Method for purifying RNA

Abstract

Disclosed are methods and systems for purifying mRNA, e.g., therapeutic mRNA, that result in one or more benefits, such as increased purification yield, reduced double stranded RNA after purification. The methods can be used in conjunction with biochips and / or automated methods.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Patent Application No. 63 / 563,990, entitled “RNA Purification Methods,” filed March 12, 2024, the disclosure of which is incorporated herein by reference.

[0002] background The topics discussed in this section should not be considered prior art simply because they are mentioned in this section. Similarly, problems mentioned in this section or related to the topics provided as background should not be considered as having been previously recognized in the prior art. The topics in this section represent only different methods, which in themselves may correspond to implementations of the claimed technology.

[0003] In vitro transcription (IVT) is commonly used to produce therapeutic polynucleotides, such as messenger RNA (mRNA). IVT uses phage DNA-dependent RNA polymerases (e.g., SP6, T3, and T7) to synthesize template-guided messenger RNA (mRNA) transcripts. However, in vitro transcription reactions can result in double-stranded contaminants generated during the reaction, which must be removed to provide a therapeutically acceptable mRNA therapeutic agent. There is a need for improved methods to isolate contaminating double-stranded RNA (dsRNA) fragments from a reaction mixture containing full-length single-stranded RNA (ssRNA) products. This disclosure aims to address one or more of these aforementioned needs in the art.

[0004] Overview Compositions and methods for separating double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) are disclosed. In one aspect, a method for separating dsRNA and ssRNA is disclosed, comprising contacting a equilibration buffer with a resin. In one aspect, the equilibration buffer may contain from about 25 mM NaCl to less than 500 mM NaCl. In one aspect, the equilibration buffer may contain from about 10 mM NaCl to about 400 mM NaCl. In one aspect, the equilibration buffer may contain from about 20 mM NaCl to about 300 mM NaCl. In one aspect, the equilibration buffer may contain from about 30 mM NaCl to about 200 mM NaCl. In one aspect, the equilibration buffer may contain from about 40 mM NaCl to about 100 mM NaCl. In one aspect, the equilibration buffer may contain from about 25 mM NaCl to about 75 mM NaCl. In one aspect, the equilibration buffer may contain from about 40 mM NaCl to about 60 mM NaCl. In one aspect, the equilibration buffer may contain about 50 mM NaCl.

[0005] In one aspect, a method for separating dsRNA and ssRNA is disclosed, the method comprising contacting a mixture containing both ssRNA and dsRNA with an oligo-dT resin to form an RNA resin mixture, and then contacting a binding buffer with the RNA resin mixture.

[0006] In one aspect, a method for separating dsRNA and ssRNA is disclosed, the method comprising contacting an oligo-dT resin bound to ssRNA with a first washing buffer. The first washing buffer may contain from about 25 mM NaCl to less than 500 mM NaCl. In one aspect, the first washing buffer may contain from about 10 mM NaCl to about 400 mM NaCl. In one aspect, the first washing buffer may contain from about 20 mM NaCl to about 300 mM NaCl. In one aspect, the first washing buffer may contain from about 30 mM NaCl to about 200 mM NaCl. In one aspect, the first washing buffer may contain from about 40 mM NaCl to about 100 mM NaCl. In one aspect, the first washing buffer may contain from about 25 mM NaCl to about 75 mM NaCl. In one aspect, the first washing buffer may contain from about 40 mM NaCl to about 60 mM NaCl. In one aspect, the first washing buffer may contain about 50 mM NaCl.

[0007] In one aspect, a method for separating dsRNA and ssRNA is disclosed, the method comprising contacting an oligo-dT resin bound to ssRNA with a second washing buffer. The second washing buffer may contain from about 5 mM NaCl to less than 100 mM NaCl. In one aspect, the second washing buffer may contain from about 10 mM NaCl to about 90 mM NaCl. In one aspect, the second washing buffer may contain from about 20 mM NaCl to about 80 mM NaCl. In one aspect, the second washing buffer may contain from about 30 mM NaCl to about 70 mM NaCl. In one aspect, the second washing buffer may contain from about 40 mM NaCl to about 60 mM NaCl. In one aspect, the second washing buffer may contain from about 45 mM NaCl to about 55 mM NaCl. In one aspect, the second washing buffer may contain about 50 mM NaCl. Brief description of the attached diagram Details of one or more embodiments are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the description, drawings, and claims, in which: Figure 1 An exemplary prior art method for purifying mRNA using functionalized solid particles without a denaturation step is described.

[0009] Figure 2 An exemplary prior art method for purifying mRNA using functionalized solid particles is described, incorporating a denaturation step (65°C for 10 minutes, followed by introduction into a column).

[0010] Figure 3 Exemplary methods according to this disclosure are described.

[0011] Figure 4 The application is described Figure 3 The method shown yields dsRNA levels (pg / μg mRNA). Compared to ampoule-only (A), cellulose + ampoule purification (B), commercial methods (D), or commercial methods without a denaturation step (E), the disclosed method (C) provides significantly improved dsRNA clearance efficiency.

[0012] Figure 5 The percentage of recovery using the method described herein is shown compared to existing techniques.

[0013] Figure 6 The percentage of purity achieved using the method described herein is shown compared to existing techniques.

[0014] Figure 7 The amount of double-stranded RNA (dsRNA) produced using the method described herein is shown compared to existing techniques.

[0015] Detailed Explanation This document discloses methods and compositions for processing therapeutic polynucleotides (e.g., mixtures produced by an IVT reaction). In one aspect, the disclosed methods and compositions can be used to minimize the amount of double-stranded RNA (dsRNA) following the IVT process. The disclosed methods and compositions can be adapted to automated closure processes, such as those for purifying single-stranded mRNA products from an IVT reaction.

[0016] I. Terminology Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” mean that various components / components may be used together in methods and articles (e.g., compositions and apparatuses and methods including devices). For example, the term “comprise” will be understood to imply the inclusion of any stated element or step, but does not exclude any other element or step. Generally, any apparatus and method described herein should be understood as inclusive, but all or a subset of components and / or steps may optionally be exclusive and may be expressed as “consisting of a plurality of components, steps, sub-components or sub-steps” or optionally “substantially consisting of a plurality of components, steps, sub-components or sub-steps”.

[0017] As used herein, unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated to “ / ”.

[0018] As used herein in the specification and claims, including in the examples, and unless expressly stated otherwise, all figures are to be interpreted as if preceded by the words “about” or “approximately,” even if the term is not explicitly stated. The phrase “about” or “approximately” may be used when describing a quantity and / or location to indicate that the described value and / or location is within a reasonably expected range of the value and / or location. For example, a numerical value may have values ​​of ±0.1% of the stated value (or range of values), ±1% of the stated value (or range of values), ±2% of the stated value (or range of values), ±5% of the stated value (or range of values), ±10% of the stated value (or range of values), etc. Any numerical value given herein should also be understood to include about or approximately that value, unless the context otherwise requires. For example, if the value “10” is disclosed, “about 10” is also disclosed. Any numerical ranges listed herein are intended to include all subranges contained therein.

[0019] It should also be understood that when a value is disclosed, "less than or equal to" that value, "greater than or equal to" that value, and possible ranges between the values ​​are also disclosed, as would be appropriately understood by a person skilled in the art. For example, if the value "X" is disclosed, then "less than or equal to X" and "greater than or equal to X" (e.g., where X is a numerical value) are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and that the data represents endpoints and starting points, as well as ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, then it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as ranges between 10 and 15, are considered to be disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0020] While the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context otherwise requires. These terms are used to distinguish one feature / element from another and do not indicate a particular order unless specifically stated. Therefore, without departing from the teachings of the invention, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element.

[0021] As used herein, the terms “system,” “apparatus,” and “equipment” are to be understood as interchangeable. A system, apparatus, and equipment may each comprise more than one component having various structural and / or functional relationships with each other.

[0022] As used herein, "polynucleotide" refers to a nucleic acid molecule containing multiple nucleotides, and generally refers to both "oligonucleotide" (a polynucleotide molecule of 18-25 nucleotides in length) and polynucleotides of 26 or more nucleotides. Aspects of this disclosure include compositions comprising: oligonucleotides of 18-25 nucleotides in length (e.g., 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer), or intermediate-length polynucleotides of 26 or more nucleotides in length (e.g., 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 3 ...9, 39, 39, 39, 39, 39, 39, 39, 39, 39, 39, 39, 39, 39, 39, 3 9, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 24 0, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides, or long polynucleotides longer than about 300 nucleotides (e.g., between about 300 and about 400 nucleotides, between about 400 and about 500 nucleotides, between about 500 and about 600 nucleotides, between about 600 and about 700 nucleotides, between about 700 and about 800 nucleotides, between about 800 and about 900 nucleotides, between about 900 and about 300 nucleotides). Polynucleotides can be described in terms of length: approximately 1000 nucleotides, approximately 300 to approximately 500 nucleotides, approximately 300 to approximately 600 nucleotides, approximately 300 to approximately 700 nucleotides, approximately 300 to approximately 800 nucleotides, approximately 300 to approximately 900 nucleotides, or approximately 1000 nucleotides, or even longer than approximately 1000 nucleotides, such as polynucleotides of approximately 1500, approximately 2000, approximately 2500, approximately 5000, or approximately 7000 nucleotides. In the case of a double-stranded polynucleotide, its length can be similarly described using base pairs.

[0023] As used herein, a “cassette” (e.g., a synthetic in vitro transcription promoter cassette) refers to a polynucleotide sequence that may include or be operatively linked to one or more expression elements, such as enhancers, promoters, leader sequences, introns, 5' untranslated regions (UTRs), 3' UTRs, or transcription termination sequences. In some aspects, the cassette contains at least a first polynucleotide sequence capable of initiating transcription of an operatively linked second polynucleotide sequence (which may contain a template) and optionally a transcription termination sequence operatively linked to the second polynucleotide sequence. As described below, the template may contain a sequence of interest, such as an open reading frame of interest (“ORF”). The cassette may be provided as a single element or as two or more unlinked elements.

[0024] As used herein, a “template” refers to a nucleic acid sequence containing a sequence of interest for preparing a therapeutic polynucleotide according to the disclosed methods. A template can be, but is not limited to, double-stranded DNA (dsDNA), engineered plasmid constructs, cDNA sequences, or linear nucleic acid sequences (e.g., linear templates generated by PCR or by annealing chemistry of oligonucleotides). In some respects, the template can be integrated into a “box” as described above.

[0025] As used herein, the term "sequence of interest" refers to a polynucleotide sequence whose use is considered desirable for a suitable purpose (particularly for the preparation of mRNA for therapeutic purposes); and includes, but is not limited to, coding sequences of structural genes and non-coding regulatory sequences that do not encode mRNA or protein products.

[0026] As used herein, “in vitro transcription” or “IVT” refers to the process by which transcription occurs in vitro in a non-cellular system to produce synthetic RNA molecules (e.g., synthetic mRNA) for various applications, including therapeutic delivery to a subject, such as as therapeutic polynucleotides, which may be part of or used to form therapeutic polynucleotide compositions as described below. The resulting therapeutic polynucleotides (e.g., synthetic RNA molecules (transcription products)) may be combined with a delivery medium to form therapeutic polynucleotide compositions. Synthetic transcription products include mRNA, antisense RNA molecules, shRNA, circular RNA molecules, ribozymes, etc. IVT reactions may use a purified linear DNA template containing a promoter sequence and an open reading frame (ORF) sequence of interest, ribonucleotide triphosphates or modified ribonucleotide triphosphates, a buffer system including DTT and magnesium ions, and phage RNA polymerase.

[0027] As used herein, "therapeutic polynucleotide" means a polynucleotide (e.g., mRNA) that may be part of a therapeutic polynucleotide composition intended for delivery to a subject to treat the subject's symptoms, disease, or condition; prevent the subject's symptoms, disease, or condition; or improve or otherwise alter the subject's health.

[0028] As used herein, a “therapeutic polynucleotide composition” (or simply “therapeutic composition”) can refer to a composition comprising one or more therapeutic polynucleotides (e.g., mRNA) encapsulated by a delivery medium, which can be administered to a subject in need using any suitable route of administration, such as intratumoral, intramuscular, or other injection. An example of a therapeutic polynucleotide composition is mRNA (therapeutic) nanoparticles comprising at least one mRNA encapsulated by a delivery medium molecule. mRNA vaccines are an example of therapeutic polynucleotide compositions.

[0029] As used herein, “delivery vehicle” means any substance that at least partially facilitates the delivery of polynucleotides (e.g., therapeutic polynucleotides) in vivo, in vitro, or ex vivo to target cells or tissues (e.g., tumors, etc.). Referring to something as a delivery vehicle does not preclude the possibility that the delivery vehicle may also have therapeutic effects. Some versions of delivery vehicles may provide additional therapeutic effects. In some versions, the delivery vehicle may be a peptide-like molecule, such as an aminoesterified peptide molecule, which can be used to at least partially encapsulate mRNA. The term “DV” will also be used herein as an abbreviation for “delivery vehicle.”

[0030] As used herein, “purification” refers to the physical and / or chemical separation of a component (e.g., particles) from other unwanted components (e.g., contaminants, debris, etc.).

[0031] II. Overview of mRNA purification methods Figure 1A prior art mRNA purification method (100) is described. In the mRNA purification method (100), a column is packed with resin and equilibrated with approximately 3 to 4 column volumes of equilibration buffer containing 10 mM Tris-HCl, 0.5 M NaCl, and 1 mM EDTA at pH 7.4. A crude IVT mixture containing mRNA is then prepared by adding binding buffer (10 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 7.4). The sample is then immediately placed on wet ice and loaded onto the column at 50 cm / h–150 cm / h, followed by washing the column with another 2–3 column volumes of equilibration buffer (10 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 7.4). The column is then washed with 3–5 column volumes of a first wash buffer (10 mM Tris-HCl, 100 mM–300 mM NaCl, 1 mM EDTA, pH 7.4) or until conductivity stabilizes. The bound mRNA is then eluted with 3–5 column volumes of a second wash buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) or 3–5 column volumes of water. Elution can be enhanced by heating the elution buffer, elution water, or column to 65°C. The mRNA is then collected.

[0032] Figure 2 An exemplary non-limiting mRNA purification method (200) according to this disclosure is described. Figure 2 In this study, a column containing oligo-dT resin was prepared and equilibrated using equilibration buffers as described herein. The crude IVT product was denatured by heating. The denatured IVT product was then mixed with binding buffer and contacted with the column. The column was then washed with washing buffer (50 mM NaCl, 10 mM Tris, 1 mM EDTA). Following the washing step, mRNA was eluted from the column and collected using water at 75°C.

[0033] Variations of the disclosed method are provided below. It should be noted that any described method may be performed wholly or in part using the apparatus described in U.S. Patent Publication No. 2021 / 0040472A1, entitled “Methods and apparatuses for manufacturing for removing material from a therapeutic composition,” published February 11, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0034] Purified resin In this respect, the disclosed method involves contacting nucleic acids (particularly mRNA produced via an IVT process) with functionalized solid particles (such as purification beads or resins). Various types of purification resins (or beads) can be used to purify and / or isolate RNA, including, for example, mRNA obtained from an IVT manufacturing process. Exemplary resins (beads) may include resins that selectively capture mRNA via a polyadenylate (polyA) tail, using a salt and water purification step to separate mRNA from components of the transcription reaction process (such as plasmid DNA, enzymes, and other IVT components). Such beads may include a rigid polymeric resin support matrix comprising cross-linked poly(styrene-divinylbenzene); and / or may have a polyhydroxyl surface coating. The surface of such beads can be multifunctionalized (dT) to form oligo-dT resins. Oligo-dT resins contain short DNA sequences containing deoxythymidine residues (dT) complementary to the polyA tail of the mRNA molecule. When a sample containing mRNA molecules (such as a crude IVT mixture) is applied to the resin, the polyA tail of the mRNA hybridizes with the oligo-dT sequence on the resin. After binding, non-mRNA molecules, such as ribosomal RNA (rRNA), transfer RNA (tRNA), and genomic DNA, can be washed away from the resin, leaving the resin-bound mRNA molecules. Subsequently, the purified mRNA can be eluted from the resin using mild conditions that disrupt hybridization between the multi-A tail and oligo-dT sequences.

[0035] The resins may each have a predetermined particle size (e.g., diameter), such as in the range of about 10 µm to about 200 µm, such as, for example, about 50 µm; and / or may each be porous to increase the surface area of ​​each resin (bead) and thus effectively increase the contact area between the resin and the fluid. An exemplary resin that can be used in conjunction with the disclosed method is POROS Oligo (dT)25 affinity resin from ThermoFisher Scientific® (Waltham, Massachusetts) (which is designed for the isolation of messenger RNA (mRNA)), a rigid 50 μm polymer resin with a cross-linked poly(divinylstyrene) backbone. The polyhydroxy surface coating provides low nonspecific binding, while the poly(dT) at the surface allows for the capture of mRNA by pairing with the polyA tail bases of the mRNA. Other examples of particles that can be used for purification include urea particles, which may also each have a predetermined particle size (e.g., diameter), such as in the range of about 10 µm to about 200 µm, such as, for example, about 50 µm.

[0036] Column preparation The resin is placed in the column. Those skilled in the art will readily understand the appropriate column size and material. Typically, the column is cleaned and free of contaminants before the resin is added. If it is in dry or concentrated form, the slurry can be prepared by suspending the resin in a solvent or buffer solution. The column can be filled via dry filling, where the resin is filled into the column in a dry state. Alternatively, the column can be filled via wet filling, where the column is filled with a solvent or buffer solution before adding the resin slurry. The column can be tapped to settle resin particles and remove any air bubbles.

[0037] Balanced buffer In some respects, oligo-dT resins can be equilibrated with equilibration buffers, such as... Figure 2 As shown. In one aspect, the equilibration buffer is a low-salt equilibration buffer (“low-salt wash buffer”) having a salt concentration of less than 100 mM, or less than 75 mM, or less than about 50 mM. The low-salt equilibration buffer may include a salt concentration of at least 20 mM, or at least 30 mM, or at least 40 mM, or at least 45 mM. In some aspects, the salts of the low-salt equilibration buffer include sodium, potassium, magnesium, manganese, calcium, sulfate, phosphate, and / or chloride salts. In some aspects, the low-salt equilibration buffer comprises sodium chloride, sodium phosphate, sodium sulfate, potassium chloride, potassium phosphate, potassium sulfate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium chloride, calcium phosphate, and / or calcium sulfate.

[0038] The equilibration buffer may also contain buffering agents, such as Tris buffer, and chelating agents, such as EDTA. For example, in one aspect, the equilibration buffer may contain about 25 mM to about 100 mM of salt, or about 35 mM to 75 mM of salt, or about 40 mM to about 65 mM of salt, or about 45 mM to about 55 mM of salt, or about 50 mM of salt; and about 5 mM to about 15 mM, or about 7 mM to about 12 mM, or about 10 mM of Tris; and about 0.5 mM to about 1.5 mM, or about 1 mM of EDTA. In one aspect, the equilibration buffer contains about 50 mM NaCl, about 10 mM Tris, and about 1 mM EDTA.

[0039] In some embodiments, equilibration buffer is applied to a column containing oligo-dT resin, and then RNA (e.g., a crude IVT mixture) is contacted with the oligo-dT resin. For example, a column containing oligo-dT resin can be equilibrated by applying one, two, three, four, or more column volumes of oligo-dT resin. After applying the equilibration buffer, the oligo-dT resin can be mixed. The equilibration buffer is then passed through the column. Column equilibration can be repeated once, twice, three times, or more than three times. In one embodiment, equilibration can be performed at room temperature.

[0040] Sample preparation Before being introduced into the column, a sample containing the RNA to be purified is prepared. Typically, the RNA-containing sample (e.g., a crude IVT sample) is denatured and applied to the column. Denaturation is performed using methods known in the art, such as thermal denaturation, wherein the sample is heated to a temperature of approximately 65°C to approximately 95°C. In one aspect, the RNA sample to be purified (e.g., a crude IVT sample) is denatured by heating the composition to 60°C to 90°C, 60°C to 80°C, 60°C to 70°C, 65°C to 85°C, 65°C to 75°C, 70°C to 90°C, 70°C to 80°C, 65°C to 70°C, 70°C to 75°C, or 75°C to 95°C. In one aspect, the RNA is denatured at a temperature of approximately 65°C. The RNA can be heated for approximately 1 minute to approximately 20 minutes, or approximately 2 minutes to approximately 15 minutes, or approximately 3 minutes to approximately 12 minutes, or approximately 5 minutes to approximately 10 minutes. In one aspect, the RNA is heated for approximately 10 minutes. In another aspect, the RNA sample was heated at 65 degrees Celsius for about 10 minutes before contacting the oligo-dT resin.

[0041] Following the denaturation step, the denatured RNA composition may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the total RNA as denatured RNA. In some aspects, at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the total RNA in the denatured RNA composition may be denatured RNA. In some aspects, 50%-70%, 45%-60%, 55%-70%, 60%-80%, 60%-100%, 75%-100%, 50%-95%, 75%-95%, 80%-100%, 80%-90%, 90%-95%, 95%-100%, 90%-99%, or 95%-99% of the total RNA in the denatured RNA composition comprises denatured RNA.

[0042] RNA purification The denatured RNA composition is then contacted with the oligo-dT resin using any method known in the art, such as by directly pipetting the sample onto the resin. In some aspects, the application of the denatured RNA composition to the oligo-dT resin can be automated.

[0043] binding buffer In this aspect, a sample containing the RNA to be purified is applied to a resin before contacting the sample with the binding buffer. After the denatured RNA composition is applied to the oligo-dT resin, the binding buffer is applied. In this aspect, the binding buffer may include a "high-salt" binding buffer containing at least 100 mM, at least 125 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, or at least 1000 mM of salt. The high-salt binding buffer may include salt concentrations of 100 mM-1000 mM, 200 mM-800 mM, 300 mM-700 mM, 400 mM-800 mM, 500 mM-700 mM, or about 500 mM of salt. In some aspects, the salts of the binding buffer include sodium, potassium, magnesium, manganese, calcium, sulfate, phosphate, and / or chloride salts. In some aspects, the binding buffer comprises sodium chloride, sodium phosphate, sodium sulfate, potassium chloride, potassium phosphate, potassium sulfate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium chloride, calcium phosphate, and / or calcium sulfate.

[0044] In one aspect, the binding buffer may contain about 250 mM NaCl to about 1000 mM NaCl, or about 350 mM to 750 mM NaCl, or about 400 mM to about 650 mM NaCl, or about 450 mM to about 550 mM NaCl, or about 500 mM NaCl.

[0045] In one aspect, the binding buffer may further comprise a buffering agent, such as about 1 mM to about 50 mM, or about 2 mM to about 40 mM, or about 3 mM to about 30 mM, or about 4 mM to about 20 mM, or about 5 mM to about 10 mM Tris; and a chelating agent, such as about 0.1 mM to about 4 mM, or about 0.2 mM to about 4 mM, or about 0.3 mM to about 3 mM, or about 0.5 mM to about 2 mM EDTA. In one aspect, the binding buffer may comprise about 500 mM NaCl, about 10 mM Tris, and about 1 mM EDTA.

[0046] Table 1 illustrates an exemplary method in which, after a denaturation step at 65°C lasting 10 minutes, the crude IVT sample is contacted with a 400 μL volume of resin slurry, and then contacted with a binding buffer containing 500 mM NaCl, 10 mM Tris, and 1 mM EDTA.

[0047] In one aspect, the denatured RNA composition is contacted with oligo-dT resin at temperatures ranging from about 4°C to about 30°C, from about 4°C to about 25°C, from about 4°C to about 20°C, from about 4°C to about 15°C, from about 4°C to about 10°C, from about 4°C to about 8°C, from about 10°C to about 30°C, from about 10°C to about 25°C, from about 10°C to about 20°C, from about 10°C to about 15°C, or from about 15°C to about 25°C. In another aspect, the denatured RNA may be bound to or contacted with the oligo-dT resin for a total residence time of less than 20 minutes, less than 18 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. In some respects, denatured RNA can bind to or be exposed to oligo-dT resin for a total residence time of 1-2 minutes, 1-5 minutes, 2-5 minutes, 2-10 minutes, 5-20 minutes, 5-10 minutes, 5-15 minutes, 8-15 minutes, 10-15 minutes, 12-20 minutes, or 15-20 minutes.

[0048] Following the oligo-dT resin contact step described above, the column containing the oligo-dT resin and denatured RNA is washed to remove components not bound to the oligo-dT resin. Those skilled in the art will understand the methods used to apply the wash buffer. For example, the wash buffer can be applied manually or using an automated process.

[0049] The method may include a first washing step with a first washing buffer and a second washing step with a second washing buffer.

[0050] In one aspect, a first washing step is performed, wherein the resin (containing bound mRNA) is washed with a first washing buffer. In one aspect, the first washing buffer is a low-salt washing buffer having a salt concentration of less than 100 mM, or less than 75 mM, or about 50 mM or lower. The washing buffer may include a salt concentration of at least 20 mM, or at least 30 mM, or at least 40 mM, or at least 45 mM, or about 50 mM. In some aspects, the first washing buffer may contain sodium, potassium, magnesium, manganese, calcium, sulfate, phosphate, and / or chloride salts. In some aspects, the first washing buffer contains sodium chloride, sodium phosphate, sodium sulfate, potassium chloride, potassium phosphate, potassium sulfate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium chloride, calcium phosphate, and / or calcium sulfate.

[0051] In one aspect, the first wash buffer comprises a salt concentration of less than 100 mM NaCl, or less than 75 mM NaCl, or about 50 mM NaCl or less. The first wash buffer may comprise a salt concentration of at least 20 mM NaCl, or at least 30 mM NaCl, or at least 40 mM NaCl, or at least 45 mM NaCl, or about 50 mM NaCl, further combined with a buffer and a chelating agent.

[0052] The first wash buffer may also contain a buffer, such as a Tris buffer, and a chelating agent, such as EDTA. For example, in one aspect, the first wash buffer may contain about 25 mM to about 100 mM of salt, or about 35 mM to 75 mM of salt, or about 40 mM to about 65 mM of salt, or about 45 mM to about 55 mM of salt, or about 50 mM of salt; and about 5 mM to about 15 mM of Tris buffer, or about 7 mM to about 12 mM of Tris buffer, or about 10 mM of Tris buffer; and about 0.5 mM to about 1.5 mM of EDTA, or about 1 mM of EDTA. In one aspect, the first wash solution contains about 50 mM NaCl, about 10 mM Tris, and about 1 mM EDTA. Table 1 illustrates the use of the first wash solution in exemplary methods.

[0053] A second washing step can then be performed, wherein the resin is washed with a second washing buffer. In one aspect, the second washing buffer is a low-salt washing buffer having a salt concentration of less than 100 mM, or less than 75 mM, or about 50 mM or lower. The second washing buffer may include a salt concentration of at least 20 mM, or at least 30 mM, or at least 40 mM, or at least 45 mM, or about 50 mM. In some aspects, the second washing buffer may contain sodium, potassium, magnesium, manganese, calcium, sulfate, phosphate, and / or chloride salts. In some aspects, the second washing buffer may contain sodium chloride, sodium phosphate, sodium sulfate, potassium chloride, potassium phosphate, potassium sulfate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium chloride, calcium phosphate, and / or calcium sulfate.

[0054] In one aspect, the second wash buffer comprises a salt concentration of less than 100 mM NaCl, or less than 75 mM NaCl, or about 50 mM NaCl or less. The second wash buffer may further comprise a salt concentration of at least 20 mM NaCl, or at least 30 mM NaCl, or at least 40 mM NaCl, or at least 45 mM NaCl, or about 50 mM NaCl, in combination with a buffer and a chelating agent.

[0055] The second wash buffer may also contain a buffer, such as a Tris buffer, and a chelating agent, such as EDTA. For example, in one aspect, the second wash buffer may contain about 25 mM to about 100 mM of salt, or about 35 mM to 75 mM of salt, or about 40 mM to about 65 mM of salt, or about 45 mM to about 55 mM of salt, or about 50 mM of salt; and about 5 mM to about 15 mM of Tris buffer, or about 7 mM to about 12 mM of Tris buffer, or about 10 mM of Tris buffer; and about 0.5 mM to about 1.5 mM of EDTA, or about 1 mM of EDTA. In one aspect, the second wash solution contains about 50 mM NaCl, about 10 mM Tris, and about 1 mM EDTA. Table 1 illustrates the use of the second wash buffer in exemplary methods.

[0056] In one aspect, the first wash buffer, the second wash buffer, and the equilibration buffer have the same salt concentration. In another aspect, the first wash buffer, the second wash buffer, and the equilibration buffer contain approximately 50 mM NaCl, approximately 10 mM Tris, and approximately 1 mM EDTA.

[0057] The washing step can be repeated, for example, once to three times. After the washing step, the mRNA bound to the oligo-dT resin is eluted. In one aspect, elution can be performed by applying water to the column. In another aspect, this step can be performed under heating. For example, the elution step can be performed at temperatures of 60°C to 100°C, 65°C to 90°C, 70°C to 80°C, or about 75°C. In one aspect, the RNA is eluted at about 75°C.

[0058] In some respects, RNA eluted from oligo-dT resin contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of RNA with poly-A tails. In some respects, RNA eluted from oligo-dT resin contains approximately 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 25%-75%, 30%-50%, 40%-60%, 50%-70%, 45%-60%, 55%-70%, 60%-80%, 60%-100%, 75%-100%, 50%-95%, 75%-95%, 80%-100%, 80%-90%, 90%-95%, 95%-100%, 90%-99%, or 95%-99% of polyA-tailed mRNA. In some respects, the RNA eluted from oligo-dT resin contains at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of polyA-tailed mRNA.

[0059] RNA eluted from oligo-dT resin can be of any size or length. In some respects, the length of RNA eluted from oligo-dT resin is 50-250, 200-500, 400-5000, 400-4000, 400-3000, 400-2000, 400-1000, 500-5000, 500-1500, 750-2000, 1000-1500, 1250-2000, 1500-2000, 1750-2500, 2000-3000, 2500-3500, 3000-4000, 3500-4500, or 4000-5000 nucleotides.

[0060] In some aspects, the RNA is unmodified and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some aspects, the nucleotides and nucleosides of this disclosure comprise standard nucleoside residues (e.g., A, G, C, or U). In some aspects, the nucleotides and nucleosides of this disclosure comprise standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT).

[0061] In some aspects, RNA is modified mRNA (mmRNA) and contains at least one modified nucleotide. In some aspects, the terms "modified" and "modified" refer to modifications relating to at least one of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides in their nucleotide position, pattern, percentage, or group. RNA may contain naturally occurring, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. RNA may include any useful modifications, such as those to sugars, nucleotides, or internucleotide bonds (e.g., phosphate ester, phosphodiester, or phosphodiester backbones).

[0062] In some respects, RNA may contain modified nucleosides and / or nucleotides. A “nucleoside” is a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof combined with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. In some embodiments, the modified nucleotides in the RNA are selected from the group consisting of: pseudouridine (Y), N1-methylpseuuridine (m1Y), N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the RNA comprises a combination of at least two (e.g., 2, 3, 4, or more) of the above-described modified nucleotides.

[0063] RNA may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C) or any percentage in between (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10%). (The percentages are listed as follows: from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is occupied by the presence of unmodified A, G, U, or C. In some embodiments, the RNA contains a cap analogue. RNA cap analogs typically enhance mRNA stability and translation efficiency. Common cap analogs include GpppG, m7GpppG, and m2,2,7GpppG. In some embodiments, the RNA cap analogs of this disclosure are dinucleotide, trinucleotide, or tetranucleotide caps. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.

[0064] Automation methods As described, any of the methods described may be carried out, in whole or in part, using the apparatus described in U.S. Patent Publication No. 2021 / 0040472A1 entitled “Methods and apparatuses for manufacturing for removing material from atherapeutic composition”, published February 11, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0065] For example, the disclosed method can be automated, employing an apparatus for in vitro transcription (IVT) using template material to form therapeutic mRNA, such as the apparatus described in U.S. Patent Publication No. 2021 / 0040472A1. For instance, an automated in vitro transcription (IVT) reaction can be performed using a system comprising more than one fluid bank configured to be in sealed fluid communication with one or more microfluidic pathway devices, wherein the apparatus performs the following operations: transporting reagents within a closed fluid pathway between one or more fluid banks and more than one reactor on one or more microfluidic devices, the closed fluid pathway being protected from atmospheric contact, to perform in vitro transcription of therapeutic mRNA from a template in one or more microfluidic pathway devices, and to purify the therapeutic polynucleotide. Automated purification of the therapeutic polynucleotide can be performed according to the method described herein.

[0066] In another example, an automated method for performing in vitro transcription (IVT) reactions using a system comprising more than one fluid bank configured to be in sealed fluid communication with a microfluidic pathway device is disclosed. The method includes: delivering a DNA template, polymerase, and nucleotides from one or more of the following into one or more IVT reactors of the microfluidic pathway device: the fluid bank in the more than one fluid bank and a site on the microfluidic pathway device; processing the DNA template and nucleotides in the one or more IVT reactors to form therapeutic mRNA; and transferring the therapeutic mRNA to one or more purification reactor regions of the microfluidic pathway device and purifying the therapeutic mRNA according to the method described herein, wherein the microfluidic pathway device and the more than one fluid bank form a closed pathway and sealed environment to prevent atmospheric exposure.

[0067] Typically, the system may include a controller to perform these methods, including, for example, the step of transporting reagents by deflecting one or more elastic layers within a microfluidic pathway device. These methods may include receiving optical sensor data from one or more sensors of the system in a controller for the system, wherein the controller controls the operation of the system based on the optical sensor data. The controller may also control the pressurization of the fluid reservoir. In any of the methods described herein, the system may include a microfluidic pathway device located within the system.

[0068] Typically, a DNA template may contain a synthetic double-stranded DNA template of the gene of interest and a synthetic in vitro transcription facilitator cassette.

[0069] III. Examples The following non-limiting embodiments are provided to further illustrate implementations of the invention disclosed herein. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent methods found to work well in the practice of the invention and can therefore be considered as examples of patterns for use in its practice. However, those skilled in the art will understand that, based on this disclosure, many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or analogous results can still be obtained.

[0070] Example 1 - Column preparation for benchtop purification The following steps are taken to prepare a column for purifying mRNA: Secure the column to the vacuum manifold and attach the vacuum line / trap to the port on the manifold.

[0071] The empty column was sterilized by filling it with denatured ethanol and then vacuum-dried.

[0072] The column was filled with water and dried under vacuum.

[0073] Determine the resin slurry percentage. Allow the resin to reach room temperature. Once the resin has fully settled, use a ruler to determine the approximate slurry percentage. Use this percentage to add 200 μL of resin (not slurry) to the column—that is, if it is 50% slurry, add 400 μL to the sterilized column for every 200 μL of resin.

[0074] Open the stopcock valve on the sample processing station to empty the column, using vacuum assistance if necessary.

[0075] Equilibrate the resin with 6 mL (2 × 3 mL) of environmental low-salt wash buffer (50 mM NaCl, 10 mM Tris, 1 mM EDTA).

[0076] a. Add 3 mL of low-salt wash buffer (equilibration buffer) to the column.

[0077] b. Pipette to mix.

[0078] c. Allow the resin to settle for 3 minutes.

[0079] d. Open the stopcock valve on the sample processing station to empty the column, using vacuum assistance if necessary.

[0080] e. Repeat steps (a) through (d) once.

[0081] Empty the sample buffer solution from the sample processing station and dispose of the waste.

[0082] Place a clean 5 mL Eppendorf tube under the column.

[0083] purification Denature the IVT product for 10 minutes at 65°C and 400 rpm (1.5 mL Thermotop) in a hot mixer. Immediately add 1 mg of mRNA to the equilibrated resin. Add a volume of binding buffer. Pipette to mix. Allow the resin to settle for 10 minutes. Open the stopcock valve on the sample processing station to empty the column, using vacuum assistance if necessary. Do not over-dry the resin. Place a clean 5 mL Eppendorf tube under the column.

[0084] Wash the resin with 6 mL (2×3 mL) of environmental low-salt wash buffer (first wash buffer, 50 mM NaCl, 10 mM Tris, 1 mM EDTA).

[0085] a. Add 3 mL of low-salt wash buffer (first wash buffer) to the column.

[0086] b. Pipette to mix.

[0087] c. Allow the resin to settle for 3 minutes.

[0088] d. Open the stopcock valve on the sample processing station to empty the column, using vacuum assistance if necessary.

[0089] e. Repeat steps (a) through (d) once.

[0090] Heat 1 mL of water to 75°C (per column) on a hot mixer.

[0091] Place a clean 5 mL Eppendorf tube under the column.

[0092] Elute with 0.8 mL (2 × 0.4 mL) of RNase-free water at 75°C.

[0093] a. Add 0.4 mL of RNase-free water at 75°C to the column.

[0094] b. Pipette to mix.

[0095] c. Allow the resin to settle for 3 minutes.

[0096] d. Open the stopcock valve on the sample processing station to empty the column, using vacuum assistance if necessary.

[0097] e. Repeat steps (a) through (d) once.

[0098] Example 2 - Recovery Rate Calculation Recovery was calculated by comparing the mass of RNA collected from dT purification with a 179 μL sample of IVT product purified solely by AMPure magnetic beads. This method does not target multiple (A) tails. The recovery from AMPure magnetic bead purification was used to determine the theoretical concentration of RNA in the IVT product (for dT column loading) and to determine the percentage recovery.

[0099] For example, if 0.75 mg of mRNA is recovered from a 179 μL IVT product sample during AMPure purification, the theoretical yield of the reaction will be determined to be 4.19 mg mRNA / mL reaction. The operator will then add 239 μL of IVT product to achieve a column loading capacity of 5 mg IVT product / mL resin.

[0100] After dT purification, the operator obtains the concentration value of 1 mL of dT eluate. In the above examples, 1 mg of IVT product was loaded onto a dT column. The recovery rate was calculated as follows: Example 3 - Purity Calculation The purity percentage is calculated using the area of ​​the "main peak" of the final purified product. This calculation is performed by adding the total areas of the peaks on the chromatogram or electrophoresis plot, then dividing the area of ​​the main peak by the total peak area and multiplying the fraction by 100. Example 4 - Calculation using double-stranded RNA (dsRNA) from immunoblotting and ELISA Both heated and non-heated elution in RNase-free water were used to test the conditions described herein against prior art conditions for batch purification of IVT products to test the reduction of dsRNA in the process detailed herein relative to prior art processes. These were repeat dT purifications. Residual dsRNA was measured using a quantitative K1 dsRNA ELISA. Additionally, a combination of cellulose and AMPure purification was tested as a standard measure of post-purification dsRNA content, and samples of material purified by AMPure alone were tested because AMPure beads are known not to be selective for dsRNA, and previous studies have shown that AMPure-purified samples contain residual dsRNA. Recovery percentages and purity percentages were calculated as described above.

[0101] Purity was measured using the Agilent Fragment Analyzer (Agilent 5300).

[0102] dsRNA was measured for each sample, and experimental replicates were combined into a single bar with an error bar for reference. The process described in this paper has the lowest measured residual dsRNA concentration. Neither the heat elution recommended in the supplier's process nor simply denaturing the IVT product with the supplier's protocol showed an effect on residual dsRNA concentration. The proposed method describes heating the IVT product and adding the heated product to dT resin equilibrated in a low-salt solution, followed by adding salt to the column or reservoir. The order of addition of the IVT product and the higher-salt binding buffer demonstrated that it provided an environment conducive to reducing residual dsRNA in the purified mRNA.

[0103] These data were converted to dsRNA clearance efficiencies, expressed as a percentage of dsRNA. The efficiencies were ~36% in samples processed using the NTX process, ~48% in samples processed using the supplier's denaturing process, and ~49% in samples processed using the non-denaturing supplier's process. Therefore, the dsRNA clearance efficiency can be expressed as ~64% clearance in samples processed using the exemplary method, ~52% clearance in samples processed using the supplier's denaturing process, and ~51% clearance in samples processed using the non-denaturing supplier's process. Thus, compared to the aforementioned supplier processes, the exemplary method process resulted in a reduction of approximately 25% in dsRNA.

[0104] Table 1. Exemplary method conditions and supplier method conditions with or without alteration steps. IV. Exemplary Combinations The following examples illustrate various non-exhaustive ways in which the teachings of this document can be combined or applied. It should be understood that the following examples are not intended to limit the scope of any claim that may appear at any time in this application or in subsequent documents thereof. No waiver of rights is intended. The following examples are provided merely for illustrative purposes. It is contemplated that the various teachings of this document may be arranged and applied in a variety of other ways. It is also contemplated that some variations may omit certain features mentioned in the examples below. Therefore, no aspect or feature mentioned below should be considered critical unless explicitly stated later by the inventor or a successor with an interest in the inventor. If any claim set forth in this application or in subsequent documents related to this application includes additional features beyond those mentioned below, such additional features shall not be presumed to have been added for any reason relating to patentability.

[0105] Example 1 A method includes: heating an RNA mixture at a temperature of about 60°C to 80°C to form a denatured RNA solution; contacting the denatured RNA solution with an oligo-dT resin to form an RNA-resin mixture; contacting the RNA-resin mixture with a salt-binding buffer; washing the RNA-resin mixture with a washing buffer; and eluting mRNA from the oligo-dT resin with a washing buffer.

[0106] Example 2 According to the method described in Example 1, the RNA mixture comprises single-stranded RNA and double-stranded RNA.

[0107] Example 3 According to the method described in Example 1, the mRNA mixture contains crude mRNA from an in vitro transcription (IVT) reaction.

[0108] Example 4 According to the method described in Example 1, the heating step is performed for 1 minute to 1 hour.

[0109] Example 5 According to the method described in Example 1, the heating step is performed for 2 to 50 minutes.

[0110] Example 6 According to the method described in Example 1, the heating step is performed for 3 to 40 minutes.

[0111] Example 7 According to the method described in Example 1, the heating step is performed for 3 to 30 minutes.

[0112] Example 8 According to the method described in Example 1, the heating step is performed for 4 to 20 minutes.

[0113] Example 9 According to the method described in Example 1, the heating step is performed for 5 to 15 minutes.

[0114] Example 10 According to the method described in Example 1, the heating step is performed for approximately 10 minutes.

[0115] Example 11 According to the method described in Example 1, the heating step is performed at a temperature of approximately 65°C.

[0116] Example 12 According to the method described in any of the foregoing examples, the oligo-dT resin contains functional groups selected from butyl, tert-butyl, methyl, ethyl, and combinations thereof.

[0117] Example 13 According to the method described in any of the foregoing examples, the oligo-dT resin comprises crosslinked poly(styrene-divinylbenzene).

[0118] Example 14 According to the method described in any of the foregoing examples, the oligo-dT resin comprises bead shapes.

[0119] Example 15 According to the method described in any of the foregoing examples, the oligo-dT resin comprises an average particle size of about 50 μm.

[0120] Example 16 According to the method described in any of the foregoing examples, the binding buffer contains approximately 25 mM NaCl to approximately 1000 mM NaCl.

[0121] Example 17 According to the method described in any of the foregoing examples, the binding buffer contains about 50 mM NaCl to about 800 mM NaCl.

[0122] Example 18 According to the method described in any of the foregoing examples, the binding buffer contains about 100 mM NaCl to about 700 mM NaCl.

[0123] Example 19 According to the method described in any of the foregoing examples, the binding buffer contains about 200 mM NaCl to about 600 mM NaCl.

[0124] Example 20 According to the method described in any of the foregoing examples, the binding buffer contains about 300 mM NaCl to about 500 mM NaCl.

[0125] Example 21 According to the method described in any of the foregoing examples, the binding buffer contains approximately 500 mM NaCl.

[0126] Example 22 According to the method described in any of the foregoing examples, the binding buffer comprises Tris and EDTA.

[0127] Example 23 According to the method described in any of the foregoing examples, the binding buffer contains approximately 10 mM Tris and approximately 1 mM EDTA.

[0128] Example 24 According to the method described in any of the foregoing examples, the oligo-dT resin is equilibrated in a low-salt solution containing about 25 mM NaCl to about 100 mM NaCl.

[0129] Example 25 According to the method described in any of the foregoing examples, the RNA eluted from the oligo-dT resin contains at least 90% polyA-tailed mRNA.

[0130] Example 26 According to the method described in any of the foregoing examples, the RNA eluted from the oligo-dT resin contains at least 95% polyA-tailed mRNA.

[0131] Example 27 A method comprising: contacting a composition comprising a denatured IVT sample and oligo-dT resin with a binding buffer comprising about 500 mM NaCl; and eluting purified mRNA from the mixture formed in (a).

[0132] Example 28 According to the method described in Example 27, the RNA eluted from the oligo-dT resin contains at least 90% of the poly-A-tailed mRNA.

[0133] Example 29 The method according to Example 27 or 28, wherein the RNA eluted from the oligo-dT resin contains at least 95% of the poly-A-tailed mRNA.

[0134] Example 30 A therapeutic polynucleotide comprising mRNA purified according to any of the foregoing examples.

[0135] Example 31 A therapeutic polynucleotide composition comprising mRNA purified according to any one of Examples 1 to 29.

[0136] V. Miscellaneous The foregoing description is provided to enable those skilled in the art to practice the various configurations described herein. While the subject matter has been specifically described with reference to various accompanying drawings and configurations, it should be understood that these are for illustrative purposes only and should not be considered as limiting the scope of the subject matter.

[0137] There may be many other ways to implement the subject matter technology. The various functions and elements described herein can be divided differently from those shown without departing from the scope of the subject matter technology. Various modifications to these embodiments may be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments. Therefore, many changes and modifications can be made to the subject matter technology by those skilled in the art without departing from its scope. For example, different numbers of given modules or units may be used, one or more different types of given modules or units may be used, given modules or units may be added, or given modules or units may be omitted.

[0138] Some versions of the examples described herein can be implemented using a processor, which may be part of a computer system and communicate with multiple peripheral devices via a bus subsystem. Versions of the examples described herein implemented using a computer system can be implemented using a general-purpose computer programmed to perform the methods described herein. Alternatively, versions of the examples described herein implemented using a computer system can be implemented using a special-purpose computer with hardware configurations arranged to perform the methods described herein. Versions of the examples described herein can also be implemented using a combination of at least one general-purpose computer and at least one special-purpose computer.

[0139] In versions implemented using computer systems, each processor may include a central processing unit (CPU), microprocessor, application-specific integrated circuit (ASIC), other types of hardware components, and combinations thereof. A computer system may include more than one type of processor. Peripheral devices of a computer system may include a storage subsystem, including, for example, memory devices and file storage subsystems, user interface input devices, user interface output devices, and a network interface subsystem. Input and output devices allow users to interact with the computer system. The network interface subsystem provides an interface to external networks, including interfaces to corresponding interface devices in other computer systems. User interface input devices may include keyboards; pointing devices, such as mice, trackballs, touchpads, or graphics tablets; scanners; touchscreens integrated into a display; audio input devices, such as speech recognition systems and microphones; and other types of input devices. Generally, the term "input device" is intended to encompass all possible types of devices and methods for inputting information into a computer system.

[0140] In versions implemented using computer systems, user interface output devices may include a display subsystem, a printer, a fax machine, or a non-visual display such as an audio output device. The display subsystem may include a cathode ray tube (CRT), a flat panel device such as a liquid crystal display (LCD), a projection device, or some other mechanism for creating visible images. The display subsystem may also provide non-visual displays, such as audio output devices. Generally, the term "output device" is intended to encompass all possible types of devices and methods for outputting information from a computer system to a user or another machine or computer system.

[0141] In versions implemented using computer systems, the storage subsystem may store programming and data structures that provide the functionality of some or all of the modules and methods described herein. These software modules can typically be executed by the computer system's processor alone or in conjunction with other processors. The memory used in the storage subsystem may include multiple memories, including main random access memory (RAM) for storing instructions and data during program execution and read-only memory (ROM) for storing fixed instructions. The file storage subsystem may provide permanent storage for program and data files and may include hard disk drives, floppy disk drives along with associated removable media, CD-ROM drives, optical drives, or removable media cartridges. Modules implementing the functionality of a particular implementation may be stored in the storage subsystem or in a file storage subsystem on another machine accessible to the processor.

[0142] In versions implemented using computer systems, the computer system itself can be of different types, including personal computers, portable computers, workstations, computer terminals, network computers, televisions, mainframes, server farms, a loosely networked group of widely distributed computers, or any other data processing system or user equipment. Due to the constantly evolving nature of computers and networks, the examples of computer systems described herein are intended only as specific examples for illustrating the disclosed techniques. Many other configurations of computer systems with more or fewer components than those described herein are possible.

[0143] As an article of manufacture rather than a method, a non-transitory computer-readable medium (CRM) may be loaded with processor-executable program instructions. When executed, the program instructions implement one or more of the computer-implemented methods described above. Alternatively, the program instructions may be loaded onto a non-transitory CRM and, when combined with suitable hardware, become one or more components of a computer-implemented system practicing the disclosed methods.

[0144] Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject matter, and are mentioned without regard to the interpretation of the subject matter description. All structural and functional equivalents of elements of the various embodiments described throughout this disclosure that are known or will be known by one of ordinary skill in the art are expressly incorporated herein by reference and are intended to be included by the subject matter. Furthermore, the disclosure herein is not intended to be made public, whether or not such disclosure is expressly stated in the foregoing description.

[0145] It should be recognized that all combinations of the foregoing concepts and other concepts discussed in more detail below (assuming that such concepts are not inconsistent with each other) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein.

Claims

1. A method comprising: (a) Heating the RNA mixture at a temperature of about 60°C to 80°C to form a denatured RNA solution; (b) The denatured RNA solution is contacted with oligo-dT resin to form an RNA-resin mixture; (c) Contact the RNA-resin mixture with a salt-binding buffer; (d) Wash the RNA-resin mixture with a washing solution; (e) Elute mRNA from the oligo-dT resin with washing buffer.

2. The method according to claim 1, wherein the RNA mixture comprises single-stranded RNA and double-stranded RNA.

3. The method of claim 1, wherein the mRNA mixture comprises crude mRNA from an in vitro transcription (IVT) reaction.

4. The method according to claim 1, wherein the heating step is performed for 1 minute to 1 hour.

5. The method according to claim 1, wherein the heating step is performed for 2 to 50 minutes.

6. The method according to claim 1, wherein the heating step is performed for 3 to 40 minutes.

7. The method according to claim 1, wherein the heating step is performed for 3 to 30 minutes.

8. The method according to claim 1, wherein the heating step is performed for 4 to 20 minutes.

9. The method according to claim 1, wherein the heating step is performed for 5 to 15 minutes.

10. The method according to claim 1, wherein the heating step is performed for approximately 10 minutes.

11. The method according to claim 1, wherein the heating step is performed at a temperature of about 65°C.

12. The method according to any of the preceding claims, wherein the oligo-dT resin comprises a functional group selected from butyl, tert-butyl, methyl, ethyl, and combinations thereof.

13. The method according to any of the preceding claims, wherein the oligo-dT resin comprises crosslinked poly(styrene-divinylbenzene).

14. The method according to any of the preceding claims, wherein the oligo-dT resin comprises beads.

15. The method according to any of the preceding claims, wherein the oligo-dT resin comprises an average particle size of about 50 μm.

16. The method according to any of the preceding claims, wherein the binding buffer comprises about 25 mM NaCl to about 1000 mM NaCl.

17. The method according to any of the preceding claims, wherein the binding buffer comprises about 50 mM NaCl to about 800 mM NaCl.

18. The method according to any of the preceding claims, wherein the binding buffer comprises about 100 mM NaCl to about 700 mM NaCl.

19. The method according to any preceding claim, wherein the binding buffer comprises about 200 mM NaCl to about 600 mM NaCl.

20. The method according to any of the preceding claims, wherein the binding buffer comprises about 300 mM NaCl to about 500 mM NaCl.

21. The method according to any of the preceding claims, wherein the binding buffer comprises about 500 mM NaCl.

22. The method according to any of the preceding claims, wherein the binding buffer comprises Tris and EDTA.

23. The method according to any of the preceding claims, wherein the binding buffer comprises about 10 mM Tris and about 1 mM EDTA.

24. The method according to any of the preceding claims, wherein the oligo-dT resin is equilibrated in a low-salt solution containing about 25 mM NaCl to about 100 mM NaCl.

25. The method according to any of the preceding claims, wherein the RNA eluted from the oligo-dT resin comprises at least 90% polyA-tailed mRNA.

26. The method according to any of the preceding claims, wherein the RNA eluted from the oligo-dT resin comprises at least 95% polyA-tailed mRNA.

27. A method comprising: (a) Contact the composition containing the denatured IVT sample and oligo-dT resin with a binding buffer containing about 500 mM NaCl; and (b) The purified mRNA eluted from the mixture formed in (a).

28. The method of claim 27, wherein the RNA eluted from the oligo-dT resin comprises at least 90% polyA-tailed mRNA.

29. The method of claim 27 or 28, wherein the RNA eluted from the oligo-dT resin comprises at least 95% polyA-tailed mRNA.

30. A therapeutic polynucleotide comprising mRNA purified according to the method of any preceding claim.

31. A therapeutic polynucleotide composition comprising mRNA purified by the method according to any one of claims 1 to 29.

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