Systems and methods for isolating mRNA

Abstract

A method for isolating mRNA molecules by a combination of magnetic particles and positive pressure affinity chromatography is provided. mRNA molecules can be obtained from in vitro transcription with high yield and purity by using a combination of magnetic particles and positive pressure affinity chromatography. In particular, the method can be adapted to an automated process to obtain high quality mRNA molecules with a high-throughput simplified workflow.

Description

Systems and methods for isolating mRNA Technical Field

[0001] The present invention relates to the field of biological purification, and in particular to a system and method for isolating mRNA. Background Art

[0002] Messenger RNA (mRNA)-based therapies have shown great promise in preventing and treating a variety of diseases. Compared to recombinant proteins expressed in mammalian cell lines, mRNA manufacturing is faster and more flexible because mRNA can be easily produced by in vitro transcription. However, the technical barriers faced by mRNA therapies are also significant. For example, the efficacy of mRNA vaccines is reduced due to poor RNA stability in solution and in vivo and limited expression of payload mRNA, which are key issues that require careful optimization for preclinical and clinical applications (Sahin, U., K.&Türeci, mRNA-based therapeutics-developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014); Jackson, NAC, Kester, KE, Casimiro, D., Gurunathan, S. & DeRosa, F. The promise of mRNA vaccines: a biotech and industrial perspective. npj Vaccines 5, 11 (2020); Weng, Y. et al. challenge and prospect of mRNA therapeutics landscape. Biotechnol. Adv. 40, 107534 (2020); Crommelin, DJ A, Anchordoquy, TJ, Volkin, DB, Jiskoot, W. & Mastrobattista, E. Addressing the cold reality of mRNA vaccine stability. J. Pharm. Sci. 110, 997–1001 (2021)).

[0003] To solve the above problems, it is necessary to optimize the design of mRNA molecules, including 5' capping, promoter, 5' and 3' untranslated regions (UTR), Kozak sequence and signal domain, enhancer element, poly (A), intron and splice site, ribosome internal entry site and CDS codon optimization (Guo, X., Wang, C., and Wang, TY (2020). Chromatin-modifying elements for recombinant protein production in mammalian cell systems. Crit. Rev. Biotechnol. 40, 1035–1043; Wang, TY, and Guo, X. (2020). Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems. Appl. Microbiol. Biotechnol. 104, 5673–5688; Mauro, VP (2018). Codon optimization in the production of recombinant biotherapeutics: potential risks and Considerations. BioDrugs. 32, 69–81; Kawabe, Y., Inao, T., Komatsu, S., Huang, G., Ito, A., Omasa, T., and Kamihira, M. (2017). Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site. J. Biosci. Bioeng. 123, 390–397). In addition, for the same vaccine project, it is necessary to screen for effective sequences, which is a huge workload, tedious and variable, and cannot be effectively completed by manpower in a short time.

[0004] In terms of technology, the modification process of in vitro transcription, the reaction process (including NTP ratio, DNA template, reaction time, Mg 2+, etc.) are all crucial to the quality of the final mRNA stock solution and are typically optimized using a Design of Experiments (DOE) approach. Manual manipulation is prone to deviations due to the tedious nature of varying conditions. Using automated systems for mRNA preparation and purification can achieve efficient and controllable parallelization results.

[0005] The mRNA product obtained through enzymatic transcription will produce impurities such as truncated RNA, double-stranded (ds) RNA, and reactants, which need to be removed during downstream purification. A variety of techniques can be used to isolate and purify mRNA, including but not limited to LiCl precipitation, chromatography, and magnetic particles.

[0006] LiCl is commonly used to precipitate RNA from in vitro transcription (IVT). LiCl precipitation uses high concentrations of lithium cations to selectively precipitate RNA. After the mRNA is precipitated, the supernatant, which contains salts, free NTPs, proteins, and DNA, is discarded. In this purification method, post-transcription DNase I treatment is optional to ensure complete removal of the template DNA. LiCl precipitation provides a simple and rapid method for recovering RNA from in vitro transcription reactions.

[0007] The mRNA purification process also often uses Oligo(dT) affinity chromatography to capture mRNA molecules and remove process-related impurities such as enzymes, NTPs, and template DNA. High-resolution hydrophobic or ionic fillers are used for fine purification to remove product-related impurities such as fragments or dsRNA. Oligo(dT) affinity chromatography binds to the Poly(A) tail and performs affinity purification, which can remove most impurities such as NTPs, enzymes, and DNA, but cannot distinguish between dsRNA and truncated RNA. In addition, ion exchange chromatography (IEC), hydrophobic chromatography (HIC), and ion pair reverse chromatography are also frequently used for mRNA purification.

[0008] The use of magnetic particles to separate biomolecules is known in the art. For example, patent publication number WO2018122246 describes a method for separating biomolecules from cell culture, comprising binding biomolecules to magnetic beads, separating the magnetic beads comprising the bound biomolecules from the rest of the cell culture using a magnetic separator, transporting the magnetic beads comprising the bound biomolecules as a slurry with added buffer to a separate elution unit, and eluting the biomolecules from the magnetic beads in the elution unit. Magnetic particles are an effective way to purify target biomolecules directly from a crude feed unit reserve, and this technology can replace clarification tools such as centrifugation, filtration, and capture chromatography. Magnetic particles are typically functionalized with ligands that have an affinity for the target biomolecule. Magnetic particles are added and mixed with the crude feed for a certain period of time to specifically bind to all target biomolecules in the feed. After binding, the magnetic particles are captured by a magnet, while cells and impurities are decanted. Washing buffer is added to the magnetic particles, the magnet is turned off, and the washing buffer and magnetic particles are mixed. After mixing, the magnet is turned on to capture the magnetic particles, and the washing buffer is decanted. After several washings, elution buffer is added to the magnetic particles in the same manner as wash buffer, and several batch elution steps are used to release the target biomolecule from the magnetic particles. All washing steps and elution steps are carried out in batch mode, and owing to the need to carry out several batch elution steps to obtain high elution yields, using this technology will dilute the target more compared to column chromatography. In addition, using batch elution is more difficult to optimize elution conditions, because elution buffer needs to titrate the wash buffer to reach correct elution conditions to release the target biomolecule from the magnetic beads, and different or large-volume elution buffers are needed for each elution step. A way to overcome this is to transfer the washed magnetic particles to a chromatographic column for the elution step, to reduce the volume of elution buffer, and use a kind of optimized elution buffer classification to collect the eluted amalgam. However, this requires additional equipment and column packing, which is time-consuming.

[0009] Currently, these technologies for isolating and purifying mRNA have certain deficiencies, making them inadequate for large-scale platform applications and making it difficult to rapidly deliver mRNA therapies in response to sudden outbreaks. New tools and scalable workflows for the development and production of mRNA vaccines are crucial for advancing this promising technology into the clinic. Therefore, there is a need in the art for improved methods for isolating mRNA molecules, including purifying high-quality mRNA molecules from feedstocks in a reduced method time and more cost-effective manner.

[0010] Summary of the Invention

[0011] The present disclosure achieves the above-mentioned object of providing an improved method for isolating and purifying mRNA molecules. The present disclosure relates to a method for isolating mRNA molecules using a combination of magnetic particles and positive pressure affinity chromatography. In particular, the method is suitable for automation to increase throughput and shorten workflows to obtain high-quality mRNA molecules for downstream applications.

[0012] After multiple rounds of testing, the present invention discovered that mRNA molecules can be obtained from in vitro transcription with high yield and purity by using a combination of magnetic particles and positive pressure affinity chromatography. Without being bound by any theory, magnetic particles and positive pressure affinity chromatography work synergistically based on different principles of action, achieving higher yield and purity than other methods or combinations of methods for purifying mRNA with less time and material costs. Those skilled in the art will understand that in the method of the present invention, the order and number of magnetic particle and positive pressure affinity chromatography separation steps are not limited, and thus the method of the present invention may include one or more magnetic particle separation steps and / or one or more positive pressure affinity chromatography separation steps in any order or alternating manner.

[0013] Specifically, in one embodiment, the method comprises one or more magnetic particle separation steps. In another embodiment, the method comprises one or more positive pressure affinity chromatography separation steps. In one embodiment, the method comprises first performing one or more magnetic particle separation steps, followed by one or more positive pressure affinity chromatography separation steps. In another embodiment, the method comprises first performing one or more positive pressure affinity chromatography separation steps, followed by one or more magnetic particle separation steps. In one embodiment, the method comprises first performing one or more magnetic particle separation steps, followed by one or more positive pressure affinity chromatography separation steps, followed by one or more magnetic particle separation steps. In another embodiment, the method comprises first performing one or more positive pressure affinity chromatography separation steps, followed by one or more magnetic particle separation steps, followed by one or more positive pressure affinity chromatography separation steps.

[0014] In a preferred embodiment, the method comprises first performing a magnetic particle separation step, then performing a positive pressure affinity chromatography separation step, and optionally thereafter performing a magnetic particle separation step. In another preferred embodiment, the method comprises first performing a positive pressure affinity chromatography separation step, then performing a magnetic particle separation step.

[0015] More specifically, the present disclosure relates to a method for isolating mRNA molecules, comprising the steps of:

[0016] (a) providing magnetic particles comprising a ligand capable of binding to an mRNA molecule;

[0017] (b) contacting a solution comprising mRNA molecules with the magnetic particles to obtain magnetic particles comprising bound mRNA molecules;

[0018] (c) separating the magnetic particles containing the bound mRNA molecules using a magnetic field;

[0019] (d) eluting the mRNA molecules from the separated magnetic particles to obtain a primarily purified solution containing mRNA molecules; and

[0020] (e) loading the solution containing mRNA molecules obtained in step (d) onto an affinity chromatography layer containing oligo(dT), and eluting the mRNA molecules under positive pressure to obtain a secondary purified solution containing mRNA molecules.

[0021] In one embodiment, the method further comprises the step of performing in vitro transcription (IVT) on the DNA template to generate a solution comprising mRNA molecules before step (b).

[0022] In one embodiment, the method further comprises the step of capping and / or tailing the mRNA molecules prior to step (b).

[0023] In one embodiment, the method further comprises the step of capping and / or tailing the mRNA molecules prior to step (e).

[0024] In one embodiment, the method further comprises the step of capping the mRNA molecule after step (e).

[0025] In one embodiment, the method further comprises the step of performing a LiCl method to concentrate the secondary purified solution containing the mRNA molecules after step (e).

[0026] In one embodiment, the method further comprises the step of performing a magnetic particle method on the secondary purified solution containing mRNA molecules after step (e). In one embodiment, the magnetic particle method comprises repeating steps (a)-(d).

[0027] In one embodiment, the method further comprises the step of sterile filtering the secondary purified solution containing the mRNA molecules after step (e).

[0028] More specifically, the present disclosure relates to a method for isolating mRNA molecules, comprising the steps of:

[0029] (a) providing magnetic particles comprising a ligand capable of binding to an mRNA molecule;

[0030] (b) performing in vitro transcription on the DNA template to generate a solution containing mRNA molecules, wherein the mRNA molecules contain a poly (A) tail;

[0031] (c) contacting a solution comprising mRNA molecules with the magnetic particles to obtain magnetic particles comprising bound mRNA molecules;

[0032] (d) separating the magnetic particles containing the bound mRNA molecules using a magnetic field;

[0033] (e) eluting the mRNA molecules from the separated magnetic particles to obtain a primarily purified solution containing mRNA molecules;

[0034] (f) capping and incubating the mRNA molecules;

[0035] (g) loading the solution containing mRNA molecules obtained in step (f) onto an affinity chromatography layer containing oligo(dT), and eluting the mRNA molecules under positive pressure to obtain a secondary purified solution containing mRNA molecules; and

[0036] (h) performing liquid replacement, concentration, and / or sterile filtration on the solution containing the mRNA molecules obtained in step (g).

[0037] In one embodiment, the liquid exchange concentration in step (g) is performed by a LiCl method or a magnetic particle method. In one embodiment, the magnetic particle method comprises repeating steps (c) to (e).

[0038] More specifically, the present disclosure relates to a method for isolating mRNA molecules, comprising the steps of:

[0039] (a) providing a solution containing mRNA molecules;

[0040] (b) loading the solution containing the mRNA molecules onto an affinity chromatography layer containing oligo(dT), and eluting the mRNA molecules under positive pressure to obtain a primary purified solution containing the mRNA molecules;

[0041] (c) providing magnetic particles comprising ligands capable of binding to mRNA molecules, by contacting the solution comprising mRNA molecules obtained in step (b) with the magnetic particles to obtain magnetic particles comprising bound mRNA molecules;

[0042] (d) separating the magnetic particles containing the bound mRNA molecules using a magnetic field; and

[0043] (e) eluting the mRNA molecules from the separated magnetic particles to obtain a secondary purified solution containing mRNA molecules.

[0044] In one embodiment, step (a) further comprises in vitro transcription of the DNA template to generate a solution comprising mRNA molecules.

[0045] In another embodiment, step (a) further comprises capping and / or tailing the mRNA molecule.

[0046] In another embodiment, step (b) is followed by a step of capping the mRNA molecules.

[0047] In another embodiment, step (e) is followed by a step of capping the mRNA molecules.

[0048] In one embodiment, the solution containing the mRNA molecules obtained in step (e) is subjected to liquid exchange, concentration and / or sterile filtration.

[0049] In one embodiment, the step of replacing the liquid and concentrating is performed by a LiCl method and / or a magnetic particle method.

[0050] In one embodiment, the magnetic particle method comprises repeating steps (c) to (e).

[0051] In any embodiment of the present disclosure, the DNA template can be single-stranded or double-stranded linear or circular DNA. In a preferred embodiment, the DNA template is linearized before in vitro transcription. In a preferred embodiment, the DNA template is a linearized plasmid.

[0052] In a preferred embodiment of the present disclosure, the magnetic particles are carboxyl magnetic beads, such as Dynabeads TM MyOne TM Carboxylic acid magnetic beads.

[0053] In a preferred embodiment of the present disclosure, the positive pressure affinity chromatography comprises Oligo(dT) 25 Affinity media, such as POROS TM Oligo(dT) 25 Affinity filler.

[0054] In any of the embodiments of the present disclosure, the method is performed in an automated, semi-automated, or manual manner.

[0055] Furthermore, the present disclosure relates to a system for isolating mRNA molecules, comprising:

[0056] a first purification module comprising magnetic particles containing ligands capable of binding to mRNA molecules;

[0057] A second purification module comprising an affinity chromatography column, wherein the affinity chromatography column is operated under positive pressure; and

[0058] A fluid module includes a container for containing a solution containing mRNA, and the fluid module is capable of fluid communication with the first purification module and the second purification module.

[0059] In one embodiment, the system further comprises a feed module for providing a source of mRNA molecules and / or a collection module for collecting mRNA molecules. In one embodiment, the feed module comprises a container for holding IVT reaction reagents. In another embodiment, the feed module comprises a container for an IVT reaction.

[0060] In one embodiment, the fluid module further comprises a liquid transfer device. In one embodiment, the liquid transfer device is any device or means capable of transferring liquid, such as a pipette, such as a manual or automatic pipette, a pipette, a pipetting workstation, etc.

[0061] In one embodiment, the fluid module further comprises conduits that facilitate fluid communication among the first purification module, the second purification module, and the fluid module, and optionally the feed module and the collection module.

[0062] In one embodiment, the first purification module comprises a magnetic separator. In another embodiment, the first purification module comprises a magnetic field.

[0063] In one embodiment, the fluid module further comprises a container for containing an elution solution for eluting mRNA from the magnetic particles, and a container for containing an elution solution for eluting mRNA from the affinity chromatography column.

[0064] In one embodiment, the fluid module further comprises a container for containing a washing solution for washing the magnetic particles, and a container for containing a washing solution for washing the affinity chromatography column.

[0065] In one embodiment, the system further comprises one or more additional modules, including but not limited to one or more means for performing the following operations:

[0066] The DNA template is transcribed in vitro to generate a solution containing mRNA molecules;

[0067] Capping and / or tailing mRNA molecules;

[0068] performing a liquid exchange and concentration on the solution containing the mRNA molecule; and / or

[0069] The solution containing the mRNA molecules is sterile filtered.

[0070] In one embodiment, the fluid exchange and concentration is performed by a LiCl method. In one embodiment, the fluid exchange and concentration is performed by a magnetic particle method. In one embodiment, the additional module for performing the fluid exchange and concentration is the same as the first purification module. In one embodiment, the additional module for performing the fluid exchange and concentration is different from the first purification module.

[0071] In one embodiment, the system is operated in an automated, semi-automated or manual manner. Therefore, in one embodiment, the system further comprises a control module, which is configured to control the various modules of the system to operate in an automated or semi-automated manner.

[0072] In some embodiments of the methods or systems described herein, the mRNA molecule comprises one or more functional nucleotide analogs. Preferably, the functional nucleotide analogs are one or more selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG1 shows two exemplary flow charts (A and B) of the method for isolating mRNA molecules according to the present invention.

[0074] Figure 2 shows a comparison of purification results using the LiCl method and magnetic particle methods. A, Comparative experimental scheme for the two purification methods; B, Purity results after the IVT reaction (before capping) using the two purification methods; C, mRNA purity and capping efficiency after the two purification methods using the two purification methods.

[0075] Figure 3 shows the results of impurity analysis using the magnetic bead method. A, IFN-β analysis of mRNA stock solution in BJ cells; B, activity analysis of mRNA stock solution in BJ cells; C, cytotoxicity analysis of magnetic beads in BC2C12 mouse muscle cells.

[0076] FIG4 shows the results of hematological and blood biochemical analyses of mice injected with magnetic beads via the tail vein.

[0077] Figure 5 shows the yield of mRNA molecules isolated by negative pressure affinity chromatography. NC: positive pressure affinity chromatography.

[0078] FIG6 shows a flow chart of various methods for isolating mRNA molecules, wherein Scheme 3 exemplifies a method according to the present invention.

[0079] Figure 7 shows the comparison of FA purity analysis results for Process 1, Process 2, and Process 3.

[0080] Figure 8 shows the comparison of capping rate results of Process 1, Process 2, and Process 3.

[0081] Figure 9 shows the comparison of protein residue detection results of Process 1, Process 2, and Process 3.

[0082] FIG10 shows a comparison of the in vitro expression results of Process 1, Process 2, and Process 3.

[0083] FIG11 shows a comparison of the protein residue detection results of Process 1 and Process 4.

[0084] FIG12 shows a comparison of mRNA purity, capping efficiency, residual DNA, residual dsRNA, and in vitro protein expression results between Process 3 and Process 5.

[0085] FIG13 shows a flow chart of a method for isolating mRNA molecules, wherein Scheme 7 exemplifies a method according to the present invention.

[0086] FIG14 shows a comparison of the FA purity analysis results of Scheme 6 and Scheme 7.

[0087] FIG15 shows a comparison of the capping rate results of Process 6 and Process 7.

[0088] FIG16 shows a comparison of the DNA residue detection results of Process 6 and Process 7.

[0089] FIG17 shows a comparison of the dsRNA residual detection results of Process 6 and Process 7.

[0090] FIG18 shows a comparison of the results of the immune factor IFN-β detection in BJ fibroblasts for Process 6 and Process 7.

[0091] FIG19 shows a comparison of the results of detecting the IFN-β content in the supernatant of BJ cell transfection in IFN-α / βReporter HEK 293 cells for Process 6 and Process 7.

[0092] 20A-20E show the comparison of the results of in vitro protein expression assays in A549 cells for Process 6 and Process 7. DETAILED DESCRIPTION

[0093] The specific embodiments of the present invention will be described in detail below.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those of skill in the art with general definitions of many of the terms used in this invention: Dictionary of Biochemistry and Molecular Biology, (2nd ed.) J. Stenesh (ed.), Wiley-Interscience (1989); Dictionary of Microbiology and Molecular Biology (3rd ed.), P. Singleton and D. Sainsbury (eds.), Wiley-Interscience (2007); Chambers Dictionary of Science and Technology (2nd ed.), P. Walker (ed.), Chambers (2007); Glossary of Genetics (5th ed.), R. Rieger et al. (eds.), Springer-Verlag (1991); and The HarperCollins Dictionary of Biology, WG Hal and JP Margham (eds.), HarperCollins (1991).

[0095] The terms "one" or "an" as used herein do not indicate a limit on quantity, but rather indicate the presence of at least one of the related items. The terms "or" and "alternatively" do not mean exclusive, but rather refer to the presence of at least one of the mentioned items (e.g., components), and include situations where a combination of the mentioned items may exist. "Include," "comprise," "comprising," "having," or "containing" and similar words used in this application mean that in addition to the items listed thereafter and their equivalents, other items may also be within the scope.

[0096] Approximate terms in this application are used to modify quantity, representing that the present invention is not limited to the specific quantity, and also include the correction part that is close to the quantity, acceptable, and will not cause the change of the relevant basic function. Accordingly, a numerical value is modified with "about", "approximately" and "about", meaning that the present invention is not limited to the precise numerical value. In certain embodiments, approximate terms may correspond to the precision of the instrument for measuring numerical value. Numerical ranges in the present invention can be merged and / or interchanged, and unless otherwise clearly stated, numerical ranges include the subranges of all numerical values ​​it encompasses.

[0097] The term "messenger ribonucleic acid", also known as "messenger RNA" or "mRNA", is a type of single-stranded ribonucleic acid transcribed from a strand of DNA as a template, which carries genetic information and can guide protein synthesis. An mRNA vaccine is a vaccine that introduces mRNA encoding an antigen protein into the human body, directly translates it, and forms the corresponding antigen protein, thereby inducing the body to produce a specific immune response and achieve the effect of preventive immunity. In this article, unless otherwise indicated, RNA molecules, mRNA molecules, or mRNA vaccines have the same meaning. In some embodiments, "messenger ribonucleic acid," "messenger RNA," or "mRNA" includes functional nucleotide analogs. In some embodiments, the functional nucleotide analogs include non-standard nucleobases. In some embodiments, the standard nucleobases in the nucleotide (e.g., adenine, guanine, uracil, thymine, and cytosine) can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modifications of the nucleobases include, but are not limited to, one or more substitutions or modifications including, but not limited to, alkyl, aryl, halogen, oxo, hydroxyl, alkoxy and / or thio substitutions; one or more fused or opened rings, oxidation and / or reduction.

[0098] In some embodiments, the nucleic acid molecules of the present disclosure include mRNA molecules. In specific embodiments, the nucleic acid molecules include at least one coding region (e.g., open reading frame (ORF)) encoding a peptide or polypeptide of interest. In some embodiments, the nucleic acid molecules further include at least one non-translated region (UTR). In specific embodiments, the non-translated region (UTR) is located upstream (5' end) of the coding region and is referred to herein as 5'-UTR. In specific embodiments, the non-translated region (UTR) is located downstream (3' end) of the coding region and is referred to herein as 3'-UTR. In specific embodiments, the nucleic acid molecules include both 5'-UTR and 3'-UTR. In some embodiments, the 5'-UTR includes a 5'-cap structure. In some embodiments, the nucleic acid molecules include a Kozak sequence (e.g., in the 5'-UTR). In some embodiments, the nucleic acid molecules include a poly-A region (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecules include a polyadenylation signal (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecules include a stabilizing region (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecule comprises a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in a 5'-UTR and / or a 3'-UTR). In some embodiments, the nucleic acid molecule comprises one or more intron regions that can be excised during splicing. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR and a coding region. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a coding region and a 3'-UTR. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR, a coding region, and a 3'-UTR.

[0099] In some embodiments, the non-standard nucleobase is a modified uracil. Exemplary nucleobases and nucleosides with modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromouracil), 3-methyluracil, 5-naphthyl- ... Uracil (m3U), 5-methoxyuracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyluracil (mcm5U), 5-methoxycarbonylmethyl-2-thiouracil (mcm5s2U), 5-Aminomethyl-2-thiouracil (nm5s2U), 5-methylaminomethyl-2-thiouracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyluracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm5s2U) , 5-propynyl uracil, 1-propynyl-pseudouracil, 5-taurine methyl uracil (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouracil (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (Et1ψ) and 5-methyl-2-thio-uracil (m5s2U). In some embodiments, the non-standard nucleobase of the functional nucleotide analogue can independently be a purine, a pyrimidine, a purine or a pyrimidine analogue. For example, in some embodiments, the non-standard nucleobase can be a modified adenine, cytosine, guanine, uracil or hypoxanthine.In other embodiments, non-canonical nucleobases can also include, for example, naturally occurring and synthetic derivatives of bases, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-Thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine and 1,3,5-triazine.

[0100] As described herein, 0% to 100% of all nucleotides of one type (e.g., all purine-containing nucleotides of one type in a payload nucleic acid molecule, or all pyrimidine-containing nucleotides of one type, or all A, G, C, T, or U) can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1% to about 20%, from about 1% to about 25%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10%% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70% , about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100% or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, functional nucleotide analogs can be present in any position of the nucleic acid molecule, including the 5'-end, the 3'-end and / or one or more internal positions. In some embodiments, a single nucleic acid molecule can include different sugar modifications, different core base modifications and / or different types of nucleoside bonds (such as backbone structures).

[0101] The terms "isolated" or "purified" refer to a substance that is substantially or essentially free of components that normally accompany the substance in its natural or original state. Thus, isolated mRNA molecules according to the present disclosure are preferably free of substances that are normally associated with the mRNA molecules in their natural or original environment. A compound or entity is considered isolated or purified when it is free of substantially all other compounds or entities, i.e., preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. In some embodiments, the mRNA molecules isolated by the methods of the present disclosure include less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of impurities other than full-length mRNA molecules. Impurities include IVT contaminants, such as proteins, enzymes, free nucleotides, and / or short polymers. In some embodiments, mRNA molecules prepared according to the present invention are substantially free of short polymers or abortive transcripts.

[0102] Any method known in the art can be used to detect and quantify the full length or abortive transcripts of mRNA. In some embodiments, the mRNA molecules separated are detected using blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectrum, ultraviolet (UV) or UPLC or a combination thereof. Other detection methods known in the art are included in the present invention. In some embodiments, mRNA molecules are detected by capillary electrophoresis separation using UV absorption spectroscopy. In some embodiments, before gel electrophoresis ("glyoxal gel electrophoresis"), mRNA is denatured by glyoxal dye. In some embodiments, the synthesized mRNA is characterized before capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing.

[0103] The term "plasmid" refers to a genetic construct consisting of genetic material (i.e., nucleic acid). Typically, a plasmid contains an origin of replication that is functional in a bacterial host cell, such as E. coli, and a selectable marker for detecting bacterial host cells containing the plasmid. In the present disclosure, a plasmid may include one or more genetic elements that are configured such that an inserted coding sequence can be transcribed and translated in a suitable expression cell. Additionally, the plasmid may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments obtained or derived from one or more natural and / or artificial sources.

[0104] The term "LiCl method," also known as "LiCl precipitation," refers to a method that uses high concentrations of lithium cations to selectively precipitate RNA. Typically, synthesized RNA is added to a high-concentration LiCl solution and incubated at -20°C. The mRNA is then purified by high-speed centrifugation at 4°C, for example, 12,000-15,000g. The LiCl method is a common method used in the art for purifying mRNA.

[0105] The term "magnetic particles" is defined herein as particles that can be attracted by a magnetic field. At the same time, the magnetic particles used in the method of the present invention should not aggregate in the absence of a magnetic field. In other words, the behavior of the magnetic particles should be similar to that of superparamagnetic particles. The magnetic particles can have any symmetrical shape, such as a sphere or a cube, or any asymmetric shape. Spherical magnetic particles are generally referred to as magnetic beads. It should be understood that the terms "magnetic particles" and "magnetic beads" are used interchangeably herein without limiting the scope to magnetic particles with a spherical shape. The volume-weighted median diameter (d50,v) of the magnetic particles suitable for the method of the present invention can be in the range of 10nm-100μm, such as 10nm, 20nm, 100nm, 1μm, 2μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 50μm, 60μm, 70μm, 80μm, 90μm, 95μm or 100μm. Preferably, the volume-weighted median diameter (d50,v) of the magnetic particles or the particle size of the magnetic beads is between 20 nm and 5 μm. Generally, the smaller the diameter of the magnetic particles, the greater the overall contact surface area. It should be understood that the diameter of the magnetic particles is not limited to this range and depends on factors such as the type, concentration, and volume of the biomolecules to be separated, as well as the separation system.

[0106] The magnetic particles contain ligands that can bind to biomolecules. The ligands can be covalently coupled to the surface or the interior of the magnetic particles. It should be understood that the type of ligand and its affinity constant k for the biomolecule off / k on The ligand is selected based on the type of biomolecule to be separated. In one embodiment, the ligand is a carboxyl group. In addition, it should be understood that the concentration of the one or more ligands per magnetic particle depends on the concentration of the biomolecule to be separated, the size of the magnetic particles, and / or the total volume of magnetic particles added to the magnetic separator. In a preferred embodiment of the present disclosure, the magnetic particles used in the method for isolating mRNA molecules are carboxyl magnetic beads.

[0107] The terms "magnetic bead method" and "magnetic particle method" used herein are used interchangeably and refer to a method for purifying biological molecules, such as mRNA molecules, using magnetic particles or magnetic beads.

[0108] The term "magnetic separator" has its conventional meaning in the field of separation methods and refers to a device for separating magnetic particles from a fluid. In a preferred embodiment of the present disclosure, the magnetic separator used in the method for separating biomolecules is a high gradient magnetic separator, or a high gradient magnetic separation system (HGMS). The magnetic separator is configured to separate the magnetic particles with bound mRNA molecules from the rest of the feed. The magnetic separator contains components of magnetic or magnetizable material inside that attract the magnetic particles when a magnetic field is applied.

[0109] In one embodiment, the magnetic bead method further comprises filtering the fluid using a 0.22 μm membrane after separating the magnetic particles from the fluid.

[0110] The term "affinity chromatography" is a chromatographic method that uses the binding properties of a stationary phase to separate molecules. Molecules that have a certain binding ability to the substance to be separated are connected to the affinity chromatography filler, and their binding is reversible. The two can be separated from each other when the mobile phase conditions are changed. The most significant structural feature of eukaryotic mRNA is that it has a cap at the 5' end and a Poly (A) tail at the 3' end. This structure provides an extremely convenient selection marker for the purification of eukaryotic mRNA. Affinity chromatography for isolating mRNA usually uses oligo (dT) to complement the Poly (A) tail to capture mRNA. In a preferred embodiment of the present disclosure, the affinity chromatography used in the method for isolating mRNA molecules uses Oligo (dT) 25 Affinity filler.

[0111] In one embodiment, the affinity chromatography used in the method for isolating mRNA molecules is operated in a positive pressure mode. The so-called "positive pressure" or "negative pressure" refers to the pressure state within the closed container of the chromatography system. The state when the pressure within the closed container is greater than atmospheric pressure is called positive pressure, while the state when the pressure is less than atmospheric pressure is called negative pressure. Positive pressure is the use of pressurized gas (compressed air or nitrogen) to allow the solution to enter the solid phase extraction column at a controllable flow rate for related operations; negative pressure is the use of a pump to extract air from the instrument and allow the solution to flow through the solid phase extraction column for related operations.

[0112] The term "in vitro transcription," also known as "IVT," refers to the process of producing RNA in a cell-free system using DNA as a template, mimicking the in vivo transcription process. In vitro transcription typically utilizes DNA containing T7, T3, or SP6 promoter sequences as a template. In the presence of T7, T3, or SP6 RNA polymerase, respectively, RNA complementary to one strand of the template DNA is synthesized using NTPs or modified bases as substrates. This allows for the large-scale production of RNA molecules in a cell-free environment. Prior to in vitro transcription, the DNA template is typically obtained through 1) Escherichia coli fermentation to amplify the DNA, purify the DNA, and linearize the plasmid DNA, or 2) enzymatic amplification and purification of the transcribed region.

[0113] During or after in vitro transcription, the resulting mRNA molecule may optionally be 5' capped and / or 3' tailed. The presence of the "cap" can provide resistance to nucleases found in most eukaryotic cells. The presence of the "tail" can be used to protect the mRNA from exonuclease degradation and / or to regulate protein expression levels. Capping and tailing techniques are well known to those skilled in the art.

[0114] The term "cap" refers to a structure comprising or consisting essentially of a 5'-terminal nucleoside-5'-triphosphate, which is typically bound to an uncapped RNA (e.g., an uncapped RNA having a 5'-diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, including, for example, but not limited to, an N7-methylguanosine cap having a structure designated as "m7G". In some embodiments, the cap is or comprises a synthetic cap analog that is similar to an RNA cap structure and has the ability to stabilize RNA if attached thereto, including, for example, but not limited to, anti-reverse cap analogs (ARCAs) known in the art. It will be understood by those skilled in the art that methods for attaching a cap to the 5' end of an RNA are known in the art. For example, in some embodiments, a capped RNA can be obtained by capping an RNA having a 5' triphosphate group or an RNA having a 5' diphosphate group in vitro with a capping enzyme system (including, for example, but not limited to, a vaccinia capping enzyme system or a Saccharomyces cerevisiae capping enzyme system). Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a DNA template, wherein the IVT system contains, in addition to GTP, a cap analog, for example, as known in the art. Non-limiting examples of cap analogs include m7GpppG cap analogs or N7-methyl-, 2'-O-methyl-GpppG ARCA cap analogs or N7-methyl-, 3'-O-methyl-GpppG ARCA cap analogs, or any commercially available cap analogs, including, for example, CleanCap (Trilink), EZ Cap, etc. In some embodiments, the cap analog is or comprises a trinucleotide cap analog. Various cap analogs are described herein and are known in the art, for example, commercially available.

[0115] The 5' cap can be added as follows: first, one terminal phosphate group is removed from the 5' nucleotide by RNA terminal phosphatase, leaving two terminal phosphate groups; then guanosine triphosphate (GTP) is added to the terminal phosphate group by guanylyltransferase to produce a 5,5,5 triphosphate bond; then the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G. More cap structures have been described in the prior art, such as U.S. patent application No. US2016 / 0032356, Ashiqul Haque et al., Chemically modified hCFTR mRNAs recuperate lung function in a mouse model of cystic fibrosis, Scientific Reports (2018) 8: 16776, and Kore et al., Recent Developments in 5'-Terminal Cap Analogs: Synthesis and Biological Ramifications, Mini-Reviews in Organic Chemistry, 2008, 5, 179-192, which are incorporated herein by reference.

[0116] The tail structure may include a poly (A) tail. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500 or more nucleotides at the 3' end of the mRNA may be a poly (A) tail. The poly (A) tail may include at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or 100% adenosine nucleotides.

[0117] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing. In some embodiments, the mRNA is purified by a LiCl method before or after or both before and after capping and tailing. In some embodiments, the mRNA is purified by a magnetic particle method before or after or both before and after capping and tailing. In some embodiments, the mRNA is purified by affinity chromatography before or after or both before and after capping and tailing.

[0118] In some embodiments, capping and / or tailing are performed concurrently with transcription. Thus, the mRNA can be purified directly after the IVT step is complete. In some embodiments, the mRNA molecules are purified before and / or after tailing. In some embodiments, the mRNA molecules are purified before and / or after capping.

[0119] The term "module" refers to a hardware or software unit composed of one or more basic functional units, which may also be referred to as a device or program. A module is typically named after its function or purpose and includes any hardware or software necessary to achieve the desired function. For example, a purification module consists of one or more devices used to achieve the purification purpose, while a control module contains a control program and the hardware configured to execute the program, such as a processor, storage, memory, disk, etc. It should be understood that those skilled in the art are familiar with the meaning and components of a "module" and can easily configure the various modules used in the methods and systems of the present invention after reading this disclosure. Exemplary configurations of modules are described in more detail below.

[0120] The term "automation" refers to the process of automatically controlling and automatically adjusting the operating device according to a predetermined pattern in a production operation. As the name suggests, the term "semi-automation" refers to an automation method that automatically performs a work cycle under human intervention. Automation, semi-automation or manual methods are the choices made based on a comprehensive consideration of factors such as operating conditions and cost-benefit ratio, and are all within the expected scope of the present invention. For example, by the method and system of the present invention, in an automation mode, 168 mRNA samples (mg level) can be processed in 24h, and by manual mode, 1 manual person can process 24 mRNA samples (mg level) in 5 days. In a preferred embodiment, the method or system according to the present invention is operated in an automation mode.

[0121] Figure 1, Scheme A, shows an exemplary flow chart of a method for isolating mRNA molecules according to the present invention. First, an in vitro transcription (IVT) reaction is performed on a linearized DNA template. The transcription template is typically obtained through processes such as 1) DNA amplification by E. coli fermentation, DNA purification, and plasmid DNA linearization, or 2) enzymatic amplification and purification of a DNA transcribed region fragment. In addition to the DNA template, the in vitro transcription system also includes reagents such as nucleoside triphosphate (NTP) substrates, RNA polymerase, inorganic pyrophosphatase, and RNase inhibitors. Optionally, the IVT reagents also include one or more of a 5'-cap analog, a capping enzyme, Poly(A) polymerase, DNase, and Proteinase K. 5' capping and / or 3' tailing can be performed during the IVT reaction or as a separate step after transcription. Strict control of DNA residues is required during the mRNA production process. DNase treatment hydrolyzes the DNA template, and Proteinase K treatment degrades proteins, facilitating subsequent purification. Depending on the reaction volume, IVT can be performed in any suitable container, such as a 24-well plate, a 96-well plate, or a microcentrifuge tube.

[0122] After the IVT incubation reaction, primary purification is performed using a magnetic bead method. Magnetic beads containing a ligand capable of binding to mRNA molecules are provided, preferably carboxyl magnetic beads. These magnetic beads bind to the mRNA via a salt bridge. In a purification buffer system containing PEG and high salt ions, the RNA is adsorbed by forming an ionic bridge between RNA, salt ions, and carboxyl groups. This binding is reversible; in TE buffer without PEG and salt ions, the ionic bridge is released, allowing the RNA molecules to elute.

[0123] The IVT-incubated solution is brought into contact with the magnetic beads to obtain magnetic beads containing bound mRNA molecules, and the magnetic beads are separated using a magnetic field; the mRNA molecules are eluted from the separated magnetic beads to obtain a primary purified mRNA solution.

[0124] The primary purified mRNA solution after the IVT reaction is quantified. Any method known in the art can be used to detect and quantify mRNA molecules. Commercially available RNA quantification kits, such as the Agilent RNA 6000 Nano Kit (#5067-1511), are preferably used. Based on the quantification results, capping and / or tailing solutions are prepared and further incubated. This step yields the full-length mRNA product, including the 5' cap structure and the 3' poly(A) tail.

[0125] Next, secondary purification is performed by affinity chromatography. Since intact mRNA contains a 3' poly (A) tail, affinity fillers with oligo (dT) as ligands (such as POROS TM Oligo(dT) 25Affinity fillers (A-fillers) capture mRNA by complementary pairing with the poly(A) tail, thereby purifying the mRNA. In this step, affinity chromatography is performed under positive pressure to elute the mRNA molecules and obtain a secondary purified mRNA solution.

[0126] If IVT uses a co-transcriptional approach, that is, transcription directly obtains a full-length mRNA product, which includes a 5' cap structure and a 3' poly(A) tail, the full-length mRNA transcript can be directly obtained by primary purification after the IVT incubation reaction is completed, and then secondary purification is performed by affinity chromatography.

[0127] At this point, the mRNA solution has reached a fairly high purity, and it is then concentrated by replacing the solution. The main purpose is to adjust the mRNA concentration in the original solution and replace the buffer system. The solution replacement and concentration can be performed using the LiCl method or the magnetic bead method. The magnetic bead method can be performed using the same or different system or device as the primary purification. To save equipment and procedures, it is preferably performed using the same magnetic bead separation device as the primary purification.

[0128] The concentrated mRNA after liquid exchange is prepared into a formula stock solution suitable for cryopreservation, and then sterilized and filtered and aseptically packaged into containers suitable for cryopreservation.

[0129] Figure 1, Scheme B, shows another exemplary flow chart of a method for isolating mRNA molecules according to the present invention. First, as described above, an in vitro transcription (IVT) reaction is performed on a linearized DNA template. 5' capping and / or 3' tailing can be performed co-transcriptionally during the IVT reaction or as a separate step after the IVT reaction.

[0130] After IVT incubation, or optionally after capping and / or tailing, the full-length mRNA product is obtained, including the 5' cap structure and the 3' poly(A) tail.

[0131] In one embodiment, the mRNA solution is capped, optionally after obtaining the full-length product of the mRNA.

[0132] The full-length mRNA product was initially purified by affinity chromatography. Since the intact mRNA contains a 3' poly (A) tail, an affinity filler with oligo (dT) as a ligand (such as POROS TM Oligo(dT) 25 Affinity fillers (A) capture mRNA by complementary pairing with the Poly(A) tail, thereby purifying the mRNA. In this step, affinity chromatography is performed under positive pressure to elute the mRNA molecules and obtain a primary purified mRNA solution.

[0133] In one embodiment, the primary purified mRNA solution is optionally capped after affinity chromatography.

[0134] Next, secondary purification is performed using a magnetic bead method. Magnetic beads containing a ligand capable of binding to mRNA molecules are provided, preferably carboxyl magnetic beads. These magnetic beads bind to the mRNA via a salt bridge. In a purification buffer system containing PEG and high salt ions, the RNA is adsorbed by forming an ionic bridge between RNA, salt ions, and carboxyl groups. This binding is reversible; in TE buffer, which is free of PEG and salt ions, the ionic bridge is released, allowing the RNA molecules to elute.

[0135] The primary purified mRNA solution is contacted with the magnetic beads to obtain magnetic beads containing bound mRNA molecules, and the magnetic beads are separated using a magnetic field; the mRNA molecules are eluted from the separated magnetic beads to obtain a secondary purified mRNA solution.

[0136] In one embodiment, the secondary purified mRNA solution is optionally capped after magnetic bead purification.

[0137] The primary purpose of performing a liquid exchange and concentration step during secondary purification using a magnetic bead method is to adjust the mRNA concentration in the stock solution and replace the buffer system. Alternatively, the secondary purified mRNA solution can be exchanged and concentrated in a separate step, such as using a LiCl method or a magnetic bead method. The magnetic bead method can be performed using the same or different systems or apparatus as the primary purification. To save equipment and procedures, it is preferred to use the same magnetic bead separation apparatus as the primary purification.

[0138] The concentrated mRNA after liquid exchange is prepared into a formula stock solution suitable for cryopreservation, and then sterilized and filtered and aseptically packaged into containers suitable for cryopreservation.

[0139] In any embodiment of the present invention, the mRNA solution is optionally purified / concentrated by LiCl method or magnetic bead method before capping.

[0140] It should be understood that the process schemes described above are merely exemplary. Not all processes and apparatus are required for the methods and systems of the present invention. The scope of the present invention is defined by the appended claims. The following examples are included to provide additional guidance to those skilled in the art for practicing the claimed invention. These examples do not limit the invention as defined in the appended claims.

[0141] Example

[0142] Example 1: Experimental Materials and Methods

[0143] 1.1 DNA linearization

[0144] Linearize the DNA plasmid template using restriction enzyme digestion. Mix 10 μg of plasmid with 10 U of Esp3I / BsmBI and incubate at 37°C for 4 hours to ensure complete linearization. Terminate the reaction by adding 1 / 10 volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, mix thoroughly, and chill at -20°C for 1 hour. Pellet the linearized DNA by centrifugation at 13,800 g for 15 minutes at 4°C, wash twice with 70% ethanol, and resuspend in nuclease-free water.

[0145] 1.2 IVT

[0146] System for 20 μL reaction mixture:

[0147] The reaction mixture was incubated at 37°C for 6 hours, and then 1 μL of DNase I (RNase-free, 1 U / μL) was added to remove the DNA template and incubated at 37°C for 30 minutes.

[0148] Capped IVT:

[0149] The purified linear plasmid was used as a template and T7 RNA polymerase was used for in vitro transcription. According to the instructions of T7 High Yield RNA Synthesis Kit (NEB, #E2040S), m7GpppAmpU or m7GpppAmpG was added for in vitro transcription to synthesize mRNA with Cap1 “cap” structure.

[0150] 1.3 LiCl method

[0151] The synthesized RNA was purified by adding 0.5 volumes of 7.5 M LiCl, 50 mM EDTA and incubating at -20°C for 45 minutes, followed by centrifugation at 13,800 g for 15 minutes at 4°C to precipitate the mRNA. The supernatant was then removed, and the pellet was washed twice with pre-chilled 70% ethanol. The mRNA was resuspended in nuclease-free H2O and stored at -20°C.

[0152] 1.4 Capping

[0153] Each 10 μg of uncapped mRNA was heated at 65°C for 10 min, placed on ice for 5 min, and then mixed with 10 U of Vaccinia capping enzyme, 50 U of mRNA Cap 2′-O-methyltransferase, 0.2 mM SAM, 0.5 mM GTP, and 1 U of RNase inhibitor and incubated at 37°C for 60 min to generate cap1-modified structures.

[0154] 1.5 Affinity chromatography

[0155] POROS using Thermo fisher TM Oligo(dT)25 (#A47383) affinity filler, RNA purification by solid phase extraction instrument (positive or negative pressure). Equilibration / loading buffer: 10mM Tris-HCl, 500mM NaCl, 1mM EDTA, pH 7.4. Wash buffer: 10mM Tris-HCl, 100-300mM NaCl, 1mM EDTA, pH 7.4. Elution buffer: 10mM Tris-HCl, 1mM EDTA, pH 7.4 or direct elution with water. For specific experimental procedures, please refer to Thermo: POROS TM Oligo(dT)25Affnity Resin.

[0156] 1.6 Quantitative detection

[0157] Aglient's Bioanalyzer RNA Kit and Reagents (Agilent RNA 6000 Nano Kit, #5067-1511) or Fragment Analyzer System RNA Kit (RNA Kit (15NT), #DNF-471-0500) were used.

[0158] 1.7 Capping Rate Detection

[0159] The capping rate was detected using RNase H enzyme, streptavidin magnetic beads and LC-MS. A biotin-TEG-labeled probe was designed at the 5' end of the mRNA. After RNase H enzyme cleavage and magnetic bead treatment, short 5' end nucleotide fragments with or without a cap structure were obtained. LC-MS was used for detection and quantitative analysis of the proportion of each component to obtain the capping rate.

[0160] 1.8 Magnetic bead purification

[0161] Using Thermo Fisher Dynabeads TM MyOne TM Purify using carboxylic acid magnetic beads (#35401). Bind the sample to the beads, transfer the beads using a magnetic rack, wash with 50% to 80% ethanol, and elute with nuclease-free water. For detailed experimental procedures, refer to the protocol provided on the Thermo website.

[0162] 1.9 Physical and chemical properties testing

[0163] mRNA samples were tested for DNA residue using a plasmid DNA residue detection kit (PCR-fluorescent probe method) according to the manufacturer's protocol. TMThe mRNA samples were tested for total protein residue using the kit. The mRNA samples were tested for dsRNA residue using the Double-stranded-RNA (dsRNA) kit.

[0164] 1.10 In vitro testing

[0165] 1.10.1 In vitro IFN-β assay

[0166] BJ fibroblasts were purchased from ATCC. EMEM (Life Technologies, Thermofisher) culture medium was supplemented with L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco-FBS). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. mRNA was transfected using Lipofectamine 2000 (Thermo Fisher Scientific). Cell culture supernatants were collected 48 hours after transfection and analyzed for IFN-β expression using an ELISA kit (R&D System).

[0167] 1.10.2 Detection of IFN-β in the supernatant of BJ cell transfection using IFN-α / βReporter HEK 293 cells

[0168] BJ fibroblasts were purchased from ATCC. EMEM (Life Technologies, Thermo Fisher Scientific) culture medium was supplemented with L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco-FBS). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. Cells were transfected with mRNA using Lipofectamine 2000 (Thermo Fisher Scientific). Cell culture supernatants were collected 48 hours after transfection.

[0169] IFN-α / βReporter HEK 293 cells were purchased from Invivogen and supplemented with L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) in DMEM medium. 20,000 cells were seeded per well in a 96-well flat-bottom cell culture plate (Corning) and cultured for 24 hours. The culture medium was discarded, and the BJ cell transfection supernatant diluted in Opti-MEM was added and culture continued. The supernatant was aspirated, and QUANTI-Blue detection reagent (Invivogen) was added. The absorbance at 620 nm (OD620) was measured using a microplate reader (MD). The IFN-β content in the BJ cell transfection supernatant sample was calculated using a standard curve drawn between the standard concentration and OD620.

[0170] 1.10.3 Cell viability assay

[0171] BJ fibroblasts were purchased from ATCC. L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) were added to EMEM medium. 20,000 cells were seeded per well in a 96-well flat-bottom cell culture plate (Corning) and transfected 24 hours later. mRNA was transfected using Lipofectamine 2000 (Thermo Fisher Scientific). 48 hours after transfection, 100 μl of Cell Culture Buffer was added. Luminescent cell viability reagent (Promega) was used, and the chemiluminescent signal was detected using a microplate reader (MD).

[0172] 1.10.4 Cytotoxicity assay

[0173] C2C12 mouse muscle cells (C2C12) were purchased from ATCC. 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) was added to DMEM medium. 5,000 to 10,000 cells were seeded per well in a 96-well flat-bottom cell culture plate (Corning) and cultured for 24 hours. 50 μl of magnetic beads of different dilutions and magnetic beads of different dilutions containing Lipofectamine 2000 (Thermo Fisher Scientific) were added. Untransfected cells were used as controls. After 72 hours of culture, 100 μl of Cell Luminescent cell viability reagent (Promega) was used to detect chemiluminescent signals using a microplate reader (MD). Inhibition rate (%) = 100% * [(control well reading - experimental well reading) / (control well reading - blank well reading)].

[0174] 1.10.5 In vitro expression detection - HEK293T analysis

[0175] eGFP: Human embryonic kidney (HEK293T) cells were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. Dulbecco's modified Eagle's medium (DMEM) was supplemented with 10% fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) mixed with mRNA encoding green fluorescent protein (eGFP). Twenty-four hours after transfection, eGFP protein expression was analyzed using a microplate reader.

[0176] Luciferase: Human embryonic kidney (HEK293T) cells were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai. Dulbecco's modified Eagle's medium (DMEM) was supplemented with 10% fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. HEK293T cells were transfected with mRNA encoding luciferase (Luc) using Lipofectamine 2000 (Thermo Fisher Scientific). 24 hours after transfection, 100 μL of Luciferase Reporter Gene Assay Reagent (Norvozymes) was added and Luc protein expression was analyzed using a microplate reader.

[0177] RBD: Human embryonic kidney cells (HEK293T) were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai. 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) was added to DMEM medium. Cells were seeded at 80,000 cells per well in 6-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. HEK293T cells were transfected with mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (S-RBD) using Lipofectamine 2000 (Thermo Fisher Scientific). Cell culture supernatants were collected 18 to 24 hours after transfection, and the expression of RBD protein was analyzed using an ELISA kit (Sino-Bio).

[0178] H1N1 HA or H3N2 HA: Human embryonic kidney (HEK293T) cells were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. Dulbecco's modified Eagle's medium (DMEM) was supplemented with 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, ThermoFisher). After cells were grown to ≥80% confluence, mRNA encoding influenza virus H1N1 HA or H3N2 HA protein was transfected into HEK293T cells using Lipofectamine Messenger MAX reagent (ThermoFisher Scientific). Cell culture supernatants were collected 18 to 24 hours after transfection and treated with H1N1 HA primary antibody working solution (Sino Biological, #11055-MM04T) or H3N2 HA primary antibody working solution (Sino Biological, #11055-MM03) and secondary antibody working solution (Jackson Immune Research, #115-095-164), and then analyzed on a flow cytometer (BD, LSR Fortessa).

[0179] 1.10.6 In vitro expression detection - A549 analysis

[0180] eGFP: Human non-small cell lung cancer cells (A549) were purchased from ATCC. They were cultured in F-12K medium (ATCC, 30-2004) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. Cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) mixed with mRNA encoding green fluorescent protein (eGFP). Twenty-four hours after transfection, eGFP protein expression was analyzed using a microplate reader.

[0181] Luciferase: Human non-small cell lung cancer cells (A549) were purchased from ATCC. F-12K medium (ATCC, 30-2004) was supplemented with 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. Lipofectamine 2000 (Thermo Fisher Scientific) was used to mix mRNA encoding luciferase (Luc) and transfect the cells. 24 hours after transfection, 100 μL of Luciferase Reporter Gene Assay Reagent (Norvozymes) was added, and Luc protein expression was analyzed using a microplate reader.

[0182] RBD: Human non-small cell lung cancer cells (A549) were purchased from ATCC. 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) was added to F-12K medium (ATCC, 30-2004). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. The cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) mixed with mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (S-RBD). Cell culture supernatants were collected 18 to 24 hours after transfection, and the expression of RBD protein was analyzed using an ELISA kit (Sino-Bio).

[0183] RSV: Human non-small cell lung cancer cells (A549) were purchased from ATCC. F-12K medium (ATCC, 30-2004) was supplemented with 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher). Cells were seeded at 35,000–40,000 cells per well in 6-well flat-bottom cell culture plates (Corning) 24 hours prior to transfection. Cells were transfected with mRNA using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were harvested 18–24 hours after transfection and analyzed by flow cytometry. Transfected cells were plated into 96-well plates, centrifuged, and blocked with 2% BSA. Following blocking, cells were incubated with pre-diluted F488 anti-RSV fusion protein antibody at 4°C for 1 hour. After incubation, the cells were washed, resuspended in DPBS, and analyzed by flow cytometry.

[0184] BA.4 / 5: Human non-small cell lung cancer cells (A549) were purchased from ATCC. 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) was added to F-12K medium (ATCC, 30-2004). Cells were seeded at 20,000 cells per well in 96-well flat-bottom cell culture plates (Corning) and transfected 24 hours later. Lipofectamine 2000 (Thermo Fisher Scientific) was used to mix with mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (S-RBD) and transfect the cells. Cell culture supernatants were collected 18 to 24 hours after transfection, and the expression of RBD protein was analyzed using an ELISA kit (Sino-Bio).

[0185] Epo: Human non-small cell lung cancer cells (A549) were purchased from ATCC. They were cultured in F-12K medium (ATCC, 30-2004) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher). Cells were seeded at 35,000 to 40,000 cells per well in 6-well flat-bottom cell culture plates (Corning) and transfected 24 hours after transfection. Cell mRNA was transfected using Lipofectamine 2000 (Thermo Fisher Scientific). Cell culture supernatants were collected 18 to 24 hours after transfection and analyzed for EPO protein expression using an ELISA kit (R&D Systems).

[0186] Rabies: Human non-small cell lung cancer cells (A549) were purchased from ATCC. 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, Thermofisher) was added to F-12K medium (ATCC, 30-2004). Cells were seeded in 6-well flat-bottom cell culture plates (Corning) at 35,000 to 40,000 cells per well and transfected 24 hours later. Cell mRNA was transfected using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were collected 18 to 24 hours after transfection and analyzed by flow cytometry. The transfected cells were collected and added to 96-well plates. After centrifugation, they were blocked with 2% BSA. After blocking, pre-diluted anti-rabies virus glycoprotein G mouse IgG1 antibody was incubated at 4°C for 1 hour. After incubation, R-phycoerythrin AffiniPure TM Incubate with goat anti-mouse IgG Fcγ fragment for 30 minutes, wash, resuspend in DPBS, and detect on flow cytometry.

[0187] 1.11 Hematology and blood biochemistry tests

[0188] Hematology Hematology samples were measured using a whole blood automatic analyzer (Sysmex xs-800i) and its supporting reagents.

[0189] Blood biochemistry samples were measured using a fully automatic biochemical analyzer (Hitachi 7060).

[0190] Example 2: Comparison of LiCl method and magnetic bead method

[0191] Using Sample 1, both the LiCl and magnetic bead methods were performed according to the experimental protocol shown in panel A of Figure 2. Purity testing of the IVT reaction solution prior to capping, using both LiCl and magnetic bead purification, is shown in panel B of Figure 2. The purity achieved by both methods was essentially identical, with the yield from magnetic bead purification being 88.4% of that achieved with LiCl.

[0192] IVT samples purified by LiCl and magnetic beads were capped separately (at the same level). The capping purity was basically the same, but the capping rate of magnetic bead purification was higher than that of the LiCl method, as shown in sub-graph C in Figure 2. This shows that the magnetic bead method has certain advantages over the LiCl method.

[0193] Example 3: Analysis of impurities introduced by magnetic bead method

[0194] After positive pressure affinity chromatography, sample 2 was concentrated by magnetic bead method. IVT, LiCl purification, capping and positive pressure affinity chromatography were performed. After affinity chromatography, the magnetic bead method was used for liquid concentration, and bottom suction was added to remove residual magnetic beads. The sample was then filtered with a 0.22 μm membrane. Sterile filtration also played a role in secondary removal of magnetic beads (process 3). Processes 1 and 2 were also performed for comparison. ICP-MS was used to detect the Fe element to evaluate the residual magnetic beads, and cell activity (see 1.10.3 Cell activity assay) and in vitro innate immunity assay (see 1.10.1 In vitro IFN-β assay) were performed. At the same time, the cytotoxicity of magnetic beads was tested in BC2C12 mouse muscle cells (see 1.10.4 Cytotoxicity assay). The specific test results are shown in Table 1 and Figure 3.

[0195] Process 1: Same as process 3, except that ultrafiltration is used instead of magnetic beads for liquid concentration.

[0196] Process 2: Add 0.02 mg of magnetic beads to the sample obtained in process 1 (magnetic bead concentration 0.01 mg / mL)

[0197] Process 3: Positive pressure affinity chromatography followed by magnetic bead-based concentration

[0198] Table 1: Fe elements detected by ICP-MS

[0199] As shown in panels A and B of Figure 3, compared to Process 1, Process 3 showed slightly lower in vitro IFN-β assay results and slightly higher cell viability assay results. According to the Fe element results (Table 1), positive pressure affinity chromatography followed by magnetic bead-based concentration and exchange of the buffer resulted in a residual magnetic bead level of less than 0.01 mg / ml. Combined with the cytotoxicity assay of magnetic beads in BC2C12 mouse muscle cells (see panel C of Figure 3), this indicates that the current residual magnetic beads are essentially non-toxic to cells.

[0200] At the same time, in order to investigate whether the introduced impurities have an effect on the mice, a certain amount of magnetic beads (200 μL) was injected into the mice through the tail vein, and blood was collected one day later. A group of mice (0.05 μg) was retained until the eighth day for a second blood draw to test hematology and blood biochemistry. The results are shown in sub-figures A and B in Figure 4.

[0201] The results showed that even the highest dose of 0.5 μg had low toxicity to the animals. On the eighth day (0.05 μg), all indicators were found to have rebounded.

[0202] Example 4: Negative pressure affinity chromatography

[0203] When affinity chromatography was performed under negative pressure, it was found that the chromatography filler was deformed due to the instantaneous force of the negative pressure, and the yield was lower than that of positive pressure affinity chromatography, as shown in Figure 5.

[0204] Example 5: Comparative Experiment 1

[0205] Samples 3, 4, 1, and 5 were selected, and two linearized plasmids with and without Poly (A) were prepared, respectively. The linearized plasmids were composed in a ratio of 8:2 for IVT. The LiCl method and the magnetic bead method were selected for purification 1. The uncapped product was capped and purified 2 by the LiCl method, the magnetic bead method, and the positive pressure affinity chromatography + magnetic bead method. The specific process was carried out according to processes 1-3 in Figure 6. The obtained samples were tested for mRNA purity (referred to as FA purity) based on the Agilent Fragment Analyzer, and the capping rate, protein residue, etc. were tested. At the same time, samples 3 and sample 4 were selected for in vitro expression in HEK293T cells. Samples 3, sample 4, sample 1, and sample 5 were tested for in vitro expression in A549 cells. The results are as follows:

[0206] FA purity was higher in Process 3 than in Process 1 and Process 2, as shown in Figure 7. Capping rates were similar, both exceeding 99%, as shown in Figure 8. Protein analysis showed that Process 2 was significantly higher than both Process 1 and Process 3, with Process 3 being the lowest, as shown in Figure 9.

[0207] The in vitro expression results showed that Process 3 was superior to Process 1 and Process 2, as shown in FIG10 .

[0208] Sample 6 was used for mRNA preparation. After IVT, the LiCl method was selected for purification 1. The uncapped product was capped and purified by the LiCl method and affinity chromatography 2. Affinity chromatography used a gravity column, which uses gravity to allow the sample and buffer to drip naturally without applying additional pressure. After affinity chromatography, the ultrafiltration tube was exchanged for liquid. The specific process followed the procedures 1 and 4 in Figure 6. The resulting sample was tested for protein residue, and it was found that Procedure 4 was less effective in removing proteins than Procedure 1 (see Figure 11).

[0209] Comparative experiments using procedures 3 and 5 were conducted using samples 7, 8, and 9. Samples 7-1, 8-1, and 9-1 were prepared using procedure 5, while samples 7-2, 8-2, and 9-2 were prepared using procedure 3 (automated). The resulting mRNA stock solutions were then tested for purity, capping efficiency, residual DNA, and residual dsRNA, as well as for innate immunity (INF-β) and in vitro protein expression. The results are shown in Figure 12 and Table 2.

[0210] Table 2: Physicochemical properties (purity, capping efficiency, residual DNA, residual dsRNA) and innate immunity (INF-β) results of the three samples

[0211] The test results showed that the mRNA purity, capping rate, residual DNA, and residual dsRNA were basically consistent, with no significant differences. The residual DNA and dsRNA in Process 3 were relatively low, as shown in Figure 12, Panel A. Innate immunity (INF-β) levels were all below the detection limit, as shown in Table 2. The in vitro expression of the three samples was similar, with no significant differences, as shown in Figure 12, Panels B, C, and D.

[0212] Example 6: Comparative Experiment 2

[0213] Eleven samples were selected and mRNA for Cap 1 was prepared according to procedures 6 and 7 in Figure 13. Samples 10-17 were added with m7GpppAmpU, and samples 18-20 were added with m7GpppAmpG. The samples were prepared by co-transcription. The prepared samples were tested for FA purity, capping efficiency, and physicochemical properties (residual DNA, residual dsRNA, and residual protein).

[0214] The FA and capping rates of the 11 samples are basically consistent. The specific data are shown in Figures 14 and 15.

[0215] Physical and chemical property testing revealed no protein residue in either process. DNA residue in Process 7 was significantly lower than in Process 6 (see Figure 16 for details). dsRNA residue in Process 7 was significantly lower than in Process 6 (see Figure 17 for details).

[0216] The samples were tested for the immune factor IFN-β in BJ fibroblasts. The results showed that the stimulation of IFN-β by process 7 was lower than that by process 6, as shown in Figure 18. Among them, samples 14, 15, 16 and 17 were all lower than the detection value in this method. These four samples were tested by another method (IFN-α / βReporter HEK 293 cells to detect the content of IFN-β in the supernatant of BJ cell transfection). The results showed that the stimulation of IFN-β by process 7 was lower than that by process 6, as shown in Figure 19.

[0217] The samples were tested for in vitro protein expression in A549 cells. The results showed that the differences between each sample were different, but overall the protein expression of Process 7 was slightly higher than that of Process 6, as shown in Figures 20A-20E.

[0218] Although the present invention has been described in conjunction with specific embodiments, it will be appreciated by those skilled in the art that many modifications and variations may be made to the present invention. Therefore, it will be appreciated that the intent of the claims is to cover all such modifications and variations that fall within the true concept and scope of the present invention.

[0219] Sequence information:

[0220] Sample 1 (coding protein: novel coronavirus delta variant RBD): SEQ ID NO: 1

[0221] Sample 2 (encoding protein: Epo): SEQ ID NO: 2

[0222] Sample 3 (encoding protein: eGFP): SEQ ID NO: 3

[0223] Sample 4 (encoding protein: Luc): SEQ ID NO: 4

[0224] Sample 5 (encoding protein: SARS-CoV-2 RBD): SEQ ID NO: 5

[0225] Sample 6 (encoding protein: murine IL-12): SEQ ID NO: 6

[0226] Sample 7 (encoding protein: H1N1 HA, 100% U in the sequence is modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 7

[0227] Sample 8 (encoding protein: H3N2 HA, 100% of U in the sequence is modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 8

[0228] Sample 9 (encoding protein: SARS-CoV-2 BA.4 / 5RBD, 100% U in the sequence modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 9

[0229] Sample 10 (encoding protein: eGFP, cap analog: m7GpppAmpU, except for the gray-marked UTP, all other U in the sequence are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 10

[0230] Sample 11 (encoding protein: RSV Pre-F, cap analog: m7GpppAmpU, except for the gray-marked UTP, all other U in the sequence are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 11

[0231] Sample 12 (encoding protein: eGFP, cap analog: m7GpppAmpU, except for the gray-marked UTP, the other U in the sequence is modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 12

[0232] Sample 13 (encoding protein: BA.4 / 5, cap analog: m7GpppAmpU, except for the gray-marked UTP, all other U in the sequence are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 13

[0233] Sample 14 (encoded protein: RABV GP, cap analog: m7GpppAmpU, except for the gray-marked UTP in the sequence, all other U are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 14

[0234] Sample 15 (encoding protein: RABV GP, cap analog: m7GpppAmpU, except for the gray-marked UTP, all other U in the sequence are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 15

[0235] Sample 16 (encoded protein: RABV GP, cap analog: m7GpppAmpU, except for the gray-marked UTP in the sequence, all other U are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 16

[0236] Sample 17 (encoded protein: RABV GP, cap analog: m7GpppAmpU, except for the gray-marked UTP in the sequence, all other U are modified with N1-methylpseudouridine (M1ψ)): SEQ ID NO: 17

[0237] Sample 18 (encoded protein: Luc, cap analog: m7GpppAmpG): SEQ ID NO: 18

[0238] Sample 19 (encoding protein: BA.4 / 5, cap analog: m7GpppAmpG): SEQ ID NO: 19

[0239] Sample 20 (encoded protein: Epo, cap analog: m7GpppAmpG): SEQ ID NO: 20

Claims

1. A method for isolating mRNA molecules, comprising one or more magnetic particle separation steps and one or more positive pressure affinity chromatography separation steps.

2. A method according to claim 1, wherein the method comprises first performing one or more magnetic particle separation steps, then performing one or more positive pressure affinity chromatography separation steps, and optionally thereafter performing one or more magnetic particle separation steps; or wherein the method comprises first performing one or more positive pressure affinity chromatography separation steps, and then performing one or more magnetic particle separation steps.

3. A method for isolating mRNA molecules, comprising the following steps: (a) providing magnetic particles comprising a ligand capable of binding to an mRNA molecule; (b) contacting a solution containing mRNA molecules with the magnetic particles to obtain magnetic particles containing bound mRNA molecules; (c) separating the magnetic particles containing the bound mRNA molecules using a magnetic field; (d) eluting the mRNA molecules from the separated magnetic particles to obtain a primarily purified solution containing mRNA molecules; and (e) loading the solution containing mRNA molecules obtained in step (d) onto an affinity chromatography containing oligo(dT), eluting the mRNA molecules by positive pressure, and obtaining a secondary purified solution containing mRNA molecules.

4. The method according to claim 3, further comprising: Prior to step (b), a step of performing in vitro transcription on the DNA template to generate a solution containing mRNA molecules; and / or A step of capping and / or tailing the mRNA molecule prior to step (b); and / or a step of capping and / or tailing the mRNA molecule prior to step (e); and / or a step of capping the mRNA molecule after step (e); and / or After step (e), the secondary purified solution containing mRNA molecules is subjected to a step of liquid replacement and concentration and / or sterile filtration, optionally wherein the liquid replacement and concentration step is performed by a LiCl method and / or a magnetic particle method, optionally wherein the magnetic particle method comprises repeating steps (a)-(d).

5. A method for isolating mRNA molecules, comprising the following steps: (a) providing a solution comprising mRNA molecules; (b) loading the solution containing mRNA molecules onto an affinity chromatography containing oligo(dT), eluting the mRNA molecules by positive pressure, and obtaining a primary purified solution containing mRNA molecules; (c) providing magnetic particles containing ligands capable of binding to mRNA molecules, by contacting the solution containing mRNA molecules obtained in step (b) with the magnetic particles to obtain magnetic particles containing bound mRNA molecules; (d) separating the magnetic particles containing the bound mRNA molecules using a magnetic field; and (e) eluting the mRNA molecules from the separated magnetic particles to obtain a secondary purified solution containing mRNA molecules.

6. The method according to claim 5, wherein Step (a) further comprises in vitro transcription of the DNA template to generate a solution comprising mRNA molecules; and / or Step (a) further comprises capping and / or tailing the mRNA molecule; and / or The method further comprises the step of capping the mRNA molecule after step (b); and / or The method further comprises the step of capping the mRNA molecule after step (e); and / or The method further comprises the steps of replacing the solution with a concentrated solution and / or sterilizing and filtering the secondary purified solution containing the mRNA molecules after step (e), optionally wherein the step of replacing the solution with a concentrated solution is performed by a LiCl method and / or a magnetic particle method, optionally wherein the magnetic particle method comprises repeating steps (c)-(e).

7. The method according to any one of claims 1 to 6, The magnetic particles are carboxyl magnetic beads, preferably Dynabeads TM MyOne TM Carboxylic acid magnetic beads; and / or The positive pressure affinity chromatography comprises Oligo(dT) 25 Affinity filler, preferably POROS TM Oligo(dT) 25 Affinity media; and / or wherein the method is performed in an automated, semi-automated or manual manner; and / or wherein the mRNA molecule comprises one or more functional nucleotide analogues, optionally wherein the functional nucleotide analogues are one or more selected from the group consisting of pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.

8. A system for isolating mRNA molecules, comprising: A first purification module, comprising magnetic particles containing ligands capable of binding to mRNA molecules; a second purification module, comprising an affinity chromatography column, wherein the affinity chromatography column is operated under positive pressure; and A fluid module includes a container for containing a solution containing mRNA, and the fluid module is in fluid communication with the first purification module and the second purification module.

9. The system according to claim 8, further comprising a feeding module for providing a source of mRNA molecules, and / or a collecting module for collecting mRNA molecules.

10. The system according to claim 8, wherein the fluid module further comprises a liquid transfer device, optionally wherein the liquid transfer device is a pipette, a pipette or a mobile workstation; and / or wherein the fluid module further comprises a conduit, which facilitates fluid communication among the first purification module, the second purification module and the fluid module; and / or The fluid module further comprises a container for containing an elution solution for eluting mRNA from the magnetic particles, and a container for containing an elution solution for eluting mRNA from the affinity chromatography column; and / or The fluid module further comprises a container for containing a washing solution for washing the magnetic particles, and a container for containing a washing solution for washing the affinity chromatography column.

11. The system of claim 8, wherein the first purification module further comprises a magnetic separator or a magnetic field.

12. The system of claim 8, further comprising one or more additional modules, including but not limited to one or more means for performing the following operations: The DNA is transcribed in vitro to generate a solution containing mRNA molecules; Capping and / or tailing mRNA molecules; replacing and concentrating the solution containing the mRNA molecule; and / or The solution containing the mRNA molecules is sterile filtered.

13. The system according to claim 12, wherein the liquid exchange concentration is performed by a LiCl method; or The fluid replacement and concentration is performed by a magnetic particle method, wherein the module for performing the fluid replacement and concentration is the same as or different from the first purification module.

14. The system according to any one of claims 8 to 13, wherein the system operates in an automated, semi-automated or manual manner, optionally wherein the system further comprises a control module configured to control the various modules of the system to operate in an automated or semi-automated manner.

15. The system according to any one of claims 8-14, wherein the mRNA molecule comprises one or more functional nucleotide analogs, optionally wherein the functional nucleotide analogs are one or more selected from the group consisting of pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.

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