Method for purifying nucleic acid molecules

Abstract

Provided is a method for purifying nucleic acid molecules, specifically relating to a method for purifying circular nucleic acid molecules. The purification method involves few chromatography steps and has a high total yield, and using the purification method to prepare LcDNA or linear DNA allows for a short production cycle and low production costs.

Description

Method for purifying nucleic acid molecules

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number CN202311049806.5, filed on August 18, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a method for purifying nucleic acid molecules, in particular to a method for purifying circular nucleic acid molecules. Background Art

[0004] Due to its molecular structure, linear plasmid DNA allows for more efficient in vitro transcription (IVT) and has been widely used as a template for mRNA in vitro transcription. Since linear closed DNA (LcDNA) carries only the target gene sequence and does not carry prokaryotic replicon or resistance gene sequences, it is widely used as a non-viral gene delivery vector.

[0005] mRNA is an emerging class of drugs used to prevent and treat a variety of diseases. In recent years, the successful commercialization of two mRNA vaccines against the novel coronavirus COVID-19, produced by Moderna and BioNtech, respectively, has highlighted the enormous potential of mRNA technology to revolutionize life sciences and pharmaceutical research. Advances in mRNA technology have stimulated the use of in vitro transcribed mRNA (IVT mRNA) in a variety of preclinical studies and clinical trials. Currently, mRNA drugs are primarily used in three therapeutic areas: immunotherapy, protein replacement therapy, and regenerative medicine. These can be categorized into cancer immunotherapy, infectious disease vaccines, allergy relief vaccines, protein replacement therapy, cell fate reprogramming, and genome editing.

[0006] The industrial production process for IVT mRNA can be broadly divided into three steps: circular plasmid production, linear plasmid production, and IVT mRNA production. The in vitro transcription and purification of mRNA is a completely cell-free process. Therefore, once a highly standardized production process is developed for the mRNA stage, batch-to-batch reproducibility is easily maintained. Currently, the challenges and challenges of industrial mRNA production primarily focus on the large-scale production of the starting materials, circular and linear plasmid DNA. The currently established production process from circular to linear plasmid DNA includes: bacterial fermentation, bacterial harvesting, bacterial lysis, lysate clarification and concentration, the classic three-step chromatography process (gel filtration, affinity, and anion chromatography), circular plasmid concentration and liquid exchange, enzyme digestion, anion chromatography, concentration and liquid exchange, sterile filtration, and linear plasmid filling. However, the complexity and length of this entire production process significantly increases the cost, cycle time, and process instability of large-scale industrial mRNA production.

[0007] Therefore, more optimized processes are still needed for the industrial production of mRNA.

[0008] Summary of the Invention

[0009] In a first aspect, the present invention provides a method for purifying a nucleic acid molecule, the method comprising:

[0010] 1) Providing a suspension containing nucleic acid molecules;

[0011] 2) The suspension from step 1 is loaded onto a porous polyacrylamide membrane for capture to obtain nucleic acid molecules.

[0012] In some embodiments, the suspension in step 1) comprises a NaCl solution with a final concentration of 50-150 mM.

[0013] In some embodiments, the step 2) further comprises adding an equilibration buffer to the porous polyacrylamide membrane before loading the sample.

[0014] In some embodiments, after the sample is loaded in step 2), the step of adding an equilibration buffer to the porous polyacrylamide membrane is further included.

[0015] In some embodiments, the equilibration buffer is a mixed solution of 1 M KAc and 150 mM NaCl.

[0016] In some embodiments, after the capture in step 2), the method further comprises performing gradient elution on the obtained nucleic acid molecules and collecting the eluate containing the nucleic acid molecules.

[0017] In some embodiments, the gradient elution buffer is a mixed solution of 100 mM Tris and 1 M NaCl, and the elution gradient is 20%.

[0018] In some embodiments, the collecting of the eluate is collecting the eluate with a UV value greater than or equal to 200 mAU at UV280 or UV260.

[0019] In some embodiments, the porous polyacrylamide membrane is a high-density, strong anionic porous polyacrylamide membrane. In some embodiments, the high-density, strong anionic porous polyacrylamide membrane is a Natrix Q membrane.

[0020] In some embodiments, the nucleic acid molecule is a circular nucleic acid molecule; preferably, it is a circular DNA.

[0021] In some embodiments, the circular nucleic acid molecule is a plasmid, and the plasmid comprises at least one restriction endonuclease cleavage site.

[0022] In some embodiments, the circular nucleic acid molecule is a plasmid, which contains two protelomerase cleavage sites connected in series in the same direction, one or more restriction endonuclease cleavage sites are independently provided at both ends of the protelomerase cleavage site, and a target gene is inserted between the protelomerase cleavage sites.

[0023] In some embodiments, the restriction endonuclease cleavage sites at each end of the plasmid are selected from any one or more of the following enzyme cleavage sites: AccI, AflII, AgeI, ApaI, AscI, AvrII, BamHI, BglI, BglII, BsaI, BspQI, BstBI, BsteII, ClaI, EcoNI, EcoRI, EcoRV, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NdeI, NheI, NotI, PacI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, StuI, SwaI, XbaI, XhoI, and XmaI.

[0024] In some embodiments, the restriction endonuclease cleavage site is selected from any one or more of the following cleavage sites: HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI and EcoRI.

[0025] In a second aspect, the present invention provides a method for preparing LcDNA, the method comprising:

[0026] a) providing a plasmid obtained by the method of the first aspect, and digesting the plasmid with protelomerase;

[0027] b) digesting the fragment using at least two restriction endonucleases and further digesting the fragment using an exonuclease;

[0028] c) Purify the cDNA containing the target gene.

[0029] In a third aspect, the present invention provides a method for preparing linear DNA, comprising:

[0030] a) providing a plasmid obtained by the method of the first aspect, and digesting the plasmid with a restriction endonuclease;

[0031] b) Purify the linear DNA containing the target gene.

[0032] In some embodiments, the method provided in the second aspect or the third aspect further comprises adjusting the pH of the eluate to pH 7.0-8.0 using 3M Tris buffer before the enzymatic cleavage step.

[0033] In some embodiments, the polishing comprises chromatography using a composite resin medium Capto Core 700.

[0034] In some embodiments, the chromatography comprises:

[0035] a) adding equilibration buffer to the resin;

[0036] b) loading the enzyme digestion reaction solution onto a chromatography column and collecting the flow-through;

[0037] The equilibration buffer is a mixed solution of 100-300 mM NaCl, 10 mM EDTA and 20 mM Tris-HCl.

[0038] In some embodiments, the purification further includes at least one process of concentration, sterile filtration, and filling.

[0039] In a fourth aspect, the present invention provides the use of the LcDNA prepared in the second aspect or the linear DNA prepared in the third aspect in preparing mRNA.

[0040] Beneficial effects of the present invention:

[0041] 1) The method for purifying nucleic acid molecules described in the present invention uses a porous polyacrylamide membrane for chromatographic capture, which reduces the number of chromatography steps and improves the overall yield without affecting the quality of linear plasmid DNA / LcDNA;

[0042] 2) The circular plasmid obtained by capture chromatography in the method for purifying nucleic acid molecules of the present invention can be directly digested without purification and used to produce LcDNA or linear DNA. Therefore, the method for preparing LcDNA or linear DNA of the present invention has a short production cycle and low production cost;

[0043] 3) The LcDNA or linear DNA preparation method of the present invention only requires one step of composite filler chromatography purification to obtain high-quality linear plasmid DNA or LcDNA, without the need for tedious purification steps and a short production cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 is a process flow chart, wherein FIG1A is a process flow chart of conventional linear plasmid DNA or LcDNA; FIG1B is a process flow chart of the linear plasmid DNA or LcDNA of the present invention.

[0045] Figure 2 is a Natrix Q plot of the first batch of linearized plasmid DNA.

[0046] Figure 3 is a Capto Core 700 image of the first batch of linearized plasmid DNA.

[0047] Figure 4 shows the AGE analysis of the first batch of linearized plasmid DNA intermediate samples and the finished product. Lane M1: Supercoiled DNA control; Lane M2: 1kb DNA control; Lane 1: Natrix Q load solution; Lane 2: Load flowthrough sample; Lane 3: Equilibration sample; Lanes 4 and 5: Natrix Q elution samples; Lane 6: Natrix Q cleanup sample; Lane 7: XbaI digestion sample; Lane 8: Capto Core 700 load solution; Lane 9: Capto Core 700 elution sample; Lane 10: Capto Core 700 cleanup sample; Lane 11: Linearized plasmid exchange sample; Lane 12: Finished linearized plasmid.

[0048] Figure 5 is a Natrix Q plot of the second batch of linearized plasmid DNA.

[0049] Figure 6 is a Capto Core 700 image of the second batch of linearized plasmid DNA.

[0050] Figure 7 shows the AGE analysis of intermediate and finished products from the second batch of linearized plasmid DNA. Lane M1: Supercoiled DNA control; Lane M2: 1 kb DNA control; Lane 1: Natrix Q loading solution; Lane 2: Loading flow-through sample; Lane 3: Equilibration sample; Lane 4: Natrix Q elution sample; Lane 5: Natrix Q cleanup sample; Lane 6: XbaI digestion sample; Lane 7: Capto Core 700 loading solution; Lanes 8 and 9: Capto Core 700 elution samples; Lane 10: Capto Core 700 cleanup sample; Lane 11: Linearized plasmid exchange sample; Lane 12: Finished linearized plasmid product.

[0051] FIG8 is a Natrix Q map of the first batch of LcDNA.

[0052] Figure 9 is a Capto Core 700 image of the first batch of LcDNA.

[0053] Figure 10 shows the AGE analysis of the first batch of LcDNA intermediate samples and finished product. In Figure 10A, lane M1: supercoiled DNA control; lane 1: Natrix Q loading solution; lane 2: loading flowthrough sample; lane 3: equilibrated sample; lane 4: Natrix Q elution sample; lane 5: Natrix Q clean sample. In Figure 10B, lane M1: supercoiled DNA control; lane M2: 1kb DNA control; lane 1: TelN digested sample; lane 2: endonuclease digested sample; lane 3: Exo III digested sample; lane 4: Capto Core 700 loading solution; lanes 5 and 6: Capto Core 700 elution samples; lane 7: Capto Core 700 clean sample; lane 8: LcDNA exchange sample; lane 9: finished LcDNA; lane M2: 1kb DNA control.

[0054] FIG11 is a Natrix Q map of the second batch of LcDNA.

[0055] Figure 12 is a Capto Core 700 image of the second batch of LcDNA.

[0056] Figure 13 shows the AGE analysis of the second batch of LcDNA intermediate samples and finished product. Lane M1: supercoiled DNA control; Lane M2: 1 kb DNA control; Lane 1: Natrix Q loading solution; Lane 2: loading flowthrough sample; Lane 3: equilibration sample; Lane 4: Natrix Q elution sample; Lane 5: Natrix Q cleanup sample; Lane 6: TelN digestion sample; Lane 7: endonuclease digestion sample; Lane 8: Exo III digestion sample; Lane 9: Capto Core 700 loading solution; Lanes 10 and 11: Capto Core 700 elution samples; Lane 12: Capto Core 700 cleanup sample; Lane 13: LcDNA exchange sample; Lane 14: finished LcDNA product.

[0057] FIG14 is a Natrix Q map of the third batch of LcDNA.

[0058] FIG15 is a Capto Core 700 image of the third batch of LcDNA.

[0059] Figure 16 shows the AGE analysis of the third batch of LcDNA intermediate samples and finished product. In Figure 16A, lane M1: supercoiled DNA control; lane 1: Natrix Q loading solution; lane 2: loading flowthrough sample; lane 3: equilibration sample; lane 4: Natrix Q elution sample; lane 5: Natrix Q cleanup sample; lane M2: 1 kb DNA control. In Figure 16B, lane M1: supercoiled DNA control; lane 1: TelN digestion sample; lane 2: endonuclease digestion sample; lane 3: Exo III digestion sample; lane 4: Capto Core 700 loading solution; lanes 5 and 6: Capto Core 700 elution samples; lane 7: Capto Core 700 cleanup sample; lane 8: LcDNA exchange sample; lane 9: finished LcDNA; lane M2: 1 kb DNA control. DETAILED DESCRIPTION

[0060] Unless otherwise defined, 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.

[0061] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0062] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0063] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0064] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0065] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0066] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0067] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0068] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0069] As used herein, the terms "buffer substance" and "buffer" refer to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffering properties of such solutions. Therefore, solutions or compositions that exhibit buffering activity are referred to as buffers or buffered solutions. The buffer that can be used in the method of the present invention is preferably selected from phosphate buffer, phosphate-buffered saline buffer (PBS), 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, TRIS-buffered saline solution (TBS) and TRIS / EDTA (TE) and the like.

[0070] As used herein, "nucleic acid molecule" refers to a molecule comprising three or more nucleotides. In some embodiments, the nucleic acid molecule refers to a deoxyribonucleic acid molecule; in some embodiments, the nucleic acid molecule refers to a ribonucleic acid molecule. In some embodiments, the nucleic acid molecule is linear; in some embodiments, the nucleic acid molecule is circular. In some embodiments, the nucleic acid molecule is single-stranded linear; in some embodiments, the nucleic acid molecule is single-stranded circular. In some embodiments, the nucleic acid molecule is double-stranded linear; in some embodiments, the nucleic acid molecule is double-stranded circular.

[0071] As used herein, "circular nucleic acid molecule" refers to circular deoxyribonucleic acid molecules and circular ribonucleic acid molecules. In some embodiments, the nucleic acid molecule is single-stranded circular; in some embodiments, the nucleic acid molecule is double-stranded circular. Circular nucleic acid molecules lack openings and include, but are not limited to, circular DNA, circular RNA, plasmid DNA, and covalently closed circular DNA.

[0072] In some embodiments, the circular nucleic acid molecule is plasmid DNA. In some embodiments, the plasmid contains at least one restriction endonuclease site; preferably, it contains two or more different restriction endonuclease sites. The restriction endonuclease site can be used to enzymatically cut the plasmid DNA into linear DNA. In other embodiments, the plasmid contains two protelomerase cleavage sites connected in series in the same direction, and one or more restriction endonuclease cleavage sites are independently provided at both ends of the protelomerase cleavage site. The protelomerase cleavage site and the restriction endonuclease cleavage site co-exist on the plasmid and are used for enzymatic cutting to produce LcDNA. The "protelomerase cleavage site" sequence includes any complete palindromic sequence, that is, any double-stranded DNA sequence with double rotational symmetry. The method of using enzyme cutting sites to perform enzymatic cutting to produce linear DNA or LcDNA has been disclosed in multiple documents, such as CN102301010B, CN103080337A, etc., and will not be repeated in this application.

[0073] In some embodiments, the circular nucleic acid molecule is produced by Escherichia coli.

[0074] A "suspension containing nucleic acid molecules" refers to a liquid containing nucleic acid molecules and other impurities. In some embodiments, the suspension is produced by fermentation of Escherichia coli. In some embodiments, circular DNA is transformed into E. coli, the circular DNA is amplified by fermentation, the E. coli is lysed after fermentation, and the lysate is clarified, resulting in a supernatant that is the suspension containing nucleic acid molecules.

[0075] The term "capture" refers to a step in the separation process. Typically, capture is the initial step of separation, and the target nucleic acid molecule is initially purified by chromatography, filtration, etc.

[0076] In the present invention, the capturing step comprises performing chromatography on a suspension containing nucleic acid molecules using a porous polyacrylamide membrane.

[0077] The term "purification" as used herein refers to the process of separating nucleic acid molecules from a suspension containing nucleic acid molecules. In some embodiments, purification comprises capturing nucleic acid molecules by chromatography, eluting the nucleic acid molecules, and further polishing. In some embodiments, the chromatography process comprises equilibrating a chromatography column with an equilibration buffer, loading a sample, and in some embodiments, equilibration with an equilibration buffer, and gradient eluting the sample. In some embodiments, the equilibration buffer is a mixed solution of 1M KAc and 150mM NaCl; in some embodiments, the pH of the equilibration buffer is between pH 5-6, for example, pH 5, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0; in some embodiments, the conductivity of the equilibration buffer is between 80-90mS / cm, for example, 81mS / cm, 82mS / cm, 83mS / cm, 84mS / cm, 85mS / cm, 96mS / cm, 97mS / cm, 88mS / cm, 89mS / cm, or 90mS / cm. In some embodiments, the volume of the equilibration buffer is 200 times the volume of the membrane. In some embodiments, the flow rate of the equilibration buffer is 10 times the membrane volume / minute.

[0078] In some embodiments, the maximum loading capacity of the sample is 10 mg / mL. In some embodiments, the suspension loaded comprises a saline solution of 100-150 mM NaCl; in some embodiments, the conductivity of the loaded suspension is between 70-80 mS / cm, such as 70 mS / cm, 71 mS / cm, 72 mS / cm, 73 mS / cm, 74 mS / cm, 75 mS / cm, 76 mS / cm, 77 mS / cm, 78 mS / cm, 79 mS / cm, or 80 mS / cm. In some embodiments, the flow rate of the suspension loading is 10 times the membrane volume / minute.

[0079] In some embodiments, after sample loading is completed, the membrane is equilibrated again using an equilibration buffer.

[0080] In some embodiments, the elution buffer is a mixture of 100mM Tris and 1M NaCl. In some embodiments, the pH of the elution buffer is 8.0-9.0; in some preferred embodiments, the pH of the elution buffer is 9.0. In some embodiments, the volume of the elution buffer is 100 times the volume of the membrane. In some embodiments, the flow rate of the elution buffer is 5 times the membrane volume / minute. In some embodiments, the elution step is a gradient elution, such as a 10% gradient, a 15% gradient, a 20% gradient, a 25% gradient, a 30% gradient, a 35% gradient, a 40% gradient, a 45% gradient, or a 50% gradient.

[0081] In some embodiments, the purification process further comprises regenerating the membrane using a regeneration buffer, wherein the regeneration buffer is a mixture of 1M NaOH and 2M NaCl. In some embodiments, the volume of the regeneration buffer is 200 times the volume of the membrane. In some embodiments, the flow rate of the regeneration buffer is 10 times the membrane volume per minute.

[0082] The term "purification" refers to the process of further purifying nucleic acid molecules obtained by chromatography capture. In some embodiments, the purification step is ion exchange chromatography, hydrophobic chromatography, or mixed-mode chromatography. Those skilled in the art can select the appropriate chromatography method and filler as needed. In some embodiments, the chromatography uses a composite resin filler Capto Core 700 chromatography. In some embodiments, the chromatography includes:

[0083] a) adding equilibration buffer to the resin;

[0084] b) loading the enzyme digestion reaction solution onto a chromatography column and collecting the flow-through;

[0085] The equilibration buffer is a mixed solution of 100-300 mM NaCl, 10 mM EDTA and 20 mM Tris-HCl. In some embodiments, the collecting of the flow-through solution is performed by collecting the flow-through solution component when the UV value of UV260 or UV280 is greater than or equal to 30 mAU.

[0086] The term "LcDNA", also known as linear closed DNA or closed linear DNA, carries the target gene and protelomerase recognition sequences at both ends. It is small in size and highly safe, and the expression level of LcDNA in vitro or in vivo is comparable.

[0087] "Porous polyacrylamide membrane" refers to a membrane whose main component is polyacrylamide, which has a three-dimensional perforated hydrogel structure. The porous polyacrylamide membrane preferably has a high density of strong anionic quaternary amine ligands that can adsorb target DNA. In some embodiments, the porous polyacrylamide membrane is a high density strong anionic porous polyacrylamide membrane. In some embodiments, the porous polyacrylamide membrane is In some embodiments, the porous polyacrylamide membrane is Q film.

[0088] As used herein, "target gene" refers to any gene of interest to the experimenter, which is operably linked to a nucleic acid molecule. In some embodiments, the target gene is a gene that expresses mRNA, a gene that expresses siRNA, a gene that expresses shRNA, a gene that expresses miRNA, or a gene that expresses a specific protein. Suitable target genes include, but are not limited to, nucleic acids encoding proteins for treating the following diseases: endocrine, metabolic, hematological, cardiovascular, neurological, musculoskeletal, urological, pulmonary, and immune disorders, including, for example, cancer, inflammatory disorders, immune disorders, chronic and infectious disorders. Cancers such as tumors arising from lesions in any of the bones, bone connections, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicles, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus gland, gonads, sublingual gland, and parotid gland; immune disorders such as systemic lupus erythematosus, multiple sclerosis, type I diabetes, psoriasis The infectious disease may be any one of viral, bacterial, fungal and parasitic infections, or a combination thereof.

[0089] Suitable genes of interest include, but are not limited to, genes encoding at least one of the following proteins: interferons (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω, IFN-τ); insulin (e.g., Novolin, Humulin, Humalog, Humalog, Lantus, etc.); erythropoietin ("EPO"; e.g., epoetin-α, darbepoetin-α, epoetin-β; etc.); antibodies (e.g., monoclonal antibodies, such as rituximab, infliximab, trastuzumab, Humira™ (adalimumab), omalizumab, tositumomab, Rapone tiva™ (efalizumab), Erbitux™ (cetuximab), bevacizumab; etc.); antigen-binding fragments including monoclonal antibodies (e.g., ranibizumab); blood factors (e.g., alteplase, tissue plasminogen activator protein, recombinant human factor VIIa, factor VIIa, factor VIII, factor IX, beta-globulin, hemoglobin; etc.); colony stimulating factors (e.g., (filgrastim; G-CSF), Neulasta (pegylated filgrastim), granulocyte colony stimulating factor (G-CSF), granulocyte monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony-stimulating factor; etc.); growth hormones (e.g., somatotropin, somatotropin; etc.); interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); growth factors (e.g., (beclapermin; PDGF), (trifermin; bFGF), (anciplostatin; stem cell factor), keratinocyte growth factor, acidic fibroblast growth factor, stem cell factor, basic fibroblast growth factor, hepatocyte growth factor; etc.); soluble receptors (e.g., TNF-α binding soluble receptors, such as (etanercept), soluble VEGF receptors, soluble interleukin receptors, soluble gamma / delta T cell receptors; etc.); enzymes (e.g., α-glucosidase, β-glucocerebrosidase, alglucosidase), enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, Mig, Groα / IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4; etc.); angiogenic agents (e.g., vascular endothelial growth factor (VEGF); anti-angiogenic agents (e.g., soluble VEGF receptor); protein vaccines;Neuroactive peptides, such as bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, cerebellofloxacin, dynorphin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; other proteins, such as thrombolytics, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human α -1 antitrypsin, leukemia inhibitory factor, transforming growth factor, insulin-like growth factor, luteinizing hormone, macrophage activating factor, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, tissue inhibitor of metalloproteinases; vasoactive intestinal peptide, angiopoietin, angiotropin, fibrinogen; hirudin; leukemia inhibitory factor; IL-1 receptor antagonist (e.g., (anakinra)); ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin (a tumor suppressor); lysosomal enzyme acid α-glucosidase (GAA); etc. Suitable target genes also include those encoding functional fragments of any of the above proteins; and nucleic acids encoding functional variants of any of the above proteins. ;

[0090] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will appreciate that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention.

[0091] Example 1 Preparation and purification of linear DNA

[0092] (1) Bacterial fermentation

[0093] The plasmid DNA (Gensher Pengbo synthesis, vector name CFL universal, vector molecular weight: 2017bp) containing the target gene (molecular weight: 3169bp) was transformed into Escherichia coli for culture and fermentation. The fermentation tank culture temperature was set at 30-37°C and the rotation speed was 200rpm. Trace elements (components 6.42g / L ferric chloride, 4.00g / L manganese chloride, 1.00g / L zinc sulfate, 0.40g / L copper sulfate, 0.50g / L sodium molybdate, 0.50g / L boric acid), 0.25g / L ammonium sulfate hydrochloride and Escherichia coli shake flask culture medium (0.1% inoculation ratio) were added in proportion to start the fermentation process. During the fermentation process, the dissolved oxygen content was maintained in the range of 20%-40%. Ammonia water was used as the alkali solution to maintain the pH at not less than 6.54. Fermentation was completed after about 24 hours.

[0094] (2) Bacteria harvest

[0095] After fermentation is stopped, the cells are collected by centrifugation at 6000-8000 rpm and 4°C for 15-20 minutes. Depending on the scale of the fermentation, a hollow fiber centrifuge can also be used to harvest the cells.

[0096] (3) Bacterial lysis

[0097] Calculate the weight of each buffer S1, S2-SDS, S2-NaOH, and S3 in the alkaline lysis process based on the weight of the cells. The calculation formula is as follows:

[0098] Thoroughly resuspend the cells in S1 buffer. Then, mix S2-NaOH and S2-SDS in a 1:1 volume ratio and add the mixture to the resuspended solution. Stir gently and slowly for 3-5 minutes. Finally, add S3 buffer using the proportions listed in the table above, stir thoroughly, and let it sit for 50-60 minutes. Depending on the production plant, equipment conditions, and production scale, you can choose to perform bacterial lysis using either a manual or automated lysis system.

[0099] (4) Lysate clarification and filtration

[0100] After standing, the lysate was filtered using a 50 μm filter (manufacturer: Hangzhou Kebet Filter Equipment Co., Ltd., item number: UFEFL0100050P) and a 0.6 μm filter (manufacturer: Hangzhou Kebet Filter Equipment Co., Ltd., item number: L30SSCHT06A1P), and the supernatant after filtration was collected.

[0101] (5) Circular plasmid capture

[0102] A final concentration of 50-150 mM NaCl solution was added to the supernatant obtained in step (4), followed by chromatography using a Natrix Q membrane (manufacturer: Merck KgaA, catalog number: NXF-10, NXF-20, NXF-50; select a membrane of appropriate specifications based on actual production scale). Circular plasmids were directly captured from the supernatant. During chromatography elution, UV260 or UV280 values ​​were monitored, and fractions with an average mass of 200 mAU or greater were collected. The membrane chromatography process parameters are shown in the following table:

[0103] You can also use Q membrane is used for chromatography process, but compared with Natrix Q membrane, the purification effect of this membrane is poor, the membrane is easily clogged, and the flow rate is slow.

[0104] (6) Circular plasmid digestion

[0105] The collected Natrix Q eluate was first adjusted to pH 7.0–8.0 with 3M Tris buffer before enzymatic digestion. The digestion process parameters are shown in the table below (using Xba I and 1000 mL of digestion system as an example). After adding the components listed below, mix thoroughly and incubate in a 37°C water bath for 12–16 hours. The digestion mixture was then incubated at 75°C for 15–30 minutes.

[0106] (7) Purification of linear plasmid DNA by chromatography

[0107] The solution obtained in step (6) was first filtered through a 0.2 μm filter membrane, and the filtrate was collected and loaded onto a chromatography column containing a composite resin filler Capto Core 700 (manufacturer: Cytiva, catalog number: 17-5481-03). The UV260 or UV280 value was monitored, and fractions with a concentration of 30 mAU or greater were collected. The chromatography process parameters are shown in the following table:

[0108] (8) Linear plasmid DNA concentration and medium replacement

[0109] The liquid collected after linear plasmid DNA chromatography was filtered through an ultrafiltration membrane (pore size 5KD or 10KD, manufacturer: Hangzhou Kebet Filter Equipment Co., Ltd., product number: UFELA0010010P, UFELA0005010P, specification: 0.01m 2 , 0.1m 2 , select a membrane package of appropriate specifications according to the actual production scale) for tangential flow filtration, concentrate the concentration of linear plasmid DNA to a specified concentration (for example, 0.5 mg / mL or 1.0 mg / mL), and replace the solution with a specified buffer (for example, sterile water for injection or Tris-EDTA buffer).

[0110] (9) Sterile filtration of linear plasmid DNA

[0111] Use a 0.2 μm filter membrane to sterilize and filter the linear plasmid DNA after concentration and liquid exchange. The pore size of the sterilizing filter is 0.2 μm. Specifications include syringe filter (manufacturer: Merck Millipore Ltd., catalog number: SLGPR33RB), 1000 mL vacuum filtration system (manufacturer: Corning Incorporated, catalog number: 431098), and capsule filter (manufacturer: Sartorius Stedim Biotech GmbH, catalog number: 5445307H7--OO--A). Select the filter of appropriate specifications according to the volume of the sample after concentration and liquid exchange.

[0112] (10) Filling and storage of linear plasmid DNA

[0113] After sterilization and filtration, the linearized plasmid DNA product is filled into designated aliquots, such as 1 mL / vial, in a clean bench or sterile isolator. The filled product is then stored in a -70 ± 10°C ultra-low temperature freezer.

[0114] (11) Linear plasmid DNA quality detection

[0115] 1. Agarose gel electrophoresis (AGE) detection of linear plasmid DNA intermediate samples and finished products

[0116] Linearized plasmid DNA intermediates and finished products were examined on a 0.7% agarose gel using 1×TAE as the electrophoresis buffer. The electrophoresis voltage was 150 V for 30 minutes. The gel was imaged using Image Lab software.

[0117] 2. High Performance Liquid Chromatography (IEC-HPLC) to Detect the Purity of Linear Plasmid DNA

[0118] The purity of the linear plasmid DNA product was tested using an Agilent 1260 high performance liquid chromatograph.

[0119] 3. Sanger sequencing to verify the detection of linear plasmid DNA

[0120] Appropriate sequencing primers were designed based on the inserted target sequence, and the linear plasmid DNA samples were verified by Sanger whole-plasmid sequencing.

[0121] 4. Nanorange fluorescence detection of total protein residues in linear plasmid DNA

[0122] The total protein residue in the linear plasmid DNA product was detected using the NanoOrange kit (ThermoFisher, Catalog No.: N10271), and the value was read using a microplate reader.

[0123] 5. Detection of host DNA (E. coli source) residues in linear plasmid DNA by fluorescence quantitative PCR (qPCR)

[0124] A real-time fluorescence quantitative PCR instrument (ThermoFisher, model: 7500) and an E. coli residual DNA detection kit (Shenke Bio, catalog number: SK030202E100) were used to detect the residual host DNA (E. coli source) in the linear plasmid DNA.

[0125] 6. Detection of bacterial endotoxins in linear plasmid DNA by gel analysis (LAL)

[0126] Bacterial endotoxins in linear plasmid DNA were detected by gel analysis using Limulus amebocyte lysate (LAL) reagent.

[0127] 7. Direct inoculation method to detect whether linear plasmid DNA is sterile

[0128] Use a disposable sterile syringe or a sterilized pipette to draw up the linear plasmid DNA sample, inoculate it into 100 mL of thioglycollate FTM medium, and culture it at 30-35°C for 14 days to test whether the linear plasmid DNA sample is sterile.

[0129] Two batches of linear plasmid DNA were prepared according to the method of Example 1. The experimental results of the first batch of linear plasmid DNA are shown in Tables 1-2 and Figures 2-4. The experimental results of the second batch of linear plasmid DNA are shown in Tables 3-4 and Figures 5-7.

[0130] From the results, we can see that

[0131] 1. This linear plasmid DNA production process is very stable. The scale of enzymatically digested circular plasmids has been gradually scaled up from 30 mg to 100 mg without any abnormal deviations during the production process. This process can also meet the requirements for linear plasmid DNA production at scales exceeding 100 mg of circular plasmids.

[0132] 2. This linear plasmid DNA production process has a high total yield. Two batches of linear plasmid DNA were produced, with a total yield of 45%-47%.

[0133] 3. This linear plasmid DNA production process has a short production cycle. At the same production scale, it is estimated that each batch of production can save 3 to 4 days.

[0134] 4. The purity and quality of the linear plasmid DNA products produced by this process can meet the requirements of the pharmacopoeia and can be used for clinical research and commercial purposes in fields such as gene therapy.

[0135] Table 1 Production data of the first batch of linear plasmid DNA

[0136] Table 2 Detection data of the first batch of linear plasmid DNA

[0137] Table 3 Summary of the second batch of linear plasmid DNA production data

[0138] Table 4 Summary of the second batch of linear plasmid DNA detection data

[0139] Example 2 Preparation and purification of LcDNA

[0140] Refer to steps (1)-(5) of Example 1 to obtain a circular plasmid.

[0141] (6) Three-step enzyme digestion of circular plasmid

[0142] The Natrix Q eluate was first adjusted to pH 7.0–8.0 with 3M Tris buffer, and then subjected to three enzymatic digestion steps:

[0143] Step 1: Telomerase TelN digestion (TelN, digestion system 1000mL as an example), add the components in the table below in sequence, mix thoroughly, place in a constant temperature water bath at 30℃ for 3 hours, and then treat the digestion mixture at 75℃ for 15-30 minutes.

[0144] Step 2: restriction endonuclease digestion. Taking KpnI-HF and SalI-HF double enzyme digestion, digestion of 1200mL system as an example, add the components in the table below in sequence, mix thoroughly, incubate in a constant temperature water bath at 37℃ for 2 hours, and then treat the digestion mixture at 75℃ for 15-30 minutes.

[0145] Step 3: Exonuclease digestion. Using Exo III digestion of 1400 mL as an example, add the following components in sequence and mix thoroughly. Incubate in a 37°C water bath for 4-12 hours. To terminate digestion, add EDTA to a final concentration of 10 mM and incubate at 75°C for 10-30 minutes.

[0146] (7) cDNA chromatography purification

[0147] The solution obtained in step (6) was first filtered through a 0.2 μm filter membrane, and the filtrate was collected and loaded onto a chromatography column containing a composite resin filler Capto Core 700 (manufacturer: Cytiva, catalog number: 17-5481-03). The UV260 or UV280 value was monitored, and fractions with a concentration of 30 mAU or greater were collected. The chromatography process parameters are shown in the following table:

[0148] (8) LcDNA concentration and medium replacement

[0149] The collected liquid after LcDNA chromatography was filtered through an ultrafiltration membrane (pore size 5KD or 10KD, manufacturer: Hangzhou Kebet Filter Equipment Co., Ltd., item number: UFELA0010010P, UFELA0005010P, specification: 0.01m 2 , 0.1m 2 , according to the actual production scale, select a membrane package of appropriate specifications) for tangential flow filtration, concentrate the concentration of LcDNA to a specified concentration (for example, 0.5 mg / mL or 1.0 mg / mL), and replace the solution with a specified buffer (for example, sterile water for injection or Tris EDTA buffer).

[0150] (9) Sterile filtration of LcDNA

[0151] After concentration and liquid exchange, the LcDNA was sterilized and filtered using a 0.2 μm filter membrane. The sterilizing filter had a pore size of 0.2 μm and was available in the following specifications: syringe filter (manufacturer: Merck Millipore Ltd., catalog number: SLGPR33RB), 1000 mL vacuum filtration system (manufacturer: Corning Incorporated, catalog number: 431098), and capsule filter (manufacturer: Sartorius Stedim Biotech GmbH, catalog number: 5445307H7--OO--A). The filter of appropriate specifications was selected according to the volume of the sample after concentration and liquid exchange.

[0152] (10) LcDNA filling and storage

[0153] After sterilization and filtration, the LcDNA product is filled into designated aliquots, such as 1 mL / vial, in a clean bench or sterile isolator. The filled product is then stored in a -70 ± 10°C ultra-low temperature freezer.

[0154] (11) LcDNA quality detection

[0155] 1. Agarose gel electrophoresis (AGE) detection of LcDNA intermediate samples and finished products

[0156] Intermediate and finished LcDNA samples were examined on 0.7% agarose gels using 1×TAE as the electrophoresis buffer. The electrophoresis voltage was 150 V for 30 minutes. The gels were then imaged using Image Lab software.

[0157] 2. High-performance liquid chromatography (IEC-HPLC) to detect the purity of LcDNA

[0158] The purity of the LcDNA product was tested using an Agilent 1260 high performance liquid chromatograph.

[0159] 3. Sanger sequencing verification and detection of LcDNA

[0160] Appropriate sequencing primers were designed according to the inserted target sequence, and the LcDNA samples were verified by Sanger whole-plasmid sequencing.

[0161] 4. Nanorange fluorescence detection of total protein residues in LcDNA

[0162] The total protein residue in the LcDNA product was detected using a NanoOrange kit (ThermoFisher, Cat. No. N10271), and the values ​​were read using a microplate reader.

[0163] 5. Detection of host DNA (E. coli source) residues in LcDNA by fluorescence quantitative PCR (qPCR)

[0164] A real-time fluorescence quantitative PCR instrument (ThermoFisher, model: 7500) and an E. coli residual DNA detection kit (Shenke Bio, catalog number: SK030202E100) were used to detect the residual host DNA (E. coli source) in LcDNA.

[0165] 6. Detection of bacterial endotoxins in LcDNA by LAL gel method

[0166] The bacterial endotoxin in LcDNA was detected by gel analysis using Limulus amebocyte lysate (LA-lysate) reagent.

[0167] 7. Direct inoculation method to detect whether LcDNA is sterile

[0168] Use a disposable sterile syringe or a sterilized pipette to draw up the LcDNA sample, inoculate it into 100 mL of thioglycollate FTM medium, and culture it at 30-35°C for 14 days to test whether the LcDNA sample is sterile.

[0169] Three batches of LcDNA were prepared according to the method of Example 2. The experimental results of the first batch of LcDNA are shown in Tables 5-6 and Figures 8-10. The experimental results of the second batch of LcDNA are shown in Tables 7-8 and Figures 11-13. The experimental results of the third batch of LcDNA are shown in Tables 9-10 and Figures 14-16.

[0170] From the results, we can see that

[0171] 1. The LcDNA production process is very stable and no abnormal deviations occur during the production process.

[0172] 2. This LcDNA production process has a high overall yield. Three batches of LcDNA were produced, with an overall yield of 22%-24%.

[0173] 3. This LcDNA production process has a short production cycle. At the same production scale, it is estimated that each batch of production can save 3 to 4 days.

[0174] 4. The purity and quality of the LcDNA products produced by this process can meet the requirements of the pharmacopoeia and can be used for clinical research and commercial purposes in fields such as gene therapy.

[0175] Table 5 Production data of the first batch of LcDNA

[0176] Table 6 The first batch of LcDNA detection data

[0177] Table 7 The second batch of LcDNA production data

[0178] Table 8 The second batch of LcDNA detection data

[0179] Table 9 The third batch of LcDNA production data

[0180] Table 10 The third batch of LcDNA detection data

[0181] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

Claims

1. A method for purifying nucleic acid molecules, the method comprising: 1) Providing a suspension containing nucleic acid molecules; 2) The suspension in step 1 is loaded onto a porous polyacrylamide membrane for capture to obtain nucleic acid molecules.

2. The method according to claim 1, wherein the suspension in step 1) comprises a NaCl solution with a final concentration of 50-150 mM.

3. The method according to claim 1 or 2, further comprising the step of adding an equilibrium buffer to the porous polyacrylamide membrane before loading the sample in step 2).

4. The method according to any one of claims 1 to 3, further comprising the step of adding an equilibrium buffer to the porous polyacrylamide membrane after step 2) loading the sample. The method according to claim 3 or 4, wherein the equilibration buffer is a mixed solution of 1 M KAc and 150 mM NaCl.

6. The method according to any one of claims 1 to 5, further comprising, after step 2) capturing, a step of gradient eluting the obtained nucleic acid molecules and collecting the eluate containing the nucleic acid molecules.

7. The method according to claim 6, wherein the gradient elution buffer is a mixed solution of 100 mM Tris and 1 M NaCl, and the elution gradient is 20%.

8. The method according to claim 6, wherein the collecting of the eluate is collecting the eluate with a UV value greater than or equal to 200 mAU at UV280 or UV260.

9. The method according to any one of claims 1 to 8, wherein the porous polyacrylamide membrane is a high-density strong anionic porous polyacrylamide membrane, preferably a Natrix Q membrane.

10. The method according to claims 1-9, wherein the nucleic acid molecule is a circular nucleic acid molecule; preferably a circular DNA. The method according to claim 10 , wherein the circular nucleic acid molecule is a plasmid, and the plasmid comprises at least one restriction endonuclease cleavage site.

12. The method according to claim 11, wherein the circular nucleic acid molecule is a plasmid, the plasmid comprises two protelomerase cleavage sites connected in series in the same direction, one or more restriction endonuclease cleavage sites are independently arranged at both ends of the protelomerase cleavage site, and a target gene is inserted between the protelomerase cleavage sites.

13. The method according to claim 11, wherein the restriction endonuclease cleavage sites at each end of the plasmid are selected from any one or more of the following restriction endonuclease cleavage sites: AccI, AflII, AgeI, ApaI, AscI, AvrII, BamHI, BglI, BglII, BsaI, BspQI, BstBI, BsteII, ClaI, EcoNI, EcoRI, EcoRV, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NdeI, NheI, NotI, PacI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, StuI, SwaI, XbaI, XhoI and XmaI.

14. The method according to claim 13, wherein the restriction endonuclease cleavage site is selected from any one or more of the following cleavage sites: HindIII, SalI, SmaI, BamHI, EcoRV, XbaI, KpnI and EcoRI.

15. A method for preparing LcDNA, the method comprising: a) providing a plasmid obtained by the method according to claim 12, and digesting it with protelomerase; b) digesting with at least two restriction endonucleases and further digesting with an exonuclease; c) Purify LcDNA containing the target gene.

16. A method for preparing linear DNA, the method comprising: a) providing a plasmid obtained according to the method of claim 11, and digesting it with a restriction endonuclease; b) Purify the linear DNA containing the target gene.

17. The method according to claim 15 or 16, further comprising adjusting the pH of the eluent to pH 7.0-8.0 using 3M Tris buffer before the enzyme cleavage step.

18. The method according to any one of claims 15 to 17, wherein the polishing comprises chromatography using a composite resin medium Capto Core 700.

19. The method according to claim 18, wherein the chromatography comprises: a) adding equilibration buffer to the resin; b) loading the enzyme digestion reaction solution onto a chromatography column and collecting the flow-through solution; The equilibration buffer is a mixed solution of 100-300 mM NaCl, 10 mM EDTA and 20 mM Tris-HCl.

20. The method according to any one of claims 15 to 19, wherein: The purification further includes at least one process of concentration, sterilization filtration and filling.

21. Use of the LcDNA prepared according to claim 15 or the linear DNA prepared according to claim 16 in preparing mRNA.

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