In vitro transcription method

Abstract

Provided are methods of producing RNA molecules in vitro. The methods include recycling or reusing components from a first RNA production reaction in a second, third or further RNA production reaction. The methods can be used to produce RNA-based vaccines and therapeutics.

Description

[0001] In Vitro Transcription Method

[0002] Field of Invention

[0003] The present invention provides methods of producing RNA molecules in vitro. The methods include recycling or reusing components from a first RNA production reaction in a second, third or further RNA production reaction. The methods can be used to produce RNA-based vaccines and therapeutics.

[0004] The manufacturing of an mRNA drug substance is carried out using an in vitro transcription (IVT) reaction. In this reaction, the RNA polymerase enzyme (e.g. T7 RNA Polymerase) assembles the nucleoside triphosphates (NTPs where N = G, A, C or II) into the ribonucleic acid (RNA) polymer based on a template DNA. The 5’ capping of the RNA can be achieved either co-transcriptionally (using capping analogues) or post-transcriptionally (using capping enzymes). Most of the components used in the IVT are not consumed during the IVT and are discarded at the end of each production run. These discarded components include: the T7 RNA Polymerase, template DNA and high-cost 5’ capping reagents. Following the IVT reaction, the RNA drug substance is purified using chromatography and filtration techniques. This often involves dilution of the reaction mix after RNA production and the salt concentration is increased to allow separation of the produced RNA by various methods such as chromatography, size exclusion and ion exchange based methods. The removal of product-related impurities, such as double-stranded RNA (dsRNA), is especially challenging. Next, the purified RNA is sterile filtered and encapsulated into lipid nanoparticles (LNPs).

[0005] The obtained RNA-LNPs are purified using methods such as tangential flow filtration and are sterile-filtered. This process is carried out in batch mode as the industry standard. The produced RNA-LNP drug product can then be filled into glass vials.

[0006] Raw materials represent 70-80% of the total manufacturing costs and out of the raw materials, the 5’ capping regents represent 70-80% of the total material costs. Most of these raw materials are discarded after every batch. It is, therefore, very important to reduce raw material costs, given the multi-product platform nature of this manufacturing technology.

[0007] Commercial purification kits may be used to purify and separate the synthesized mRNA, followed by precipitation using ethanol or isopropanol, which can remove most contaminants and obtain high-purity mRNA, and then the mRNA may be precipitated with high concentrations of LiCI or alcohol-based precipitation, chromatographic methods (molecular exclusion chromatography, ion-exchange chromatography, or affinity chromatography with immobilized oligo-dT), or elution from a silica membrane column, which removes proteins, free nucleotides or other components but not dsRNA impurities. To remove dsRNA contaminants from the transcription reaction solution, reversed-phase HPLC may be used.

[0008] WO2022 / 212710 A1 discloses a method purification and recycling nucleotide messenger RNA (mRNA) caps from the preparation of mRNA. However, the method does not recycle other components of the IVT reaction leading to loss of other components of the reaction mix.

[0009] There is a need for improved methods of IVT that increase yields of RNA.

[0010] There is a need for improved methods of IVT that improve the RNA yield to cost ratio.

[0011] There is a need for improved methods of IVT that reduce the material costs.

[0012] There is a need for improved methods of IVT that reduce unwanted by products of RNA production.

[0013] Brief summary of the disclosure

[0014] The inventors have developed an RNA manufacturing process that allows for the reuse or recycling of all of the high-cost IVT raw materials that are not consumed during RNA synthesis to produce higher yields of RNA product. The methods may provide at least 5-fold reduction in raw material costs. The methods include selectively removing RNA from the reaction mix, using downstream purification methods.

[0015] The methods provided herein also allow for the continuous production of RNA products. The methods provided herein also allow for the continuous extraction of RNA products. For example, without the need for additional steps and / or buffer exchange.

[0016] The inventors have also surprisingly found that optimisation of chaotropic agent concentrations in the IVT reaction mix and during separation of RNA from the reaction mix may allow for a cost effective and scalable method for recycling IVT reaction components.

[0017] The methods provided herein may provide for a 5 fold decrease in the costs of raw materials for mass production of RNA.

[0018] In a first aspect of the invention there is provided a method of producing RNA using recycled components for in vitro transcription (IVT), the method comprising;

[0019] (a) producing RNA in a first composition comprising components for IVT;

[0020] (b) separating the RNA from the first composition to provide a second composition comprising unconsumed components for IVT;

[0021] (c) collecting the second composition; and (d) producing RNA in the second composition.

[0022] In certain embodiments, the method further comprises repeating at least once the steps of :

[0023] (e) separating the RNA from the second composition to provide a further composition comprising unconsumed components for IVT;

[0024] (f) collecting the further composition; and

[0025] (g) producing RNA in the further composition.

[0026] In certain embodiments, the method consists of: a) producing RNA in a first composition comprising components for IVT;

[0027] (b) separating the RNA from the first composition to provide a second composition comprising unconsumed components for IVT;

[0028] (c) collecting the second composition;

[0029] (d) producing RNA in the second composition;

[0030] (e) separating the RNA from the second composition to provide a further composition comprising unconsumed components for IVT;

[0031] (f) collecting the further composition; and

[0032] (g) producing RNA in the further composition.

[0033] In certain embodiments, steps (a) to (g) are repeated. In some examples, steps (a) to (g) are continuously repeated. For example, production of RNA by an IVT reaction using recycled components, and the collection of the produced RNA and recycled components is continuous.

[0034] In certain embodiments, producing RNA is an IVT reaction. For example, the step of producing may be carrying out an IVT reaction to produce an RNA product.

[0035] In certain embodiments, the method does not include any additional steps.

[0036] In certain embodiments, the method does not require dilution of the second and / or further compositions.

[0037] In certain embodiments, the first composition comprise one or more conducting agents at a total conducting agent concentration of at most 250mM. In certain embodiments, the first composition comprise a conducting agent at concentration of at most 250mM. In certain embodiments, the first composition comprises one or more conducting agents at a total concentration of conducting agents of at most 250mM. In certain embodiments, the conducting agent comprises a chaotropic agent. In certain embodiments, the conducting agent comprises a kosmotropic agent. In certain embodiments, the conducting agent comprises a kosmotropic anion and chaotropic cation. In certain embodiments, the conducting agent comprises a chaotropic anion and kosmotropic cation.

[0038] In certain embodiments, the conducting agent is selected from one or more of ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate, potassium sorbate, sodium sorbate, sodium benzoate, potassium benzoate, potassium sulphite, sodium sulphite see, sodium bisulsulphite, sodium metabisulphite, potassium metabisulphite, potassium sulphite, sodium formate, potassium nitrite, potassium ascorbate, sodium ascorbate, potassium lactate, sodium lactate, sodium citrates, potassium citrates, sodium tartrates, potassium tartrates, potassium malate, sodium malates, sodium fumarate, potassium fumarate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, triammonium citrate, ammonium citrate, sodium gluconate, potassium gluconate, disodium guanylate, dipotassium guanylate, NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate and / or Tween-20.

[0039] In certain embodiments, the conducting agent is selected from one or more of ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate, , NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate and / or Tween-20.

[0040] In certain embodiments, the components for IVT comprise: ribonucleotide triphosphates (rNTPs); a transcription template; an RNA polymerase; a buffer solution; a pyrophosphatase; an RNAase inhibitor; a conducting agent (e.g. a chaotropic agent); a polyamine; and a source of magnesium ions.

[0041] In certain embodiments, the components for IVT comprise in the first composition (i.e. initial reaction mix): ribonucleotide triphosphates (rNTPs) at a concentration of about 0.5 to 10mM for each ribonucleotide; a transcription template at a concentration of about 1 to 250 nm or about 0.025 to 2mg / ml; an RNA polymerase at a concentration of about 1 to 1000 nM or about 1 to 1000 ll / g of transcription template; a buffer solution at a concentration of about 10 to 200 mM; a pyrophosphatase at a concentration of about 1 to 100 units / ml or 0.5 to 6% v / v of the total reaction volume; an RNAase inhibitor at a concentration of 0.5 to 2.5% v / v of the total reaction volume; a conducting agent (e.g. a chaotropic agent) at a concentration of at least 50 mM; a polyamine at a concentration of about 0.5 to 25 mM; and a source of magnesium ions at a concentration of about 8 to 80mM.

[0042] In certain embodiments, the components for IVT comprise in the first composition (i.e. initial reaction mix): ribonucleotide triphosphates (rNTPs) at a concentration of about 0.5 to 10mM for each ribonucleotide; a transcription template at a concentration of about 1 to 250 nm or about 0.025 to 2mg / ml; an RNA polymerase at a concentration of about 1 to 1000 nM or about 1 to 1000 ll / g of transcription template; a buffer solution at a concentration of about 10 to 200 mM; a pyrophosphatase at a concentration of about 1 to 100 units / ml or 0.5 to 6% v / v of the total reaction volume; an RNAase inhibitor at a concentration of 0.5 to 2.5% v / v of the total reaction volume; a conducting agent (e.g. a chaotropic agent) at a concentration of at least 50 mM; a polyamine at a concentration of about 05 to 25 mM; a source of magnesium ions at a concentration of about 8 to 80mM; and a cap analogue at a concentration of about 1 to 20 mM.

[0043] In certain embodiments, the first composition comprises a conducting agent (e.g. a chaotropic agent) concentration of 50 mM or more. In certain embodiments, a conducting agent (e.g. a chaotropic agent) concentration of about 50mM to about 150mM. In certain embodiments, the first composition comprises a conducting agent (e.g. a chaotropic agent) concentration of 100mM. In certain embodiments, the first composition comprises a conducting agent (e.g. a chaotropic agent) concentration of 150mM. In certain embodiments, the first composition comprises a conducting agent (e.g. a chaotropic agent) concentration of at most 250mM.

[0044] In certain embodiments, the second and / or further composition comprises a conducting agent (e.g. a chaotropic agent) concentration of 50 mM or more. In certain embodiments, a conducting agent (e.g. a chaotropic agent) concentration of about 50mM to about 150mM. In certain embodiments, the second and / or further composition comprises a conducting agent (e.g. a chaotropic agent) concentration of 100mM. In certain embodiments, the second and / or further composition comprises a conducting agent (e.g. a chaotropic agent) concentration of 150mM. In certain embodiments, the second and / or further composition comprises a conducting agent (e.g. a chaotropic agent) concentration of at most 250 mM.

[0045] In certain embodiments, the separating is carried out at a conducting agent (e.g. a chaotropic agent) concentration of less than 500mM. In certain embodiments, a conducting agent (e.g. a chaotropic agent) concentration of about 100 mM to about 200 mM. In certain embodiments, a conducting agent (e.g. a chaotropic agent) concentration of at most 250 mM.

[0046] In certain embodiments, the method is carried out at a conducting agent (e.g. a chaotropic agent) concentration from about 50 to 200 mM. In certain embodiments, the method is carried out at a conducting agent (e.g. a chaotropic agent) concentration of about 100mM. In certain embodiments, the method is carried out at a conducting agent (e.g. a chaotropic agent) concentration of about 150mM. In certain embodiments, the method is carried out at a conducting agent (e.g. a chaotropic agent) concentration of at most 250mM.

[0047] In certain embodiments, the producing and separating are carried out at the same conducting agent (e.g. a chaotropic agent) concentration.

[0048] In certain embodiments, the method further comprises adding one or more conducting agents (e.g. a chaotropic agent) and / or magnesium ions to the first composition and / or second composition before the separating.

[0049] In certain embodiments, the method does not include buffer exchange. In certain embodiments, the method does not comprise adding extrinsic conducting agent (e.g. a chaotropic agent)s to or removing conducting agent (e.g. a chaotropic agent)s from the second and / or further composition.

[0050] In certain embodiments, the separating comprises affinity separation and / or phase separation. In certain embodiments, the separating comprises affinity separation such as oligo-dT separation. In certain embodiments, the separating comprises phase separation. In certain embodiments, the separating comprises the method comprises phase and oligo-dT separation.

[0051] In certain embodiments, separating comprises bead based separation or column based separation. In certain embodiments, separating comprises monolith based separation. In certain embodiments, separating comprises membrane based separation.

[0052] In certain embodiments, the affinity separation comprises affinity chromatography.

[0053] In certain embodiments, the separating comprises oligo-dT or oligo-dll separation.

[0054] In certain embodiments, the phase separation comprises temperature induced phase separation.

[0055] In certain embodiments, temperature induced phase separation comprises cooling the first, and / or further composition after production of RNA thereby forming a first phase (upper phase) comprising the unconsumed components for IVT and a second phase (lower phase) comprising the produced RNA; optionally wherein the produced RNA precipitates in the second phase.

[0056] In certain embodiments, cooling comprises freezing the first and / or further composition after production of RNA and thawing the first, and / or further composition before collecting.

[0057] In certain embodiments, the phase separation further comprises centrifugation. In certain embodiments, the separating comprises or further comprises: a) cooling the first and / or further composition after production of RNA; b) optionally centrifuging the cooled first and / or further composition to form the first phase comprising unconsumed components for IVT and the second phase comprising the produced RNA.

[0058] In certain embodiments, the separating comprises or further comprises: a) separating the RNA from the first and / or further composition to form a second composition or further second composition by affinity separation after production of RNA; b) cooling the second and / or further second composition; and

[0059] Optionally the separating includes centrifuging the cooled second and / or further second composition to form the first phase comprising unconsumed components for IVT and the second phase comprising the produced RNA.

[0060] In certain embodiments, the phase separation comprises cooling at rate of 1 Kelvin per minute.

[0061] In certain embodiments, the centrifugation is carried out a temperature of 0°C to 8°C.

[0062] In certain embodiments, the conducting agent is selected from one or more of ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate, potassium sorbate, sodium sorbate, sodium benzoate, potassium benzoate, potassium sulphite, sodium sulphite see, sodium bisulsulphite, sodium metabisulphite, potassium metabisulphite, potassium sulphite, sodium formate, potassium nitrite, potassium ascorbate, sodium ascorbate, potassium lactate, sodium lactate, sodium citrates, potassium citrates, sodium tartrates, potassium tartrates, potassium malate, sodium malates, sodium fumarate, potassium fumarate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, triammonium citrate, ammonium citrate, sodium gluconate, potassium gluconate, disodium guanylate, dipotassium guanylate, NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate and / or Tween-20. In certain embodiments, the conducting agent comprises one or more of NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, and / or Tween-20. In certain embodiments, the conducting agent comprises one or more of NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, and / or Tween-20. In certain embodiments, the conducting agent comprises one or more of potassium glutamate, NaCI, potassium acetate, and / or Guanidine HCI. In certain embodiments, the conducting agent comprises NaCI. In certain embodiments, the conducting agent comprises Guanidine HCI. In certain embodiments, the conducting agent comprises potassium acetate. In certain embodiments, the conducting agent comprises potassium glutamate. In certain embodiments, the conducting agent comprises a combination of two or more of potassium glutamate, NaCI, potassium acetate, and / or Guanidine HCI.

[0063] In certain embodiments, the method further comprises collecting the RNA in a third composition.

[0064] In certain embodiments, the components for IVT further comprise one or more of: a reducing agent, optionally wherein the reducing agent is DTT; a surfactant; an amine; and / or DMSO.

[0065] In certain embodiments, the components for IVT further comprise an RNA capping agent.

[0066] In certain embodiments, the rNTPs comprise one or more modified rNTPs.

[0067] In certain embodiments, the RNA polymerase comprises a modified RNA polymerase.

[0068] In certain embodiments, the first composition comprises the components for IVT in excess.

[0069] In certain embodiments, the method further comprises adding one or more of a source of magnesium ions, ribonucleotide triphosphates, a pyrophosphatase and / or an RNA capping agent to the second and / or further composition prior to step (d) and / or step (g).

[0070] In certain embodiments, the method is a batch method, a batch fed method or a continuous method.

[0071] In certain embodiments, the method reduces a level of dsRNA produced during the step of producing.

[0072] In certain embodiments, the transcription template and / or RNA polymerase are immobilised and the second and / or further composition are contacted with the immobilised transcription template and / or RNA polymerase. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0073] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0074] The methods described herein can be used to rapidly develop and mass-manufacture RNA- based (candidate) vaccines and therapeutics against multiple diseases, including infectious diseases and cancer

[0075] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0076] Various aspects of the invention are described in further detail below.

[0077] Brief description of the Figures

[0078] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0079] Figure 1 shows the efficient use of IVT reagents by recycling unconsumed reaction components in consecutive IVT reactions. The invention couples IVT with separation methods such as affinity chromatography for extraction of RNA products and recycling the flowthrough of the separation in consecutive IVT reactions. The methods of the invention couple IVT with affinity chromatography without any intermediary purification step by utilising a reaction buffer compatible with IVT and affinity chromatography.

[0080] Figure 2 shows the efficient use of IVT reagents by recycling unconsumed IVT reaction reagents in consecutive batches. The reaction titer (first composition after production of RNA) is subjected to freezing or a combination of freezing and centrifugation to achieve a phase separation. The upper phase contains unconsumed reaction components and is used for consecutive IVT reactions.

[0081] Figure 3 shows effect of chaotropic salts (a. NaCI and / or Gu-HCI; b. Urea) on IVT mRNA yield.

[0082] Figure 4 shows total RNA yield in fractions of the recycling process (IVT= In-vitro transcription; FT= Flow through , E= Elution). Figure 5 shows electropherogram of purified RNA from initial IVT ( A.) and recycling IVT ( IVT2) process. All fractions of the recycling process showed an integrity of over 90%.

[0083] Figure 6 shows a conceptual diagram of the methods described herein.

[0084] Figure 7 shows an example recycling experimental set-up. Flow through (FT) from the oligo- dT column (cycle n) was collected and used in subsequent IVT (cycle n+1) replenished with components consumed / lost during the original IVT-Oligo-dT process. Recycling experiment were performed with eGFP template. Each cycle is defined by one IVT followed by Oligo-dT purification and five cycles were performed.

[0085] Figure 8 shows (A) Amount of mRNA post-IVT (input) and in elution after oligo-dT purification (output). (B) Integrity of mRNA. (C) Total mRNA yield and integrity determined post-IVT and post chromatography purification. Five IVT- oligo-dT purification cycles were performed. The mRNA yield (both generated within the IVT and purified after oligo-dT) and integrity were comparable between cycles. Results are plotted for n=3.IVT-OligodT cycles (5 cycles) were performed (n=3) in NaCI-GuHCI containing buffer and the purified mRNA yield determined. IVT volume was 8 mL per IVT

[0086] Figure 9 shows the effect of buffering agents on IVT mRNA yield. IVT was performed in different buffering agents for two templates (enhanced green fluorescent protein (eGFP) and Coronavirus spike protein (CSP); n=2). . The Tris buffering agent at pH 7 was selected for the recycling experiment in Fig 10.

[0087] Figure 10 shows the effect of NaCI and K-acetate on IVT mRNA yield. IVT was performed in 50mM Gu-HCI Tris pH 7 buffer for two templates (eGFP and CSP). The yield was determined at 10 minute intervals during the reaction. IVT were performed with 2 templates in different recycling buffers to determine adequate reaction time for recycling experiments (n=1).

[0088] Figure 11 shows the influence of top-up compositions on second cycle ( IVT2) mRNA yield in different purification column configurations. (A) Membrane configuration. (B) Monolith configuration. (C) Beads configuration . The compositions were: C1= FT+10mM NTP+2mM MgCI2; C2= FT+10mM NTP+4mM MgCI2; C3: FT+10mM NTP+6mM MgCI2; C4= FT+10mM NTP+8mM MgCI2; C5= FT+10mM NTP+10mM MgCI2; C6= C1+27nM template; C7= C2+27nM template; C8= C3+27nM template; C9= C4+27nM template; and C10= C5+27nM template. Recycling experiment were performed with eGFP template. The Oligo-dT purification step was performed using different column configuration and various composition for replenishing the second IVT ( “Top-up”) were tested. This allows determination for each column configuration which component would need to be supplemented. The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0089] Figure 12 shows conducting agents conductivity evaluation. Solutions of the conducting agents ranging in concentrations from 50-200mM were tested to assess their conductivity (conductivity shown is in mS / cm).

[0090] Figure 13 shows classification of buffer components as chaotropic or kosmotropic and relation to Hofmeister series (adapted from https: / / water.lsbu.ac.uk / water / kosmotropes_chaotropes.html).

[0091] Various aspects of the invention are described in further detail below.

[0092] Detailed Description

[0093] Producing RNA

[0094] The methods described herein relate to methods including in vitro transcription (IVT). IVT is a process that permits template-directed (e.g. via an IVT DNA template) synthesis of a ribonucleic acid (RNA) (for example, messenger RNA (mRNA)). It is based, generally, on the engineering of a DNA template that includes a bacteriophage promoter sequence upstream of the sequence of interest, followed by transcription using a corresponding RNA polymerase. In vitro mRNA transcripts, for example, may be used as therapeutics in vivo to direct ribosomes to express protein therapeutics within targeted tissues.

[0095] Methods for RNA in vitro transcription are known in the art (see for example Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101- 14). Reagents used in said methods may include: a linear DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase; ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); DNA- dependent RNA polymerase (e.g., T7, T3 or SP6 RNA polymerase); ribonuclease (RNase) inhibitor to inactivate any contaminating RNase; pyrophosphatase to degrade pyrophosphate, which inhibits transcription; a source of magnesium ions (e.g. MgCh, which supplies Mg2+as a cofactor for the RNA polymerase); polyamines such as spermidine; and a buffer to maintain a suitable pH value. The reaction mix for IVT may optionally include one or more of a cap analogue (if the RNA is co-transcriptionally capped); modified ribonucleotides; reducing agents (e.g. DTT); surfactants (e.g. Tween 20 and / or Triton X100); and / or DMSO.

[0096] All the components for IVT may be included in the reaction mix in excess. In excess refers to an amount of each component that is greater than required to carry out the IVT reaction.

[0097] When components are in excess the concentration of components does not lead to limitations on the IVT reaction rate as well as limitations in terms of RNA production yield, time and / or efficiency.

[0098] Capping Agents and Enzymes

[0099] IVT is performed by linearizing a transcription template (e.g. plasmid DNA templates or PCR templates) requiring at least a promoter and the corresponding RNA construct sequence.

[0100] IVT may be carried out by adding polymerases (e.g. T7, T3, or SP6) but requires additional capping. Uncapped RNA is rapidly degraded by RNase and contains a 5'-ppp group, which causes greater immune stimulation and can be treated with phosphatase to reduce undesirable efficacy. Two methods may be implemented for the capping of IVT RNA: Co- transcriptional capping and post-transcriptional capping.

[0101] During co-transcriptional capping, cap dinucleotide mixtures containing four nucleoside triphosphates (NTPs) are incorporated at the 5' end of the RNA with RNA polymerase. Co- transcriptional capping processing permits coordinated transcription with mRNA capping.

[0102] A 5' cap is typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an RNA molecule. Preferably, the 5' cap is added using a 5'-5'-triphosphate linkage. A 5' cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5'-end of an RNA. The naturally occurring 5' cap is m7GpppN.

[0103] Further examples of 5'cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4 '-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alphanucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'- 3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. A 5' cap structure may be formed by a cap analog. A cap analogue refers to a non- extendable di-nucleotide that has cap functionality which means that it facilitates translation or localization, and / or prevents degradation of the RNA molecule when incorporated at the 5' end of the RNA molecule. Non-extendable means that the cap analogue will be incorporated only at the 5'terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.

[0104] Cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or anti reverse cap analogues (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10):1486- 95).

[0105] The capping agent may be a co-transcriptional capping agent. In some examples, the capping agent may be a cap analogue. In some examples, cap analogue may be added to the initial reaction mix (i.e. first composition). The cap analogue may be added to the initial reaction mix (i.e. first composition) in the range of about 1 to 20 mM, 1 to 17.5 mM, 1 to 15 mM, 1 to 12.5 mM, 1 to 10 mM, 1 to 7.5 mM, 1 to 5 mM or 1 to 2.5 mM. In some examples, the cap analogue may be added to the initial reaction mix (i.e. first composition) in the range of about 1 to 10 mM. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM.

[0106] As capping agents for co-transcriptional capping (such as cap analogues) are at least partially consumed during IVT due to incorporation into the produced RNA, after each round of IVT, the capping agent may be topped up to the initial reaction mix concentration by addition of capping agent to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions. The amount of capping agent required is reduced using methods of the invention due to recovery or recycling of the unconsumed capping agent which is often included in the initial reaction mix in excess.

[0107] There are three cap structures in total: 0, 1 , and 2. Cap-0 is essential for efficient translation of the mRNA that carries the cap, while cap-1 is important in evading the cellular innate immune response in vivo. In humans, cap-0 and cap-1 methylations are present on all mRNA molecules, while about half of the capped poly(A) molecules contain a 2'-O-ribose methylated residue on the second transcribed nucleotide. This sequence, also called cap-2, is required alongside cap-1 for spliceosomal E-complex formation and, consequently, for efficient pre-mRNA splicing. Post-translational capping involves enzyme-based capping following the transcription reaction. The enzymes used in vitro originate from capping apparatuses of different eukaryotic organisms or viruses and can be produced recombinantly in E. coli. Enzymatic formation of cap-0 comprises three consecutive reactions targeted to nascent 5'- triphosphorylated pre-mRNAs. First, a 5'-triphosphatase (TPase) hydrolyzes the y- phosphate of pre-mRNA. Next, the p-phosphate of the resulting 5'-diphosphate end is coupled to GMP to form 5'-5'-linked Gppp-RNA. Finally, the cap structure is methylated at the N7-position by an RNA (guanine-N7) methyltransferase using S-adenosyl-L-methionine (SAM) as a co-substrate. In nature, the capping enzymes used in post-translational capping act co-transcriptionally once the transcript has reached a length of 20-30 nucleotides. These enzymes can be harnessed to produce capped RNA in vitro by adding them and their respective co-substrates to the IVT reaction. It is important to note that IVT reactions need to be purified to remove unused nucleoside triphosphates (NTPs) for enzymatic capping to work.

[0108] The term “capping enzyme” refers to any enzyme able to add a m7GpppG cap at 5'-end of mRNA and / or to modify the ultimate or penultimate bases of a RNA sequence, including cap-0 canonical or non-canonical capping enzymes and cap-1 or cap-2 nucleoside 2' methyltransferases, N6-methyl-adenosine transferase. As used herein, the term “cap-0 canonical capping enzymes” refers to enzymes able to add cap-0 structure at the 5'end of RNA molecules by involving a series of three enzymatic reactions: RNA triphosphatase (RTPase) that removes the y phosphate residue of 5' triphosphate end of nascent pre- mRNA to diphosphate ppRNA, RNA guanylyltransferase (GTase) that transfers GMP from GTP to the diphosphate ppRNA nascent RNA terminus, and RNA N7-guanine methyltransferase (N7-MTase) that adds a methyl residue on nitrogen 7 of guanine to the GpppRNA cap (Furuichi and Shatkin 2000).

[0109] The enzymatic domains of eukaryotic organisms and viruses, which are involved in the canonical formation of cap-0 structure, can be assembled in a variable number of protein subunits.

[0110] Single subunit capping enzymes with all three critical enzymatic domains, i.e. RTPase, GTase and N7-MTase. These enzymes include, but are not limited to: (I) Acanthamoeba polyphaga mimivirus capping enzyme R382 (Raoult, Audio et al. 2004, Benarroch, Smith et al. 2008) (NCBI APMV genomic sequence NC_006450; UniProtKB / Swiss-Prot accession number Q5LIQX1), (ii) ORF3 capping enzyme from yeast Kluyveromyces lactis linear extra- chromosomal episome pGKL2 (Tommasino, Ricci et al. 1988, Tiggemann, Jeske et al. 2001) (NCBI Kluyveromyces lactis CB 2359 pGKL2 genomic sequence NC_010187; UniProtKB / Swiss-Prot accession number P05469), (iii) African swine fever virus NP868R capping enzyme (Pena, Yanez et al. 1993, Jais 2011, Dixon, Chapman et al. 2013, Jais, Decroly et al. 2018) (NCBI ASFV genomic sequence strain BA71V NC_001659; UniProtKB / Swiss-Prot accession number P32094), VP4 Bluetongue virus capping enzyme (NCBI BTV serotype 10 genomic sequence Y00421; UniProtKB / Swiss-Prot accession number P07132).

[0111] Capping enzymes having two subunits include, but are not limited to: (i) the mammalian capping enzymes that consists of the RNGTT subunit having both RTPase and GTase enzymatic activities (Yue, Maldonado et al. 1997, Pillutla, Yue et al. 1998, Tsukamoto, Shibagaki et al. 1998, Yamada-Okabe, Doi et al. 1998) (also named HCE1; human and mouse UniProtKB / Swiss-Prot accession number 060942 and 055236, respectively) and RNMT having N7-MTase enzymatic activity (Pillutla, Yue et al. 1998, Tsukamoto, Shibagaki et al. 1998) (human and mouse UniProtKB / Swiss-Prot accession number Q05D80 and D3YYS7, respectively), (ii) the vaccinia capping enzyme that consists of the D1R gene product having RTPase, GTase and N7-MTase enzymatic domains (Cong and Shuman

[0112] 1993, Niles and Christen 1993, Mao and Shuman 1994, Cong and Shuman 1995, Mao and Shuman 1996, Myette and Niles 1996, Yu and Shuman 1996, Yu, Martins et al. 1997, Gong and Shuman 2003) (genomic sequence strain Western Reserve NC_006998.1; UniProtKB / Swiss-Prot accession number P04298) and D12L gene product that has no intrinsic enzymatic activity but enhances drastically the RNA N7-MTase activity of the D1 R subunit (Higman, Bourgeois et al. 1992, Higman, Christen et al. 1994, Mao and Shuman

[0113] 1994, Schwer, Hausmann et al. 2006, De la Pena, Kyrieleis et al. 2007) (genomic sequence strain Western Reserve NC_006998.1 ; Gene 3707515; UniProtKB / Swiss-Prot accession number P04318).

[0114] Capping enzymes that have three subunits, such Saccharomyces cerevisiae CET 1 with RTPase (Tsukamoto, Shibagaki et al. 1997, Gu, Rajashankar et al. 2010) (UniProtKB / Swiss- Prot accession number 013297), CEG1 with GTase (Shibagaki, Itoh et al. 1992, Yamada- Okabe, Doi et al. 1998, Gu, Rajashankar et al. 2010) (UniProtKB / Swiss-Prot accession number Q01159), and ABD1 having N7-MTase catalytic activities (Mao, Schwer et al. 1995, Schwer, Saha et al. 2000) (UniProtKB / Swiss-Prot accession number P32783). As used herein, the term “cap-0 non canonical capping enzymes” refers to enzymes able to add a cap-0 structure at the 5' end of RNA molecules but in a pathway which differs from the canonical enzymatic process. These include the m7GTP RNA capping pathway of various ss(+)RNA viruses of the alphavirus (e.g. Semliki Forest virus and Sindbis virus), potexvirus (e.g. Bamboo mosaic virus), tobamovirus (e.g. Tobacco mosaic virus), Togaviridae (e.g. Rubella virus and Chikungunya virus) and Hepeviridae (e.g. Hepatitis E virus) families (Decroly, Ferron et al. 2011). This RNA capping pathway relies on three sequential enzymatic reactions: (a) RTPase similar to the conventional pathway (for example, nsP2 protein of Semliki Forest virus resulting from the apparent cleavage of the non-structural P123 polyprotein; UniProtKB / Swiss-Prot accession number P08411), hydrolyzes the y-P bond at the 5'-end of the RNA, (b) methylation of GTP molecule by an atypical N7-MTase (for example, nsP1 protein of Semliki Forest virus also resulting from the apparent cleavage of the non-structural P123 polyprotein for example; UniProtKB / Swiss-Prot accession number P08411), (c) m7GTP is then recognized as a substrate by an atypical GTase (also nsP1 of protein of Semliki Forest virus for example) and transferred onto the 5'-end of the acceptor ppRNA, to yield a typical m7GpppN cap-0 structure (Decroly, Ferron et al. 2011). These three enzymatic activities, in addition to a RNA-dependent RNA polymerase catalytic domain, can be found in a single viral protein, i.e. the Bamboo Mosaic Virus mRNA capping enzyme ORF1 (Li, Shih et al. 2001, Huang, Han et al. 2004, Huang, Hsu et al. 2005, Han, Tsai et al. 2007) (NCBI BMV isolate BaMV-0 genomic sequence NC_001642;

[0115] UniProtKB / Swiss-Prot accession number Q65005). The GDP RNA capping pathway of many ss(-)RNA viruses of the Rhabdoviridae (e.g. vesicular stomatitis virus and Rabies virus), paramyxoviridae (e.g. human respiratory syncytial virus and Measles virus), Bornaviridae (e.g. bornavirus), and Filoviridae (e.g. Ebola virus and Marburg virus) families (Decroly, Ferron et al. 2011), which catalyzes the formation of a cap-0 / cap-1 structure in four enzymatic steps. For instance, the single subunit large L protein from the human respiratory syncytial virus (UniProtKB / Swiss-Prot accession number P28887) can complete these four enzymatic steps by itself, in addition of having an RNA dependent RNA polymerase activity: (a) the NTPase activity is responsible for the hydrolysis of a GTP into a GDP, (b) the L protein hydrolyzes the a-p bond of the pppRNA triphosphate moiety, thereby releasing pyrophosphate and creating a covalent enzyme-pRNA intermediate (i.e. RNA with monophosphate 5'-end), (c) the pRNA moiety is then transferred onto the GDP to form a GpppN block RNA. In this case, only the a-phosphate originates from the RNA whereas both the and y-phosphates are contributed by the GDP, (d) finally, synthesis of the cap-0 then cap-1 structures is completed by two successive methylations at m7pppN and 2'-residue on the first transcribed nucleotide, respectively (Grdzelishvili, Smallwood et al. 2005, Li, Fontaine-Rodriguez et al. 2005, Grdzelishvili, Smallwood et al. 2006, Ogino and Banerjee 2007, Li, Rahmeh et al. 2008, Ogino and Banerjee 2008, Rahmeh, Li et al. 2009).

[0116] RNA cap snatching, which is a process by which some viruses unable to synthesize their own cap structures, acquire capping by stealing it from host mRNA. Viruses belonging to this class include representatives of the Orthomyxoviridae (e.g. Influenza virus, Thogoto virus), Arenaviridae (e.g. Lassa virus, Machupo virus) and Bunyaviridae families (e.g. Hantaan virus, La Crosse virus, Tomato Spotted Wilt virus) (Decroly, Ferron et al. 2011). To acquire their cap structure, nucleotide sequence between 10 and 20 nucleotides in size is cleaved from the 5' end of host capped mRNAs by an endonuclease activity encompassed within the viral RNA dependent RNA polymerase and transferred to the viral genomic RNA. The capped leader obtained is subsequently used to prime transcription on the viral genomic RNA, which ultimately leads to the synthesis of capped, translatable viral mRNAs. The Arenaviridae and Bunyaviridae express a large monomeric polymerase to ensure cap snatching. Orthomyxoviridae influenza virus have heterotrimeric polymerase, consisting of PB1 (UniProtKB / Swiss-Prot accession number strain A / Puerto Rico / 8 / 1934 H1 N1 P03431), PB2 (UniProtKB / Swiss-Prot accession number strain A / Puerto Rico / 8 / 1934 H1 N1 P03428) and PA (UniProtKB / Swiss-Prot accession number strain A / Puerto Rico / 8 / 1934 H1 N1 P03433). All these three subunits are required for endonuclease activity but the enzymatic activity is thought to reside in the amino-terminal domain of the PA subunit (Ohlmann, Rau et al. 1995).

[0117] As used herein, the term “cap-1 capping enzymes” refers to enzymes able to add cap-1 structure at the 5'end of RNA molecules. As used herein, the term “cap-2 capping enzymes” refers to enzymes able to add cap-2 structure at the 5'end of RNA molecules.

[0118] In mammalians, cap-1 and cap-2 modifications are performed by two ribose-2'-O methyltransferases, (also named nucleoside-2'-methyltransferase or 2'-O-MTases) (Belanger, Stepinski et al. 2010). Firstly, MTR1 (cap-1 ribose-2'-O MTase activity, also named FTSJD2, KIAA0082 or ISG95; UniProtKB / Swiss-Prot accession number Q8N1G2), which is exclusively found in the nucleus and contains a putative nuclear localization signal and a G-patch domain that is potentially involved in RNA binding (Haline-Vaz, Silva et al. 2008). Noticeably, MRT1 associates with the CTD of RNA polymerase II, which indicate that cap-1 formation occurs early in the synthesis of mRNA (Langberg and Moss 1981). Secondly, MTR2 (cap 2 ribose-2'-O MTase, also named FTSJD1 or FLJ11171; UniProtKB / Swiss-Prot accession number Q8IYT2) transfers a methyl group from S- adenosylmethionine to the 2'-O-ribose of the second nucleotide of mRNA and small nuclear RNA. Nor N7methylation of the guanosine cap-0 or cap-1 modification is required for MTR2, but the presence of cap-1 increases MTR2 activity. The MTR2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear MTR1 (Keith, Ensinger et al. 1978).

[0119] Some eukaryotic viruses have their own cap-1 and / or cap-2 2'-O-MTases, including VP39 from the vaccinia virus (NCBI genomic sequence NC_006998.1 ; UniProtKB / Swiss-Prot accession number YP_232977) that has both cap-1 and cap-2 2'-O-MTase enzymatic activities (Schnierle, Gershon et al. 1994, Shi, Yao et al. 1996, Hu, Gershon et al. 1999), Orf69 from the Autographa californica Nucleopolyhedrovirus (NCBI genomic sequence NC_001623.1; UniProtKB / Swiss-Prot accession number P41469) (Wu and Guarino 2003), nsp16 from coronavirus (residues 6776-7073 of the polyprotein 1 ab of the human SARS coronavirus NCBI genomic sequence NC_004718.3; UniProtKB / Swiss-Prot accession number POC6X7) (Reinisch, Nibert et al. 2000, Decroly, Imbert et al. 2008, Chen, Cai et al. 2009, Lugari, Betzi et al. 2010), A2 protein from Reovirus (e.g. mammalian orthoreovirus type 3, strain Dearing; NCBI genomic sequence J03488; UniProtKB / Swiss-Prot accession number P11079), which has 2'-O-MTase in addition to GTase and N7-MTase enzymatic activities (Bujnicki and Rychlewski 2001), VP4 from the bluetongue virus (NCBI BTV serotype 10 genomic sequence ID Y00421 ; UniProtKB / Swiss-Prot accession number P07132), NS5 from the flaviviruses that include dengue virus yellow fever virus, Zika virus, West Nile virus, Meaban virus, Yokose virus, St. Louis encephalitis virus, Japanese encephalitis virus, tick-borne encephalitis virus (e.g. polyprotein from Dengue virus type 1 strain Nauru / West Pac / 1974; NCBI genomic sequence U88535; UniProtKB / Swiss-Prot accession number P17763) can also methylate internal adenosine residues of mRNA (Dong, Chang et al. 2012).

[0120] As capping agents for co-transcriptional capping (such as cap analogues) are at least partially consumed during IVT, after each round of IVT, the capping agents may be topped up to the initial reaction mix concentration by addition of capping agents to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions. The amount of capping agents required to be added for subsequent IVT reactions is reduced using methods of the invention due to recovery or recycling of the unconsumed capping agents which are often included in the initial reaction mix in excess.

[0121] In some example, when using a capping enzyme, the capping enzyme may be immobilised. For example, immobilised on support such as a bead. When the capping enzyme is immobilised, the second and / or further compositions may not need to have capping enzyme topped up (i.e. added) or require less capping enzyme added after subsequent rounds of IVT. Without being bound by theory this may reduce costs by reducing the amounts of capping enzyme used. In such cases, the second and / or further compositions may be collected and applied to the support comprising the immobilised capping enzyme.

[0122] Template

[0123] A “transcription template” as used herein, refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some examples, a transcription template encodes a 5' untranslated region, contains an open reading frame, and encodes a 3' untranslated region and a polyA tail. The particular nucleotide sequence composition and length of a transcription template will depend on the mRNA of interest encoded by the transcription template. IVT mature mRNA preparation includes several steps, transcription template obtainment, IVT, 5' capping, and poly(A) tail adding.

[0124] A “5' untranslated region (UTR)” refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a protein or peptide.

[0125] A “3' untranslated region (UTR)” refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a protein or peptide.

[0126] Poly(A) tails of IVT mRNAs are normally encoded in the transcription template or attached to IVT mRNA by enzymatic polyadenylation. The former may have more precise control of the length of the poly(A) tail. IVT mRNAs are mixed with RNA polymerase and transcription templates after synthesis; thus, purification of IVT mRNA may be needed, including removing immunostimulatory contaminants, free ribonucleotides, short mRNA and transcription template. Generally, Dnase is used to degrade excess transcription template.

[0127] A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some examples, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo, etc.), the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation. However, in some examples, mRNA molecules provided herein do not comprise a polyA tail (such molecules are referred to as “tailless”).

[0128] The concentration of the transcription template in the initial reaction mix (i.e. first composition) may be in a range from about 1 to 250 nM, 1 to 200 nM, 1 to 150 nM, 1 to 100 nM,1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. In some examples, the transcription template in the initial reaction mix may be in a range from about 25 to 250 nM. In some examples, the transcription template in the initial reaction mix may be in a range from about 50 to 250 nM. In some examples, the transcription template in the initial reaction mix may be at least 25 mM. For example, the transcription template may be at a concentration of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 nM. In some examples, the concentration of the transcription template is at least 0.025 mg / ml. In some examples, the concentration of the transcription template is preferably from about 0.025 to 0.2 mg / ml. For example 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.105, 0.11 , 0.115, 0.12, 0.125, 0.13, 0.135, 0.14, 0.145, 0.15, 0.155, 0.16, 0.165, 0.17, 0.175, 0.18, 0.185, 0.19, 0.195, or 0.2 mg / ml. More preferably, the concentration of the transcription template is from about 0.025 to 0.1 mg / ml. For example, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031 , 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.05, 0.051 , 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.06, 0.061 , 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.07, 0.071 , 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.08, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.09, 0.091 , 0.092, 0.093, 0.094, 0.095, 0.096, 0.097, 0.098, 0.099, or 0.1 mg / ml.

[0129] As described herein, after each round of IVT, the transcription template may be topped up to the initial reaction mix concentration by addition of transcription template to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions. In some example, the transcription template may be immobilised. For example, immobilised on support such as a bead. When the transcription template is immobilised, the second and / or further compositions may not need to have transcription template topped up (i.e. added) or require less transcription template added after subsequent rounds of IVT. Without being bound by theory this may reduce costs by reducing the amounts of transcription template used. In such cases, the second and / or further compositions may be collected and applied to the support comprising the immobilised transcription template.

[0130] RNA Polymerases

[0131] The RNA polymerase may be selected from T3, T7 and SP6 RNA polymerase. The concentration of the RNA polymerase in the initial reaction mix (i.e. first composition) may be from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. The person skilled in the art will understand that the choice of the RNA polymerase concentration is influenced by the concentration of the transcription template. For example, the concentration of the RNA polymerase is between 1 and 1000 ll / g transcription template, preferably between 10 and 100 ll / g transcription template. In some examples, the concentration of RNA polymerase may be at least 500 nM. For example, when the IVT reaction comprises conducting agent s as described herein at a greater than standard concentration (standard being less than 50mM conducting agent ) the concentration of RNA polymerase may be at least 500 nM. In some examples, the concentration of RNA polymerase may be from about 500 nM to about 1000 nM. For example, the concentration of RNA polymerase may be 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nM. In some examples, the concentration of RNA polymerase may be greater than 1000 nM.

[0132] The RNA polymerase in the initial reaction mix (i.e. first composition) may be at a concentration of about 2.5 to 10% v / v of the total reaction volume. For example, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% v / v of the total reaction volume. In some examples, the RNA polymerase may be determined by the enzymatic activity of the RNA polymerase. For example, the RNA polymerase may be at a concentration of 50 to 1000 units / pL of the total reaction volume. For example, 50, 60, 70, 80, 90, 100, 110, 120, 130,

[0133] 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,

[0134] 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,

[0135] 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 LI / pL of total reaction volume.

[0136] The RNA polymerase may be a modified RNA polymerase. As used herein “modified RNA polymerase” refers to an RNA polymerase that includes one or more protein modifications such as mutations (e.g. amino acid residue insertions, deletions or substitutions). Modified RNA polymerases may also or alternatively include post-translational modifications such as one or more covalent modifications made to a polypeptide during or after protein synthesis. For example, glycosylation, oxidation, deamidation, isomerization, glycation, hydroxylation, methylation, ubiquitination, pyroglutamic acid, myristoylation and sulfation. For example, the RNA polymerase may be modified to impart salt tolerance, temperature tolerance, higher fidelity or other useful or beneficial properties. Such modified RNA polymerases will be known in the field.

[0137] As described herein, after each round of IVT, the RNA polymerase may be topped up to the initial reaction mix concentration by addition of RNA polymerase to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions. In some example, the RNA polymerase may be immobilised. For example, immobilised on support such as a bead. When the RNA polymerase is immobilised, the second and / or further compositions may not need to have RNA polymerase topped up (i.e. added) or require less RNA polymerase to be added after subsequent rounds of IVT. Without being bound by theory this may reduce costs by reducing the amounts of RNA polymerase used. In such cases, the second and / or further compositions may be collected and applied to the support comprising the immobilised RNA polymerase.

[0138] Pyrophosphatases

[0139] In vitro transcription may be performed in the presence of pyrophosphatase.

[0140] Pyrophosphatases degrade pyrophosphate, which may inhibit transcription. As used herein, the term “pyrophosphatase” refers to a polypeptide having pyrophosphatase activity, i.e. a polypeptide that catalyses the following reaction:

[0141] PPi+H2O^2Pi wherein PPi refers to pyrophosphate and Pi to phosphate. The pyrophosphatase belongs to EC classes 3.6.1.1. In this context, the term “diphosphatase” refers to pyrophosphatase polypeptide which catalyses the hydrolysis of diphosphate to phosphate.

[0142] The concentration of the pyrophosphatase in the initial reaction mix (i.e. first composition) may be from about 1 to 100 units / ml, 1 to 80 units / ml, 1 to 70 units / ml, 1 to 60 units / ml, 1 to 50 units / ml, 1 to 40 units / ml, 1 to 30 units / ml, 1 to 20 units / ml, 1 to 15 units / ml, 1 to 10 units / ml, 1 to 5 units / ml, or 1 to 2.5 units / ml. In some examples, the concentration of the pyrophosphatase in the initial reaction mix may be about 50 units / ml.

[0143] In some examples, the concentration of pyrophosphatase may be determined by the enzymatic activity of the pyrophosphatase. For example, the concentration of pyrophosphatase in the initial reaction mix (i.e. first composition) may be from about 0.5 to about 6% v / v of the total reaction volume. For example, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6% v / v of the total reaction volume.

[0144] As described herein, after each round of IVT, the pyrophosphatase may be topped up to the initial reaction mix concentration by addition of pyrophosphatase to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions.

[0145] In some example, pyrophosphatase may be immobilised. For example, immobilised on support such as a bead. When the pyrophosphatase is immobilised, the second and / or further compositions may not need to have pyrophosphatase topped up (i.e. added) or require less pyrophosphatase added after subsequent rounds of IVT. Without being bound by theory this may reduce costs by reducing the amounts pyrophosphatase used. In such cases, the second and / or further compositions may be collected and applied to the support comprising the immobilised pyrophosphatase. RNase Inhibitors

[0146] Ribonuclease (RNase) inhibitors are recombinant enzymes used to inhibit RNase activity during experiments. RNase inhibitors are commonly used as a precautionary measure in enzymatic manipulations of RNA to inhibit and control for RNases. Small amounts of RNases can co-purify with isolated RNA, leading to compromised experimental results in downstream applications. Such contamination can also be introduced via tips, tubes, and other reagents used in procedures.

[0147] RNase inhibitors include, but are not limited to, 2'-cytidine monophosphate free acid (2 - CMP), aluminon, adenosine 5'-pyrophosphate, 5'-diphosphoadenosine 3'-phosphate (ppA-3 - p), 5'-diphosphoadenosine 2'-phosphate (ppA-2'-p), Leucine, poly-L-aspartic acid, tyrosineglutamic acid polymer, oligovinysulfonic acid, 5'-phopho-2'-deoxyuridine 3'-pyrophosphate P' 5'-esterwith adenosine 3'-phosphate (pdllppAp).

[0148] The concentration of RNase inhibitors in the initial reaction mix (i.e. first composition) may be from about 0.5 to 2.5% v / v of the total reaction volume. For example, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, or 2.5 % v / v of the total reaction volume.

[0149] As described herein, after each round of IVT, the RNase inhibitors may be topped up to the initial reaction mix concentration by addition of RNase inhibitors to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions.

[0150] In some example, RNase inhibitors may be immobilised. For example, immobilised on support such as a bead. When the RNase inhibitors is immobilised, the second and / or further compositions may not need to have RNase inhibitors topped up (i.e. added) or require less RNase inhibitors added after subsequent rounds of IVT. Without being bound by theory this may reduce costs by reducing the amounts RNase inhibitors used. In such cases, the second and / or further compositions may be collected and applied to the support comprising the immobilised RNase inhibitors.

[0151] Polyamines

[0152] Polyamines interact with inhibitory polyanions in the IVT reaction mix and have been shown to enable the RNA polymerase to dissociate from the transcription template and initiate new RNA chain synthesis. The polyamine may one or more of putrescine, spermine or spermidine. Preferably, the polyamine is spermidine. Preferably the concentration of the polyamine in the initial reaction mix (i.e. first composition) is from about 0.5 to 25 mM, 0.5 to 10 mM, 0.5 to 5 mM, or preferably about 0.5 to 2.5 mM. For example, the polyamine concentration may be from about 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, or 2.5 mM.

[0153] As described herein, after each round of IVT, the polyamine may be topped up to the initial reaction mix concentration by addition of polyamine to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions.

[0154] Buffer Solution

[0155] The buffer used for IVT should be capable of maintaining the pH of the reaction and provide a suitable aqueous environment of the enzymatic production of RNA. The buffer may be 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) or tris(hydroxymethyl)aminomethane hydrochloride (Tris HCI). The buffer may be used at a concentration from about 10 to 200 mM, 10 to 100 mM, 10 to 75 mM, 10 to 50 mM, 10 to 40 mM, 10 to 30 mM or 10 to 20 mM. In some examples, the concentration of the buffer may be 40 to 100 mM. In some examples, the concentration of the buffer is 40mM. The pH value of the buffer can be adjusted with, for example, NaOH, KOH or HCI. Preferably the buffer has a pH value from about 6 to 8.5, from about 6.5 to 8.0, from about 7.0 to 7.5, even more preferred about 7.5.

[0156] In some examples, the buffer is HEPES. In some examples, the buffer is HEPES at a pH of 7.4.

[0157] In some examples, the buffer it Tris-HCl. In some examples, the buffer is Tris-HCI at pH 7.

[0158] Other suitable buffers that may be used include those described in W02017109161A1 as well as those commercially available.

[0159] Source Of Magnesium Ions

[0160] Magnesium ions (Mg2+) act as a cofactor in the catalytic centre of the RNA polymerase and are therefore critical for the RNA polymerization reaction. In diffuse binding, fully hydrated Mg ions also interact with the RNA product via nonspecific long-range electrostatic interactions. The magnesium ions may be provided by any suitable source, of which many are known in the field. In some examples, the source of magnesium ions is magnesium acetate and / or magnesium chloride.

[0161] In some examples, the initial reaction mix (i.e. first composition) may include a source of magnesium ions at a concentration from about 8 to 80mM. For example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37,

[0162] 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

[0163] 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 mM.

[0164] As magnesium ions are at least partially consumed during IVT, after each round of IVT, the magnesium ions may be topped up to the initial reaction mix concentration by addition of a source of magnesium ions to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions. The amount of magnesium ions required to be added for subsequent IVT reactions is reduced using methods of the invention due to recovery or recycling of the unconsumed magnesium ions which are often included in the initial reaction mix in excess.

[0165] Ribonucleotides

[0166] The reaction mix includes ribonucleotides. These ribonucleotides may be naturally occurring ribonucleotides and / or non-naturally occurring ribonucleotides (e.g. canonical nucleotides) such as chemically modified nucleotides. In some examples, the modified therapeutic mRNAs provided herein may include at least one chemically modified ribonucleotide. In some examples, the chemically modified ribonucleotide may be selected from the group consisting of pseudouridine, N1 -methylpseudouridine (l-methyl^P), 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methoxyuridine, and 2'-O-methyl uridine.

[0167] The total concentration of ribonucleotides in the initial reaction mix (i.e. first composition) may be from about 0.5 to 10mM for each ribonucleotide. For example, about, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

[0168] 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 ,

[0169] 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,

[0170] 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1 , 9.2, 9.3, 9.4, 9.5,

[0171] 9.6, 9.7, 9.8, 9.9, or 10 mM for each ribonucleotide. As ribonucleotides are at least partially consumed during IVT due to incorporation into the produced RNA, after each round of IVT, the ribonucleotides may be topped up to the initial reaction mix concentration by addition of ribonucleotides to the second composition or further compositions (i.e. composition produced after each round of IVT) before carrying out subsequent IVT reactions. The amount of ribonucleotides required to be added to subsequent IVT reactions is reduced using methods of the invention due to recovery or recycling of the unconsumed ribonucleotides which are often included in the initial reaction mix in excess.

[0172] Optional Components

[0173] The initial reaction mix (i.e. first composition) may further include one or more of a reducing agent, a surfactant, an amine and / or additional additives.

[0174] A “reducing agent” refers to any agent or compound that is an electron donor or that contributes electrons to an atom. Suitable reducing agents include antioxidants and preferably DTT.

[0175] The amount of reducing agent may be from about 1 to 40 mM. For example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mM.

[0176] A “surfactant” to a chemical compound that lowers the interfacial tension between two liquids. Examples of surfactants include Tween, Triton-X, Brij, CHAPS and CHAPSO. Preferably, the surfactant is Tween 20 and / or Triton X100. The amount of surfactant may be about 0.001% v / v of the total reaction volume.

[0177] In some examples, the amine is a betaine. A betaine is a neutral chemical compound with a positively charged cationic functional group such as a quaternary ammonium or phosphonium cation which bears no hydrogen atom and with a negatively charged functional group such as a carboxylate group which may not be adjacent to the cationic site.

[0178] Preferably, the betaine is trimethylglycine, i.e. the amino acid glycine which has three methyl groups bound to the nitrogen atom of the amino acid.

[0179] Other examples of amines that may be used include spermine and spermidine.

[0180] The amount of amine may be from about 2 to 10 mM.

[0181] The reaction mix may also include additional additives such as DMSO. Suitable inclusion of DMSO in IVT reaction mix has been reported to afford an increase in RNA yield and lower 3’-heterogeneity. Continuous and Batch Fed

[0182] IVT reactions are typically performed as batch reactions in which all components are combined and then incubated to allow the synthesis of RNA molecules until the reaction terminates. Fed-batch reactions were developed to increase the efficiency of the RNA in vitro transcription reaction (Kern et al. (1997) Biotechnol. Prog. 13: 747-756; Kern et al. (1999) Biotechnol. Prog. 15: 174-184). In a fed-batch system, all components are combined, but then additional amounts of some of the reagents are added over time (e.g., NTPs, MgCh) to maintain constant reaction conditions.

[0183] IVT reactions can also be carried in continuous reactions. For example, the use of a bioreactor (transcription reactor) for the synthesis of RNA molecules by in vitro transcription has been reported (WO 95 / 08626). The bioreactor is configured such that reactants are delivered via a feed line to the reactor core and RNA products are removed by passing through an ultrafiltration membrane (having a nominal molecular weight cut-off, e.g., 100,000 daltons) to the exit stream.

[0184] The term “continuous” as used herein means a process wherein reactants, such as components for IVT, are introduced and products, such as produced RNA are withdrawn over a period of time during which the reaction (i.e. IVT and separation of produced RNA) continues without significant interruption. “Continuous” is not meant in any way to prohibit normal interruptions in the continuity of the process due to, for example, start-up, reactor maintenance, or scheduled shut down periods. In addition, the term “continuous” may include processes, wherein some of the components for IVT are added at the beginning of the process and the remaining reactants are recycled after separation of the produced RNA into the reactor in which the IVT takes place such that the levels of reactants support continuing reaction processes. “Continuous” further includes processes where one or more reactants are intermittently added. For example, components for IVT such as magnesium ions, ribonucleotide triphosphates, a pyrophosphatase and / or an RNA capping agent as these components may be at least partially consumed by each round of IVT.

[0185] As such, in some examples, the methods described herein may be carried out with or without interruption.

[0186] As described below, the invention allows for the simple and efficient recycling of IVT components while also separating out the RNA produced. The recycling of components therefore, is particularly suited to continuous methods of IVT or batch fed methods, wherein the unconsumed IVT products can be re-introduced into the reaction vessel for subsequent rounds of RNA production. In some examples, there is provided a continuous method of RNA production. This is at least in part due to the methods of the invention not requiring dilution after production (to allow for separation) and / or do not require the produced RNA to be exchanged into a separate and distinct buffer for separation to take place. That is to say that the steps of the method, producing, separating and subsequent production does not require any intervening steps.

[0187] As such, the methods of the invention can be carried out without interruption. For example, the methods of the invention may consist of producing RNA, separating the RNA, collecting the unconsumed components for IVT (i.e. second composition and further composition) and producing RNA in a composition including the recycled unconsumed components for IVT (i.e. in a second or further composition).

[0188] In some examples, the immobilisation of at least some of the components for IVT, such as the transcription template and / or RNA polymerase, may be particularly suited for continuous methods. For example, after an initial IVT reaction, the first composition may have the RNA produced removed, for example by a column based method, where the column is directly connected to the IVT reaction vessel. The flow through of the RNA removal (i.e. second or further compositions), may then be returned to the initial IVT reaction vessel that includes the immobilised transcription template and / or RNA polymerase. For example, by use of pumps or other systems for flowing the second and / or further compositions. Therefore, there may be provided a closed or loop system than allows for continuous return of the components for IVT from each IVT reaction back to vessel including the immobilised transcription template and / or RNA polymerase.

[0189] The methods of the invention may also reduce the amount of dsRNA produced during IVT. Without being bound by theory, this may be in part due to the continuous nature of the methods. dsRNA can form due to increased levels of produced RNA in the IVT reaction mix leading to RNA polymerase utilising the produced RNA as a template. The probability of this occurring increases over time (due to increasing concentrations of RNA in the reaction mix). As the methods described herein reduce the time period between RNA production and separation of the produced RNA, they may reduce the levels of dsRNA produced. For example, the methods of the invention may allow for separation of produced RNA within 2 hours or less of initiating each IVT reaction. In some examples, each IVT reaction is run (i.e. RNA is produced for) at most 2 hours, at most 1.5 hours, or at most 1 hour before RNA separation occurs or RNA production is stopped.

[0190] Reaction conditions

[0191] The reaction conditions for each round of IVT may be selected depending on the RNA polymerase being used and / or the transcription template. In some examples, the IVT reaction is carried out a temperature from about 37°C to 42°C. For example, 37, 38, 39, 40, 41 or 42°C.

[0192] The time of the IVT reaction may also be selected depending on the RNA polymerase being used, the RNA being produced and / or the transcription template. In some examples, each IVT reaction may be carried out for a time period of about 0.16 hours to about 2 hours (where the IVT reaction is defined as the period of time from initiating the production of RNA to the point when production of RNA is stopped or RNA is separated from the composition). For example, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1 , 1.01 , 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1 , 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41 , 1.42,

[0193] 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51 , 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58,

[0194] 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74,

[0195] 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81 , 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9,

[0196] 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 hours.

[0197] RNAs Produced

[0198] The RNAs produced by the methods described herein may be therapeutic RNAs. “Therapeutic RNA” refers to any RNA mediating a change in a physiological and / or metabolic state of a host cell comprising said therapeutic RNA. Therapeutic RNAs include any RNAs that are known to have any therapeutic effect in ameliorating, preventing or treating a disease or condition. For example, therapeutic RNAs may include non-coding RNAs such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs, tsRNA, and long ncRNAs such as Xist and HOTAIR. Therapeutic RNAs may also include coding RNAs, i.e. therapeutic mRNAs.

[0199] “Therapeutic mRNA” refers to an mRNA molecule (e.g., an in vitro transcribed (IVT) mRNA) that encodes a therapeutic protein. Therapeutic proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or ameliorate the signs and symptoms of a disease. For example, a therapeutic protein can replace a protein that is deficient or abnormal, augment the function of an endogenous protein, provide a novel function to a cell (e.g., inhibit or activate an endogenous cellular activity, or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNA may be useful for the treatment or prevention of the following diseases and conditions: infectious diseases (such as bacterial infections, viral infections, parasitic infections), cell proliferation disorders (such as cancer), genetic disorders, inflammatory disease, cardiovascular disorders, metabolic diseases, allergic disease, neurodegenerative diseases, protein or enzyme deficiency disorder and / or autoimmune diseases.

[0200] Examples of therapeutic mRNAs are Pfizer and BioNtech’s BNT162b2 (Covid-19), Moderna’s mRNA-1273 (Covid-19), mRNA-2416 (solid tumour or lymphoma), MRT5005 (cystic fibrosis), mRNA-2752 (solid tumour or lymphoma), AZD-8601 (heart failure), NY- ESO-1 (multiple myeloma, synovial sarcoma, melanoma), CTX001 (P-thalassemia), SB- 728mR-HSPC (HIV ), SB-728mR-T (HIV), BNT163 (HSV2), BNT164 (tuberculosis), BNT165 (malaria), BNT167 (shingles), BNT161 (influenza), BNT153 (undisclosed cancers), BNT152 (undisclosed cancers), BNT142 (undisclosed cancers), BNT141 (undisclosed cancers), BNT131 (undisclosed cancers), BNT122 (melanoma), colorectal cancer, BNT116 (non-small cell lung carcinoma), BNT115 (ovarian cancer), BNT113 (head and neck cancer), BNT112 (prostate cancer), BNT111 (melanoma), mRNA-1345 (Respiratory syncytial virus), mRNA- 1010 (influenza), mRNA-1647 (cytomegalovirus), mRNA-4157 / V940 (melanoma), mRNA- 3927 (Propionic acidemia), mRNA-0184 (heart failure), VX-522 (cystic fibrosis). The uses of each mRNA therapy are shown in brackets.

[0201] The RNAs produced by the methods described herein may comprise naturally occurring ribonucleotides and / or non-naturally occurring ribonucleotides (e.g. canonical nucleotides) such as chemically modified nucleotides. In some examples, the modified therapeutic mRNAs provided herein may include at least one chemically modified ribonucleotide. In some examples, the chemically modified ribonucleotide may be selected from the group consisting of pseudouridine, N1 -methylpseudouridine (l-methyl^P), 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methoxyuridine, and 2'-O-methyl uridine. Other exemplary chemical modifications useful in mRNAs described herein include those listed in US Published patent application 2015 / 0064235 which is incorporated herein.

[0202] Conducting agents The methods described herein may include the use of one or more conducting agents in the IVT reaction composition (e.g. first composition, second composition and / or third composition). Conducting agents may be added to the first composition before carrying out the initial IVT reaction (i.e. before carrying out an initial IVT reaction).

[0203] A conducting agent may be any agent that has an electrolyte conductivity. In some examples, conducting agents are salts. In some examples, conducting agents are ionic salts. In some examples, conducting agents are any compounds or agents that have a conductivity of at least 5 mS / cm at a concentration of 50mM.

[0204] In some examples, the conducting agent may be one or more of a chaotropic agent, a kosmotropic agent, a kosmotropic anion and chaotropic cation, and / or a chaotropic anion and kosmotropic cation.

[0205] In some examples, the conducting agent is selected from one or more of ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate, potassium sorbate, sodium sorbate, sodium benzoate, potassium benzoate, potassium sulphite, sodium sulphite see, sodium bisulsulphite, sodium metabisulphite, potassium metabisulphite, potassium sulphite, sodium formate, potassium nitrite, potassium ascorbate, sodium ascorbate, potassium lactate, sodium lactate, sodium citrates, potassium citrates, sodium tartrates, potassium tartrates, potassium malate, sodium malates, sodium fumarate, potassium fumarate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, triammonium citrate, ammonium citrate, sodium gluconate, potassium gluconate, disodium guanylate, dipotassium guanylate, NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate and / or Tween-20.

[0206] In some examples, the conducting agent is selected from one or more of ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate, , NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, ethanol, sodium perchlorate, n-butanol, 2-propanol, SDS, propylene glycol, sodium carbonate , potassium carbonate , calcium chloride , perchloride compounds, Hepes K-OH, ammonium citrate , sodium citrate, potassium citrate and / or Tween-20.

[0207] In some examples, the conducting agent is selected from one or more of NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, and / or Tween-20. Preferably, the conducting agent is potassium acetate, potassium glutamate, NaCI and / or Guanidine HCI. Preferably, the conducting agent is NaCI and / or Guanidine HCI. In some examples, NaCI is preferred. In some examples, Guanidine HCI is preferred. In other examples, a combination of Guanidine HCI and NaCI is preferred. In some preferred examples, the conducting agent is potassium acetate. In some examples, potassium glutamate, is preferred. In some examples, a combination of potassium glutamate, potassium acetate, Guanidine HCI and NaCI is preferred. In some examples, a combination of NaCI and one other conducting agent is preferred.

[0208] For more details of suitable conducting agents, kosmotropic agents and chaotropic agents see Liu, Lvdan, Ran Kou, and Guangming Liu. "Ion specificities of artificial macromolecules." Soft Matter 13.1 (2017): 68-80, Kunz, Werner. "Specific ion effects in colloidal and biological systems." Current Opinion in Colloid & Interface Science 15.1-2 (2010): 34-39, Zongo, Luc, Heiko Lange, and Claudia Crestini. "A study of the effect of kosmotropic and chaotropic ions on the release characteristics of lignin microcapsules under stimuli-responsive conditions." ACS omega 4.4 (2019): 6979-6993, Gregory, Kasimir P., et al. "Understanding specific ion effects and the Hofmeister series." Physical Chemistry Chemical Physics 24.21 (2022): 12682-12718, Mazzini, Virginia, and Vincent SJ Craig. "Specific-ion effects in non-aqueous systems." Current opinion in colloid & interface science 23 (2016): 82-93, Mazzini, Virginia, and Vincent SJ Craig. "Corrigendum to “Specific-ion Effects in Non-Aqueous Systems”[Curr Opin Colloid Interface Sci 23 (June 2016) 82-93] (S135902941630067X)( 10.1016 / j. cocis. 2016.06. 009)." Current Opinion in Colloid and Interface Science 38 (2018): 214-222, and Kang, Beibei, et al. "Hofmeister series: Insights of ion specificity from amphiphilic assembly and interface property." ACS omega 5.12 (2020): 6229-6239.

[0209] A “kosmotropic agent” to an agent which contributes to the stability and structure of waterwater interactions in solutions. Kosmotropes cause water molecules to favourably interact, which stabilizes intramolecular interactions in macromolecules such as proteins, contrary to chaotropic salts, which have the opposite effect, in that they disrupt water structure, increase the solubility of nonpolar solvent particles, and destabilize solute aggregates. Ionic kosmotropes tend to be small or have high charge density. Some ionic kosmotropes are CO32", SO42", HPO42", Mg2+, Lit, Zn2+and Al3+. A scale can be established if one refers to the Hofmeister series or looks up the free energy of hydrogen bonding (AGHB) of the salts, which quantifies the extent of hydrogen bonding of an ion in water. For example, the kosmotropes CO32", and OH" have AGHB between 0.1 and 0.4 J / mol, whereas the chaotrope SON- has a AGHB between -1.1 and -0.9.

[0210] In some examples, the kosmotropic agent is a weakly kosmotropic agent. For example, see Figure 13 A and B.

[0211] The concentration of conducting agents in the initial IVT reaction mix (i.e. first composition) may be at least 50 mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 50 mM to about 2M. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 50 mM to about 1M. In some examples, concentration of conducting agents in the initial IVT reaction mix may be from about 600mM to about 1 ,2M.

[0212] In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 50 mM to about 500mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 50 mM to about 200mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 50 mM to about 150mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 100mM to about 200mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 80 mM to about 120mM. For example, concentration of conducting agents in the initial IVT reaction mix may be 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 200, 250, 300, 350, 400, 450 or 500 mM.

[0213] In some examples, the concentration of conducting agents in the initial IVT reaction mix may be from about 80mM to about 120mM. For example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120 mM.

[0214] In some examples, the concentration of conducting agents in the initial IVT reaction mix may be about 100mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be about 150mM.

[0215] In some example, the concentration of conducting agent is at most 250mM. For example, from 5mM to 250mM. For example, from 50mM to 250mM. In some examples, the concentration of conducting agents in the initial IVT reaction mix may be about 600mM.

[0216] The initial IVT reaction mix may include a combination of conducting agents. For example, the initial IVT reaction mix may include at least two, three, four or more different conducting agents. In such cases, the concentration of conducting agents described above refers to the total concentration of all conducting agents together.

[0217] Without being bound by theory, the concentration of the conducting agents in the IVT reaction of the invention is higher than a standard IVT reaction. A standard IVT conducting reaction mix is around 50mM or less. In addition, the methods of the invention provide for separation of IVT components and RNA using a lower conducting agent concentration than is standard. Standard separation of RNA, for example by Oligo-dT affinity separation, from an IVT reaction mix is around 500mM or more.

[0218] In some examples, wherein the separation method is solely a phase separation method as described herein, the conducting agent concentration in the initial reaction mix may be a standard conducting concentration. For example, when the separation method is solely a phase separation method as described herein, the concentration of conducting agents in the initial IVT reaction mix may be 50 mM or less.

[0219] A “chaotropic agent” refers to a molecule that disrupts non- covalent bonds (for example, without limitation, hydrogen bonds, van der Waals forces, and hydrophobic interactions). Chaotropic agents can disrupt the structure of / denature macromolecules such as proteins and nucleic acids.

[0220] Chaotropic agents generally create a denaturing environment for nucleic acids and proteins. Thus, it is contemplated herein to use chaotropic agents to reduce the nucleic acid interactions that cause the formation of dsRNA during in vitro transcription (IVT). Chaotropic agents that may be used include one or more of NaCI, KCI, urea, formamide, sodium salicylate, ethanol, sodium perchlorate, arginine, n-butanol, thiourea, 2-propanol, guanidinium chloride, guanidine hydrochloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, propylene glycol, phenol, sodium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, Tween-20 and / or DMSO.

[0221] In some examples, the chaotropic agent is one or more of NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate, and / or Tween-20. Preferably, the chaotropic agent is potassium acetate, potassium glutamate, NaCI and / or Guanidine HCI. Preferably, the chaotropic agent is NaCI and / or Guanidine HCI. In some examples, NaCI is preferred. In some examples, Guanidine HCI is preferred. In other examples, a combination of Guanidine HCI and NaCI is preferred. In some examples, potassium glutamate, is preferred. . In some examples, a combination of potassium glutamate, potassium acetate, Guanidine HCI and NaCI is preferred.

[0222] The concentration of chaotropic agents in the initial IVT reaction mix (i.e. first composition) may be at least 50 mM. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 50 mM to about 2M. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 50 mM to about 1M. In some examples, concentration of chaotropic agents in the initial IVT reaction mix may be from about 600mM to about 1 ,2M.

[0223] In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 50 mM to about 500mM. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 50 mM to about 200mM. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 50 mM to about 150mM. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 100mM to about 200mM. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 80 mM to about 120mM. For example, concentration of chaotropic agents in the initial IVT reaction mix may be 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 200, 250, 300, 350, 400, 450 or 500 mM.

[0224] In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be from about 80mM to about 120mM. For example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120 mM.

[0225] In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be about 100mM. In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be about 150mM.

[0226] In some example, the concentration of chaotropic agent is at most 250mM. For example, from 5mM to 250mM. For example, from 50mM to 250mM.

[0227] In some examples, the concentration of chaotropic agents in the initial IVT reaction mix may be about 600mM. The initial IVT reaction mix may include a combination of chaotropic agents. For example, the initial IVT reaction mix may include at least two, three, four or more different chaotropic agents. In such cases, the concentration of chaotropic agents described above refers to the total concentration of all chaotropic agents together.

[0228] Without being bound by theory, the concentration of the chaotropic agents in the IVT reaction of the invention is higher than a standard IVT reaction. A standard IVT chaotropic reaction mix is around 50mM or less. In addition, the methods of the invention provide for separation of IVT components and RNA using a lower chaotropic agent concentration than is standard. Standard separation of RNA, for example by Oligo-dT affinity separation, from an IVT reaction mix is around 500mM or more.

[0229] In some examples, wherein the separation method is solely a phase separation method as described herein, the chaotropic agent concentration in the initial reaction mix may be a standard chaotropic concentration. For example, when the separation method is solely a phase separation method as described herein, the concentration of chaotropic agents in the initial IVT reaction mix may be 50 mM or less.

[0230] Immobilization of Components

[0231] In some examples, components of the IVT reaction may be immobilised. For example, the template DNA and / or one more of the enzymes used may be immobilised. For example, the transcription template, a capping enzyme (e.g. Faustovirus capping enzyme or Vaccina capping enzyme), RNAase inhibitors, pyrophosphatase and / or RNA polymerase may be immobilised. In some examples, the DNA template is immobilised. In some examples, the RNA polymerase is immobilised. In some examples, the DNA template and RNA polymerase are immobilised. In some examples, a capping enzyme is immobilised. In some examples, pyrophosphatase is immobilised. In some examples, RNAase inhibitors are immobilised.

[0232] Immobilization may be achieved by attachment of the component to a support. For example, the support may be a bead, surface or membrane. Methods of immobilising DNA and proteins will be well known by those skilled in the art. For example, DNA and proteins may be immobilised using cognate functional groups or by incorporating binding regions or tags that are capable of binding to support.

[0233] In the case that components, such as the transcription template and / or RNA polymerase, are immobilized, these components will not need to be “topped up” in subsequent rounds of IVT or require less topping up (i.e. lower amounts of the immobilised components may need adding to subsequent IVT reactions). For examples, of immobilisation of IVT components and IVT reactions using immobilised components see Kithmie MalagodaPathiranage, Ruptanu Banerjee, Craig T Martin, A new approach to RNA synthesis: immobilization of stably and functionally co-tethered promoter DNA and T7 RNA polymerase, Nucleic Acids Research, Volume 52, Issue 17, 23 September 2024, Pages 10607-10618, https: / / doi.org / 10.1093 / nar / gkae599, Garcia-Marquina, Guillermo, et al. "Streamlined DNA template preparation and co-transcriptional 5' capped RNA synthesis enabled by solid-phase catalysis." bioRxiv (2023): 2023-10, Lin J J, Carey M. In vitro transcription and immobilized template analysis of preinitiation complexes. Curr Protoc Mol Biol. 2012 Jan;Chapter 12:Unit 12.14.. doi: 10.1002 / 0471142727.mb1214s97. PMID: 22237857; PMCID: PMC4302722, WO2016174227A1 and EP3289077B1.

[0234] SEPARATING

[0235] The methods provided herein allow for the separation of RNA produced by an IVT reaction and any components used for the production of the RNA not consumed in the initial or subsequent IVT reactions to be recycled and utilized in further IVT reactions. For example, separation of RNA may be by affinity, or phase, based separation methods. For example, see “Feng, Xue, et al. “Messenger RNA chromatographic purification: advances and challenges.” Journal of Chromatography A (2023): 464321”, “Zhang, Jingjing, et al. “Recent Advances and Innovations in the Preparation and Purification of In Vitro-Transcribed-mRNA- Based Molecules.” Pharmaceutics 15.9 (2023): 2182” and “Rosa, Sara Sousa, et al. “mRNA vaccines manufacturing: Challenges and bottlenecks.” Vaccine 39.16 (2021): 2190-2200” and “Wadsworth, Gable M., et al. “RNAs undergo phase transitions with lower critical solution temperatures.” bioRxiv (2022): 2022-10”.

[0236] In particular, the separation step of methods of the invention do not require addition of extrinsic conducting agents after RNA production in order to separate or improve separation of the RNA. In addition, the methods of the invention do not require the removal of conducting agents from the reaction mix after production of RNA or before a subsequent round of IVT. Furthermore, the methods of the invention do not require the exchange of buffers in order to carry out separation and / or to carry out subsequent rounds of IVT.

[0237] Even though the methods of the invention do not require addition of conducting agents, in some cases conducting agents may be added before or during separation. However, as mentioned above, the concentration of conducting agents before or during separation are less than the standard conducting agent concentration (which is normally 500mM or more, for example for Oligo-dT affinity separation methods). For example, a standard separation buffer for affinity separation such as Olig-dT separation may be 250 to 1M NaCI / Guanidine- HCL; 50mM Sodium Phosphate; 1 to 5mM EDTA or 250 to 1M NaCI / Guanidine-HCL; 10mM Tris; 1-5mM EDTA.

[0238] In some examples, the conducting agent concentration during separation may be 250mM or more. In some examples, the conducting agent concentration during separation may be 500mM or more. For example, when the separation method is or includes a phase separation the conducting agent may be included at any concentration, as phase separation as described herein may not be affected by conducting agent concentration. In some examples, such as when the production step is carried out at a concentration of conducting agent 500 mM or more, the concentration of conducting agent for separation may be 500 mM or more. For example, the conducting agent concentration used for production and separation may be substantially equal.

[0239] In some examples, the concentration of conducting agents during separation may less than 50 mM. For example, when the separation method solely consists of a phase separation method as described herein.

[0240] In some examples, the concentration of conducting agents during separation may be at least 50 mM. In some examples, the concentration of conducting agents during separation may be at least 100 mM. In some examples, the concentration of conducting agents during separation may be from 50 mM to 2M. In some examples, the concentration of conducting agents during separation may be from 100mM to 2M. In some examples, the concentration of conducting agents during separation may be from 50mM to 1M. In some examples, the concentration of conducting agents during separation may be from 100mM to 1M. In some examples, the concentration of conducting agents during separation may be 2M or less. In some examples, the concentration of conducting agents during separation may be 600mM or less. In some examples, the concentration of conducting agents during separation may be about 600mM. In some examples, the concentration of conducting agents during separation may be about 1.2M. In some examples, the concentration of conducting agents during separation may be from about 600mM to about 1 ,2M.

[0241] In some examples, during separation the conducting agent concentration may be less than 250mM. In some example, the concentration of conducting agent is at most 250mM. For example, from 5mM to 250mM. For example, from 50mM to 250mM.

[0242] In some examples, during separation the conducting agent concentration may be less than 500mM. In some examples, the conducting agent concentration during separation may be between 50 mM and 500 mM. In some examples, the conducting agent concentration during separation may be between 50 mM and 250 mM. In some examples, the conducting agent concentration during separation may be from about 100mM to about 200mM. For example, the concentration of conducting agents during separation may be 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 200, 250, 300, 350, 400, 450 or 500 mM.

[0243] In some examples, the conducting agent concentration during separation may be from about 50mM to about 200mM. For example, concentration of conducting agents during separation may be 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, or 200 mM.

[0244] In some examples, the conducting agent concentration during separation may be from about 80mM to about 120mM. In some examples, the concentration of conducting agents during separation may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120mM.

[0245] In some examples, the concentration of conducting agents during separation may be 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 200 mM.

[0246] In some examples, the concentration of conducting agents during separation may be about 100 mM. In some examples, the concentration of conducting agents during separation may be about 150 mM.

[0247] In particular, the separation step of methods of the invention do not require addition of extrinsic conducting agents after RNA production in order to separate or improve separation of the RNA. In addition, the methods of the invention do not require the removal of conducting agents from the reaction mix after production of RNA or before a subsequent round of IVT. Furthermore, the methods of the invention do not require the exchange of buffers in order to carry out separation and / or to carry out subsequent rounds of IVT.

[0248] Even though the methods of the invention do not require addition of chaotropic agents, in some cases chaotropic agents may be added before or during separation. However, as mentioned above, the concentration of chaotropic agents before or during separation are less than the standard chaotropic agent concentration (which is normally 500mM or more, for example for Oligo-dT affinity separation methods). For example, a standard separation buffer for affinity separation such as Olig-dT separation may be 250 to 1M NaCI / Guanidine- HCL; 50mM Sodium Phosphate; 1 to 5mM EDTA or 250 to 1M NaCI / Guanidine-HCL; 10mM Tris; 1-5mM EDTA.

[0249] In some examples, the chaotropic agent concentration during separation may be 250mM or more. In some examples, the chaotropic agent concentration during separation may be 500mM or more. For example, when the separation method is or includes a phase separation the chaotropic agent may be included at any concentration, as phase separation as described herein may not be affected by chaotropic agent concentration. In some examples, such as when the production step is carried out at a concentration of chaotropic agent 500 mM or more, the concentration of chaotropic agent for separation may be 500 mM or more. For example, the chaotropic agent concentration used for production and separation may be substantially equal.

[0250] In some examples, the concentration of chaotropic agents during separation may less than 50 mM. For example, when the separation method solely consists of a phase separation method as described herein.

[0251] In some examples, the concentration of chaotropic agents during separation may be at least 50 mM. In some examples, the concentration of chaotropic agents during separation may be at least 100 mM. In some examples, the concentration of chaotropic agents during separation may be from 50 mM to 2M. In some examples, the concentration of chaotropic agents during separation may be from 100mM to 2M. In some examples, the concentration of chaotropic agents during separation may be from 50mM to 1M. In some examples, the concentration of chaotropic agents during separation may be from 100mM to 1M. In some examples, the concentration of chaotropic agents during separation may be 2M or less. In some examples, the concentration of chaotropic agents during separation may be 600mM or less. In some examples, the concentration of chaotropic agents during separation may be about 600mM. In some examples, the concentration of chaotropic agents during separation may be about 1.2M. In some examples, the concentration of chaotropic agents during separation may be from about 600mM to about 1 ,2M.

[0252] In some examples, during separation the chaotropic agent concentration may be less than 250mM. In some example, the concentration of chaotropic agent is at most 250mM. For example, from 5mM to 250mM. For example, from 50mM to 250mM. In some examples, during separation the chaotropic agent concentration may be less than 500mM. In some examples, the chaotropic agent concentration during separation may be between 50 mM and 500 mM. In some examples, the chaotropic agent concentration during separation may be between 50 mM and 250 mM. In some examples, the chaotropic agent concentration during separation may be from about 100mM to about 200mM. For example, the concentration of chaotropic agents during separation may be 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,

[0253] 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103,

[0254] 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,

[0255] 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139,

[0256] 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 200, 250, 300, 350, 400, 450 or 500 mM.

[0257] In some examples, the chaotropic agent concentration during separation may be from about 50mM to about 200mM. For example, concentration of chaotropic agents during separation may be 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

[0258] 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95,

[0259] 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,

[0260] 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,

[0261] 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, or

[0262] 200 mM.

[0263] In some examples, the chaotropic agent concentration during separation may be from about 80mM to about 120mM. In some examples, the concentration of chaotropic agents during separation may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120mM.

[0264] In some examples, the concentration of chaotropic agents during separation may be 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118,

[0265] 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136,

[0266] 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 200 mM.

[0267] In some examples, the concentration of chaotropic agents during separation may be about

[0268] 100 mM. In some examples, the concentration of chaotropic agents during separation may be about 150 mM.

[0269] After separation of RNA from the IVT reaction mix, the reaction mix (i.e. without the produced RNA and containing unconsumed components for IVT) is collected and recycled or reused for subsequent IVT reactions. This collected reaction mix may be referred to as a second composition (i.e. an IVT reaction mix which has undergone an IVT reaction and had the RNA produced removed) or a further composition. In comparison, reference to a first or initial IVT reaction mix refers to an IVT reaction mix prior to carrying out an IVT reaction optionally before any production of RNA has occurred.

[0270] The method of separation may be a chromatography separation method. Chromatography refers to a process in which a mixture carried by a liquid or gas (e.g. IVT reaction mix after production of RNA) is separated into components (e.g. unconsumed IVT components and RNA) as a result of differential distribution of the components as they flow around or over a stationary liquid or solid phase.

[0271] In some examples, the method of separation may be liquid chromatography (LC). Liquid chromatography is a process of selective retardation of one or more components of a fluid solution (e.g. IVT reaction mix) as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). “Liquid chromatography” includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).

[0272] High performance liquid chromatography refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. In some examples, the separation method is HPLC.

[0273] In column based methods, the solid or stationary phase may include a binding partner that specifically binds to the RNA. Thus allowing separation of the RNA from the IVT reaction mix and the IVT reaction components to be eluted.

[0274] In some examples, the separation method is a Hydrophobic Interaction Chromatography (HIC) based method. Hydrophobic interaction chromatography refers to a method of separating a target based on the strength of its relative hydrophobic interactions with a hydrophobic separation matrix. In this context, "hydrophobicity" is defined as the repulsion between a non-polar compound and a polar environment. HIC may be carried out using any suitable separation matrix, such beads, fibres (e.g. electrospun fibres), membranes or monolith based matrices. Hydrophobic groups such as, phenyl, octyl, or butyl are attached to the stationary matrix (e.g. within a column). Examples of hydrophobic interaction chromatography resins include: Butyl FF, Butyl HP, Octyl FF, Phenyl FF, Phenyl HP, Phenyl FF (high sub), Phenyl FF (low sub), Capto Phenyl ImpRes, Capto Phenyl (high sub), Capto Octyl, Capto ButyllmpRes, Capto Butyl (GE Healthcare, Uppsala, Sweden); TOYOPEARL® Super Butyl-550C, TOYOPEARL® Hexyl-6500, Butyl-650C, Phenyl-650C, Butyl 600 M, Phenyl-600M, PPG-600M, Butyl-650M, Phenyl-650M, Ether-650M, Butyl-650S, Phenyl- 650S, Ether-650S, TSKgel Pheny-5PW, TSKgel Ether-5PW (Tosoh Bioscience, Tokyo, Japan); MACRO-PREP®-butyl, MACRO-PREP®-methyl (Bio-Rad); and SARTOBIND® Phenyl (Sartorius corporation, New York, USA). For example, see WO2018096179A1.

[0275] In some examples, the separation method is an electrospun fibre-based column separation method. Examples of electrospun fibre-based that may be used in separation methods include cellulose nanofibers. For example, functionalised cellulose nanofibers. For example, see Dewar EA, Guterstam P, Holland D, et al. Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent. J Chromatogr A. 2024 and US9802979B2. In some examples, the separation method is a membrane-based separation method. Membrane chromatography refers to a process used to purify biomolecules, in which a planar porous membrane is used so that convection is greater than diffusion in terms of proportion, and separation efficiency of solution is relatively high. Commercially available membranes include, Mustang Q (Pall Corporation), Sartobind Q (Sartorius Stedim Biotech GmbH) and Purexa™ OdT. In some example, the membrane based method may use a stacked disc, cross-flow flat sheet, hollow fibre, spiral wound, or pleated sheet membrane arrangement. In some examples, the separation method is a membrane-based column separation method.

[0276] In some examples, the separation method is a monolith structure-based separation method. In some examples, the separation method is a monolith structure column-based separation method. For example, a column based separation method that utilises a monolith as the stationary phase. Monolithic columns, in contrast to traditional HPLC columns that comprise packed particles, contain a single, solid compound as the stationary phase. This stationary phase is usually made up of a network of polymethacrylate or polystyrene copolymers, or bonded silica forming pores of varying size. Thus, in monolithic columns the mobile phase must flow through the pores of the solid stationary phase. Molecules within the mobile phase are then retained to a greater or lesser extent within the pores of the stationary phase (small molecules diffuse into the pores and are retained while large molecules are excluded from the pores and are not retained). Retention of small molecules may be further enhanced by binding to specific compounds incorporated into the interior of the pores. A monolithic column comprises a continuous bed consisting of a single piece of a highly porous solid material (see e.g., Tennikova T B, Svec F (1993) J Chromatogr 646:279). Monolithic supports commercially available include: silica gel based monolithic beds, polyacrylamide based monolithic beds, and rigid organic gel based monolithic beds. For example, see US10317377B2.

[0277] Examples of monolith structures that may be used in separation methods include CIMmultus® Oligo dT18.

[0278] In some examples, the chromatography method may be a continuous chromatography method. “Continuous chromatography” refers to a process by which a conventional batch chromatography process is carried out continuously. Specifically, a solid phase and a liquid phase (e.g. IVT reaction mix including produced RNA and oligo-dT or oligo-dll comprising resin respectively) can be continuously supplied to the chromatography apparatus, and the solid phase and the liquid phase move in opposite directions to each other to cause countercurrent contact, thereby enabling the separation of substances more efficiently. Continuous chromatography may be true moving bed (TMB) chromatography and simulated moving bed chromatography (SMB).

[0279] Without being bound theory, the methods of the invention may be particularly suited to continuous chromatography as the methods recycle the liquid phase of the IVT reaction including the components for IVT, allowing a cyclic and continuous method of producing RNA.

[0280] In some examples, the separation method is a bead or particle based separation method. For example a method utilising beads such as magnetic beads. In such methods, a bead including a binding partner that specifically binds to the RNA produced in the IVT reaction mix is contacted with the RNA. The RNA is then bound to the bead which can be separated from the IVT components in the reaction mix. For example, the bead and RNA may be separated by centrifugation, enabled by the mass of the bead which allows the bead bound to the RNA to form a pellet while leaving the IVT components in the supernatant. In the case of magnetic beads, the beads bound to the RNA may be separated from the IVT reaction mix by application of a magnetic field. For example, see Berensmeier, Sonja. “Magnetic particles for the separation and purification of nucleic acids.” Applied microbiology and biotechnology 73 (2006): 495-504.

[0281] In some examples, the separation method is a porous bead separation method. For example, a column based separation method that utilises porous beads as the stationary phase. Examples of porous beads that may be used in separation methods include POROS™ resin beads, and CIMmultus® Oligo dT18.

[0282] In some examples, the separation method is a non-porous bead separation method. For example, a column based separation method that utilises non-porous beads as the stationary phase. In some examples, the method of separation may be an affinity separation method. The term “affinity separation” relates to a separation technique based on binding interaction, preferably a specific binding interaction, between a desirable compound, such as an RNA, and a binding partner. For example, in the case of IVT, affinity separation may be a nucleic acid based affinity separation method. For example, the RNA produced can include a specific sequence which a binding partner comprising a complimentary nucleic acid to the specific sequence can be produced. The IVT reaction mix including produced RNA is then mixed so that the RNA is contacted by the binding partner comprising the a complimentary nucleic acid and the RNA is bound to the a complimentary nucleic acid. The binding partner, bound to the RNA can then be separated from the other components of the reaction mix.

[0283] In some examples, the binding partner is a poly- deoxythymidilic acid or poly- deoxyuridine oligonucleotide (i.e. oligo-dT or oligo-dll nucleic acid). The produced RNA can include a poly-adenosine sequence, such as a poly-A tail, which specifically binds to the oligo-dT or oligo-dll sequence.

[0284] The oligo-dT or oligo-U nucleic acid may be conjugated to a solid support. For example, a resin such as agarose, cellulose, cross-linked poly(styrene-divinylbenzene), polymethacrylate-based or to a particle or bead such as a magnetic bead as described above.

[0285] In some examples, the affinity separation comprises a column based method as described above such as affinity chromatography. In some examples, the method of separation is affinity HPLC. For example, HPLC utilising a column including oligo-dT or oligo-dU.

[0286] In some examples, the separation method includes or is a phase separation method. In the broadest sense the expression “phase separation” refers to the separation of at least one liquid phase from other phases. Thus phase separation refers to the separation of at least two phases and particularly to the separation of at least one first liquid phase from at least another phase selected from a second liquid phase, solid phase and semi-solid phase. In the context of the invention, a first phase (e.g. upper phase) includes unconsumed components for IVT and a second phase (e.g.. lower phase) includes the produced RNA. Without being bound by theory, phase separation differs from chromatography separation techniques; as it involves separation of nucleic acids from an aqueous solution (i.e. the IVT reaction mix after production of RNA). The inventors have found that phase separation may be enhanced or improved by altering the temperature of the reaction mix and / or use of centrifugation.

[0287] In some examples, the phase separation is induced by changes in temperature of the IVT reaction after production of RNA. For example, the phase separation is temperature or thermally induced phase separation. In some examples, the temperature induced phase separation includes cooling the IVT reaction mix after production of RNA. For example, the reaction mix may be cooled to a temperature of at most 0°C. In some examples, the IVT reaction mix is cooled to a temperature from 0°C and - 80°C. In some examples, the IVT reaction mix is cooled to a temperature from 0°C to - 20°C. In some examples, the IVT reaction mix after production of RNA is frozen. In some examples, the IVT the IVT reaction mix after production of RNA does not freeze (i.e. remains a liquid and does not solidify).

[0288] In some examples, the IVT reaction mix after production of RNA is cooled at a specific cooling rate. For example, at a cooling rate of 1 Kelvin per minute.

[0289] In some examples, after freezing the IVT reaction mix after production of , the reaction mix may be thawed so that the IVT reaction mix is a liquid.

[0290] In addition to cooling, temperature phase separation may also include centrifugation after cooling of the IVT reaction mix after RNA production. For example, centrifugation at a force of 5000 to 15000g. For example, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000g. In some examples, centrifugation at a force of 10000g.

[0291] The centrifugation may be carried out at a defined temperature. For example, the cooled IVT reaction mix after production of RNA may be centrifuged while being maintained at a temperature from about 0°C to about 8°C. In some examples, the cooled IVT reaction mix after production of RNA may be centrifuged while being maintained at a temperature from about 2°C to about 4°C. For example, maintained at 0, 1 , 2, 3, 4, 5, 6, 7, or 8 °C. In some examples, the cooled IVT reaction mix after production of RNA may be centrifuged while being maintained at a temperature of about 4°C.

[0292] In some examples, the separation method may be or include an ion-pair separation method, such as ion-pair chromatography. This is a type of ion chromatography that is used to separate hydrophilic or charged analytes on columns using reversed phase or “neutral” stationary phases that do not carry charges. It involves modifying the polarity of the charged analytes through their interaction with an ion-pairing reagent that is added to the mobile phase. These reagent molecules carry charges opposite to that of the analyte ions with which they are able to form electrostatic bonds. The pairs formed between the analytes and reagent ions behave like neutral, hydrophobic moieties that can be separated on C18 or C8 columns. I PC is used for the separation of polar organic acids, bases and zwitterions as well as inorganic ions.

[0293] In some examples, separating comprises a combination of separation methods. For example, separating may include at least one, two three four or more separation methods as described herein. When a combination of separation methods are used, they may be carried in any order. In particular, affinity separation methods may be carried out prior to phase separation methods. In some examples, affinity separation methods may be carried after phase separation methods. In some examples, separation includes affinity separation and phase separation or vice versa. In some examples, separation include phase separation and ion-pair separation methods or vice versa. In some examples, separation includes affinity separation, ion-pair separation and / or phase separation methods.

[0294] Collection of RNA and Recycled IVT reaction mix

[0295] Collection of the RNA and recycled IVT reaction mix is determined based on the separation method or methods used. If multiple separation methods are used then the RNA and recycled IVT reaction mix may be collected after each separation method.

[0296] In general, the chromatography based methods (such as affinity chromatography) lead to the IVT reaction mix comprising unconsumed components for IVT being eluted while the RNA is bound to the selected binding partner.

[0297] The eluate can be collected and re-used for subsequent IVT reactions. As some of the components for IVT are inherently consumed by production of RNA, such as NTPs, magnesium ions, capping agents (when present for co-transcriptional capping) and pyrophosphatase, the eluted and collected IVT reaction mix (i.e. second and / or further composition) may be supplemented with additional NTPs, magnesium ions, pyrophosphatase and / or RNA capping agents prior to carrying out subsequent IVT reactions.

[0298] In the case of affinity separation methods the separated RNA is generally immobilised within the column by virtue of binding to the resin or matrix therein. The RNA may be collected by disrupting the interaction between the RNA and its binding partner. This leads to elution of the RNA.

[0299] Disruption of the RNA and binding partner interaction may be achieved by any suitable means. For example, by altering the salt concentration. For example, by applying an elution solution to the RNA and binding partner.

[0300] For example in the case of affinity separation or where the methods employ affinity separation as the final separation step, the RNA may be eluted using any suitable solution. The elution solution preferably has a low salt concentration. That is to say, a salt concentration lower than the IVT reaction mix after production of RNA and / or a salt concentration lower than the buffer in which the produced RNA was initially applied to the affinity binding entity (i.e. column or bead). For example, the elution buffer may be RNAase free water, or any buffer for downstream applications. In the case of case of phase separation or where the methods employ phase separation as the final separation step, the RNA is precipitated or when centrifugation is used pelleted. As such the RNA can be extracted from the residual fluid phase or supernatant and be resuspended in any suitable buffer.

[0301] The eluted RNA may be referred to herein as a third composition.

[0302] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper

[0303] Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms “a”, “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0304] Aspects of the invention are demonstrated by the following non-limiting examples.

[0305] EXAMPLES

[0306] Example 1

[0307] Materials and Methods

[0308] In-vitro transcription (General)

[0309] The mRNA transcripts were prepared in an in-vitro transcription reaction that utilized template DNA at 0.25-2 mg / mL mM (linearised plasmid with gene of interest and RNA polymerase promoter sequence). DNA-dependent RNA polymerase (2.5-10% v / v of the total reaction volume depending on the enzymatic activity between 50 to 1000 Units / pL) and ribonucleotides (naturally occurring or modified) ATP, CTP, GTP and UTP at concentration of 0.5-1 OmM each, source of Magnesium ions (such as Magnesium acetate or Magnesium chloride) at 8-80mM, the buffering agent (trisaminomethane hydrochloride (TRIS-HCI) or 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES)) maintaining the IVT reaction pH between 6.5 to 8, Dithiothreitol (DTT 1-40mM), chaotropic salt such as sodium chloride (NaCI) and Guanidine HCI (50-200mM ) and 2 mM Spermidine (0.5-2.5mM) and inorganic pyrophosphatase (0.5-6%v / v depending on enzymatic activity ) were added to the reaction mixture. Rnase inhibitor (0.5 to 2.5 % v / v) and surfactants (e.g. Tween 20, Triton X100; 0.001% v / v) can be added to the reaction mixture. The reaction was incubated at 37-42 °C for 0.16-2 hours. mRNA purification(General)

[0310] Chromatography was applied for the recycling of raw materials. Crude IVT product was loaded into the column. Purified RNA was eluted in nuclease-free water and collected from the elution phase, and the flow through during the load phase was collected and reused for recycling the raw materials.

[0311] Investigation of IVT Reaction Mix for Recycling IVT components

[0312] IVT reactions were performed in order to screen the effect of chaotropic salts on mRNA yield. Varied concentrations of NaCI and / or Gu-HCI (Table 1) were used (Figure 3a.) keeping all other components at standard concentrations.

[0313] Table 1 - IVT reaction mix chaotropic agent titrations Condition 2 (C2) was selected for initial recycling experiments as it did not significantly reduce the yield, while at the same time gave reasonable oligo-dT binding properties. Furthermore, the effect of urea concentration in combination with condition 2 on mRNA yield was investigated (Figure. 3b). Increasing Urea concentration was shown to have a detrimental effect on IVT mRNA yield. RNA concentration of each fraction was determined by UV absorbance.

[0314] In-vitro transcription (Figure specific)

[0315] The mRNA transcripts were prepared in an in-vitro transcription reaction that utilized 1.52E- 04 mM template DNA (linearised plasmid with eGFP gene of interest and RNA polymerase promoter sequence). DNA-dependent RNA polymerase of T7 bacteriophage (Roche, Germany) at 3.01 E-04 mM and ribonucleotides (Roche, Germany) ATP, CTP, GTP and UTP in an equimolar ratio at 1 mM concentration were added to the reaction mixture. The reaction was further supplemented with 10 mM Magnesium ion, 40 mM HEPES buffer (pH 7), 10 mM Dithiothreitol (DTT), 100 mM Sodium Chloride, 50 Mm Guanidine HCI and 2 mM Spermidine. Inorganic pyrophosphatase (Roche, Germany) at 3.49E-03 mM was added to the reaction mixture to prevent Magnesium pyrophosphate precipitation. RNase inhibitor (Roche, Germany) was added at 2.05E-04 mM to maintain RNase free environment in the reaction mixture. The reaction was incubated at 37 ° C for 0.16 hours. mRNA purification by continuous process (Figure specific)

[0316] Oligo-dT continuous chromatography using AKTA PCC (Cytiva, Sweden) was applied for the recycling of raw materials, four CIMmultus Oligo dT18 1 mL monolithic columns (Sartorius, Gottingen, Germany) were utilised. The chromatography method started from equilibration, washing all four columns at 5 mL / min for 30 mL. The main loop started after equilibration, allowing four columns to work simultaneously to make the chromatography continuous. The load phase started from the first two columns at 2 mL / min for 10 mL, and the flow through was collected and reused for the subsequent IVTs based on the UV levels from 50 to 40 mAU. Meanwhile, column three was undergoing wash and elution at 10 mL / min for 12.5 mL and 10 mL respectively, and column four was performing cleaning in process (CIP) at 10 mL / min for 30 mL. The four columns switched and the load was moved to the second to third columns, similarly other columns were switched to conduct different phases in sequence as programmed. After loading and washing, the column loaded with mRNA was eluted at 10 mL / min for 10 mL, and the collection was determined by the UV levels from 40 to 35 mAU. The flow through containing unconsumed reagents (such as enzymes, DNA template and capping reagent) was pumped back to of the bioreactor via inlet B of the continuous bioreactor, together with the top-up NTPs and MgCI2 pumped from inlet D for the subsequent IVT. The produced mRNA mixture is then fed back again to continuous oligo-dT chromatography after incubation to selectively isolate mRNA from the IVT reaction mix. This process went continuously and repetitively until recycled materials display a substantial degradation.

[0317] Results

[0318] Three IVTs were performed. The initial IVT was performed and run through affinity chromatography for RNA purification. The flow through of the first IVT (FT1) was used to set a second IVT ( IVT2) by supplementing with NTPs and Magnesium. Similarly, IVT2 was run through the affinity column and the flow through ( FT2) used to set-up the third IVT. The RNA was eluted for each affinity purification ( E1,2). RNA concentration of each fraction was determined by UV absorbance. Results are shown in Figures 4 and 5 (n=3).

[0319] Example 2 (figures 6 to 12)

[0320] IVT-OliqodT recycling experiments

[0321] In vitro transcription immediately followed by oligodT purification using a common buffer (for both unit operation) was done to recycle unconsumed IVT components for five consecutive cycles. The following steps were followed:

[0322] 1. 8 mL IVT with the following composition was setup in a 15 mL Falcon tube.

[0323] ATP: 2.5 mM (millimolar)

[0324] CTP: 2.5 mM

[0325] GTP: 2.5 mM

[0326] UTP: 1.875 mM

[0327] T7 RNA polymerase: 555 nM (nanomolar)

[0328] Pyrophosphatase: 3 pM (micromolar)

[0329] RNase inhibitor: 200 nM

[0330] Cleancap AG: 2.5 mM

[0331] MgCh: 16 mM NaCI: 100 mM

[0332] Gu-HCI (Guanidine hydrochloride): 50 mM

[0333] DNA: 26 nM

[0334] Reaction Buffer: 1X

[0335] RNase free water: 5035 pL (to make up the reaction volume up to 8 mL)

[0336] 1X reaction buffer has the following composition

[0337] TRIS-HCI (pH 7): 40 mM

[0338] Spermidine: 2 mM

[0339] DTT: 100 mM

[0340] TritonX 100: 0.01% Once the reaction mixture was assembled, it was mixed and incubated at 37 ° C for 1.5 h. After incubation, the IVT reaction mixture was loaded on to an oligo dT column. The loading buffer had the following composition:

[0341] TRIS-HCI (pH 7): 40 mM

[0342] NaCI: 100 mM

[0343] Gu-HCI: 50 mM Flow through from the loading and washing phase of oligodT purification step was collected. This Flow through contains the unused IVT components that are not adsorbed by the oligodT column. The flow through was analyzed with Nanodrop for UV absorbance and 7520 pL of this was used to setup the next IVT. In addition to the flowthrough, NTPs at initial concentration (9.375 mM), MgCh at 6 mM and template DNA at 10 nM final concentration were assembled in the second IVT. Step 2 to 6 were repeated four times. Elution from the oligodT purification (purified mRNA in RNase free water) was collected and stored for further analysis. The oligodT purification was done on an AKTA pure™ system. The oligo dT column was manufactured by Donaldson (Purilogics™, 100003049). It had a column volume of 2 mL.

[0344] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0345] Examples of Initial IVT compositions (e.g. first composition)

[0346] Table 2: 8 mL IVT

[0347] Table 3: 6mL IVT (Donaldson column) Table 4: 7mL IVT (Donaldson column)

[0348] Table 5: 4mL IVT (monolith)

[0349] Table 6: 4 mL IVT (monolith)

[0350] Table 7: 5ml IVT (initial IVT)

[0351] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

Claims1. A method of producing RNA using recycled components for in vitro transcription (IVT), the method comprising;(a) producing RNA in a first composition comprising components for IVT;(b) separating the RNA from the first composition to provide a second composition comprising unconsumed components for IVT;(c) collecting the second composition; and(d) producing RNA in the second composition wherein the first composition comprises one or more conducting agents at a total concentration of conducting agents of at most 250mM.

2. The method of claim 1, wherein the method further comprises repeating at least once the steps of :(e) separating the RNA from the second composition to provide a further composition comprising unconsumed components for IVT;(f) collecting the further composition; and(g) producing RNA in the further composition.

3. The method of any preceding claim, wherein the components for IVT comprise: ribonucleotide triphosphates (rNTPs); a transcription template; an RNA polymerase; a buffer solution; a pyrophosphatase; an RNAase inhibitor; a chaotropic agent; a polyamine; and a source of magnesium ions.

4. The method of any of claims 1 to 3, wherein the first composition comprises a conducting agent concentration of at least 50 mM, optionally a conducting agent concentration of about 50mM to about 150mM.

5. The method of any preceding claim, wherein the separating is carried out at a conducting agent concentration of less than 500mM, optionally a conducting agent concentration of about 100 mM to about 200 mM.

6. The method of any preceding claim, wherein the method is carried out at a conducting agent concentration from 50 to about 200 mM.

7. The method of any preceding claim, wherein the producing and separating are carried out at the same conducting agent concentration.

8. The method of any preceding claim, wherein the method further comprises adding one or more conducting agents and / or magnesium ions to the first composition and / or second composition before the separating.

9. The method of any preceding claim, wherein the method does not include buffer exchange and / or wherein the method does not comprise adding extrinsic conducting agents to or removing conducting agents from the second and / or further composition.

10. The method of any preceding claim, wherein the separating comprises affinity separation and / or phase separation.

11. The method of any preceding claim, wherein the separating comprises bead based separation, column based separation, monolith based separation, membrane based separation or combinations thereof.

12. The method of claim 10 or 11 , wherein the affinity separation comprises affinity chromatography.

13. The method of any preceding claim, wherein the separating comprises oligo-dT or oligo- dll separation.

14. The method of any of claims 10 to 13, wherein the phase separation comprises temperature induced phase separation.

15. The method of claim 14, wherein temperature induced phase separation comprises cooling the first, and / or further composition after production of RNA thereby forming a first phase (upper phase) comprising the unconsumed components for IVT and a second phase (lower phase) comprising the produced RNA; optionally wherein the produced RNA precipitates in the second phase.

16. The method of claim 15, wherein cooling comprises freezing the first and / or further composition after production of RNA and thawing the first, and / or further composition before collecting.

17. The method of any of claims 10 to 16, wherein the phase separation further comprises centrifugation.

18. The method of any of claims 15 to 17, wherein the separating comprises or further comprises:(i) a) cooling the first and / or further composition after production of RNA; b) optionally centrifuging the cooled first and / or further composition to form the first phase comprising unconsumed components for IVT and the second phase comprising the produced RNA; or(ii) a) separating the RNA from the first and / or further composition to form a second composition or further second composition by affinity separation after production of RNA; b) cooling the second and / or further second composition; and c) optionally centrifuging the cooled second and / or further second composition to form the first phase comprising unconsumed components for IVT and the second phase comprising the produced RNA.

19. The method of any of claims 14 to 18, wherein the phase separation comprises cooling at rate of 1 Kelvin per minute.

20. The method of any of claims 17 to 19, wherein the centrifugation is carried out a temperature of 0°C to 8°C.

21. The method of any preceding claim, wherein the conducting agent comprises: a chaotropic agent, a kosmotropic agent, a kosmotropic anion and chaotropic cation, or a chaotropic anion and kosmotropic cation; optionally wherein the conducting agent is selected from one or more of NaCI, Urea, Guanidine HCI, Formamide, Arginine, Sodium Salicylate, DMSO, Thiourea, MgCh, KCI, sodium acetate, potassium acetate, sodium nitrate, sodium bicarbonate, potassium glutamate, ammonium acetate and / or Tween-20.

22. The method of any preceding claim, wherein the method further comprises collecting the RNA in a third composition.

23. The method of any preceding claim, wherein the components for IVT further comprises one or more of: a reducing agent, optionally wherein the reducing agent is DTT; a surfactant; an amine; and / or DMSO.

24. The method of any preceding claim, wherein the components for IVT further comprise an RNA capping agent.

25. The method of any of claims 3 to 24, wherein the rNTPs comprise one or more modified rNTPs.

26. The method of any of claims 3 to 25, wherein the RNA polymerase comprises a modified RNA polymerase.

27. The method of any preceding claim, wherein the first composition comprises the components for IVT in excess.

28. The method of any preceding claim, wherein the method further comprises adding one or more of a source of magnesium ions, ribonucleotide triphosphates, a pyrophosphatase and / or an RNA capping agent to the second and / or further composition prior to step (d) and / or step (g).

29. The method of any preceding claim, wherein the method is a batch method, a batch fed method or a continuous method.

30. The method of any preceding claim, wherein the method reduces a level of dsRNA produced during the step of producing.

31. The method of any of claims 3 to 30, wherein one or more of the components for IVT are immobilised, optionally wherein the pyrophosphatase, transcription template and / or RNA polymerase are immobilised and wherein the second and / or further composition are contacted with the immobilised pyrophosphatase, transcription template and / or RNA polymerase.

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