concentrations of NTP, cap analog and mRNA were calculated according to the linear fitting equation, and the IVT process curve was drawn. Figure 7 .

[0234] According to the IVT process detection curve, it can be observed that when the IVT reaction is carried out for 80 minutes, the remaining amount of N1-methyl-pseudouridine triphosphate is 24%. When the reaction is carried out for 100 minutes, N1-methyl-pseudouridine triphosphate is almost consumed, and the remaining amount is only 4%. In order to avoid the reduction of the integrity of the product mRNA due to NTP shortage, the termination time of the IVT reaction should be controlled at 80 minutes.

[0235] In Example 3, NTPs, cap analogs, and product mRNA in the IVT reaction system were effectively separated, with good and stable separation. Within the concentration range investigated, the linearity was good, and the content of the components to be tested in the system could be accurately calculated according to the linear fitting equation, effectively monitoring the IVT reaction process and providing intuitive data support for the IVT reaction kinetics.

Claims

1. A method for analyzing NTP, cap analog and mRNA components in in vitro transcription product components based on UPLC, comprising the following steps: (1) preparing a sample of in vitro transcription product for UPLC detection and (2) detecting using the UPLC method; Wherein step (2) adopts UPLC method to carry out detection and comprises: a) Mobile phase preparation: Mobile phase A is an aqueous solution containing 150-250 mM triethylamine-acetic acid (TEAA) and 60-120 mM dibutylene glycol-acetic acid (DBAA), pH 6.90-7.10, and mobile phase B is an aqueous solution containing 150-250 mM triethylamine-acetic acid and 60-120 mM dibutylene glycol-acetic acid in a volume concentration of 30%-60% acetonitrile, pH 6.90-7.10; b) Chromatographic separation was performed using a C18 column: the separation gradient program was as follows: initial 95% mobile phase A + 5% mobile phase B; 1 minute: 95% mobile phase A + 5% mobile phase B; 2 minutes: 90% mobile phase A + 10% mobile phase B; 11 minutes: 84% mobile phase A + 16% mobile phase B; 22 minutes: 0% mobile phase A + 100% mobile phase B; 25 minutes: 0% mobile phase A + 100% mobile phase B; 25.1 minutes: 95% mobile phase A + 5% mobile phase B; 30 minutes: 95% mobile phase A + 5% mobile phase B; and c) Collect chromatograms of NTPs, cap analogs and mRNA in the in vitro transcription product components.

2. The method according to claim 1, wherein: The analytical method further comprises the following quantitative step: d) collecting chromatograms of NTP, cap analog and mRNA in the mixed standard solution at no less than three concentration levels; e) After taking the log of the concentrations and peak areas of NTP, cap analog and mRNA in the mixed standard solution, a standard curve is drawn to obtain a linear fitting equation; and f) Calculate the concentration of each NTP and cap analog based on the linear fitting equation of the standard.

3. The method according to claim 1 or 2, characterized in that: The NTP is natural CTP, UTP, GTP, or ATP.

4. The method according to claim 1 or 2, characterized in that: The NTP comprises two or more natural NTPs, wherein the natural NTPs include: CTP, UTP, GTP, and ATP.

5. The method according to claim 1 or 2, characterized in that: The NTP comprises a modified NTP.

6. The method according to claim 5, characterized in that: The modified NTP is N1-methyl-pseudouridine triphosphate, N6-methyl-adenosine triphosphate, N1-methyl-adenosine triphosphate, 2-amino-adenosine triphosphate, 2-oxymethyl-adenosine triphosphate, 5-methyl-cytidine triphosphate, 5-hydroxymethyl-cytidine triphosphate, N4-acetyl-cytidine triphosphate, 2-oxymethyl-cytidine triphosphate, N7-methyl-guanosine triphosphate, 2-oxymethyl-guanosine triphosphate, N1-methyl-pseudouridine triphosphate, pseudouridine triphosphate, 2-thio-uridine triphosphate, 5-methoxy-uridine triphosphate, or 2-oxymethyl-uridine triphosphate.

7. The method according to claim 1 or 2, characterized in that: The NTP comprises two or more NTPs, wherein the NTP is selected from: CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

8. The method according to claim 1 or 2, characterized in that: The NTPs are CTP, UTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

9. The method according to claim 1 or 2, characterized in that: The NTPs are CTP, GTP, ATP and N1-methyl-pseudouridine triphosphate.

10. The method according to claim 1 or 2, characterized in that: The cap analog is a dinucleotide cap analog, a trinucleotide cap analog, or a tetranucleotide cap analog.

11. The method according to claim 1 or 2, characterized in that: The hat analog is m 7 (3'OMeG)(5')ppp(5')G(ARCA)、m 7 G(5')ppp(5')(2'OMeA)pG、m 7 (3'OMeG)(5')ppp(5')(2'OMeA)pG、m 7 G(5')ppp(5')(2'OMeA)pU、 m 7 G(5')ppp(5')(2'OMeG)pG, m 7 (3'OMeG)(5')ppp(5')(2'OMeG)pG, or m 7 G(5')ppp(5')(2'OMeA)pG。 12. The method according to claim 1 or 2, characterized in that: a) The mobile phase A contained 200 mM triethylamine-acetic acid and 100 mM butanediamine-acetic acid.

13. The method according to claim 1 or 2, characterized in that: a) The mobile phase B is an acetonitrile aqueous solution containing 200 mM triethylamine-acetic acid and 100 mM butanediamine-acetic acid with a volume concentration of 30%-60%.

14. The method according to claim 1 or 2, characterized in that: The C18 column in b) is a hybrid particle bonded C18 column with an inner diameter of 2.1 mm and a particle size of 1.7 μm.

15. The method according to claim 1 or 2, characterized in that: b) The total flow rate of mobile phase A and phase B is: 0.2-0.6 mL / min.

16. The method according to claim 1 or 2, characterized in that: b) The total flow rate of mobile phase A and phase B is 0.4 mL / min.

17. The method according to claim 1 or 2, characterized in that: b) The column temperature of the C18 column is 50-70°C.

18. The method according to claim 1 or 2, characterized in that: b) The column temperature of the C18 column was 60°C.

19. The method according to claim 1 or 2, characterized in that: Step (1) adopts a method comprising the following steps: i. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; ii. The termination mixture described in i was precipitated with 2-4M LiCl, washed with 70% ethanol, and the precipitate was redissolved in enzyme-free water to obtain an in vitro transcribed mRNA stock solution sample for UPLC detection.

20. The method according to claim 1 or 2, characterized in that: Step (1) adopts a method comprising the following steps: i. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; ii. Purify the termination mixture described in i by chromatography or other means to obtain a sample of the in vitro transcription product for UPLC detection.

21. The method according to claim 1 or 2, characterized in that: Step (1) adopts a method comprising the following steps: i. The in vitro transcription product was terminated by 20-100 mM EDTA (final concentration) to obtain a termination mixture; and ii. Dilute the termination mixture described in i by 20-100 times with enzyme-free water to obtain a sample of the in vitro transcription product for UPLC detection.

22. The method according to claim 1 or 2, characterized in that The in vitro transcription product in step (1) is any mRNA in vitro transcription product.

23. The method according to claim 1 or 2, characterized in that The in vitro transcription product in step (1) is an mRNA in vitro transcription product with a length of 500-1000nt, 1000-2000nt, 2000-3000nt, 3000-4000nt, or 4000-10000nt.

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