BE1030203B1 - METHODS AND SYSTEMS FOR CAPPING NUCLEIC ACID MOLECULES - Google Patents

Abstract

Described herein are nucleic acid capping systems. Also described herein are methods of nucleic acid capping with the systems described herein.

Description

1 BE2022/5035

ACID MOLECULES COATING METHODS AND SYSTEMS

NUCLEI C

CONTEXT

In vitro nucleic acid processing is widely used in biomedical or bioscience fields. One of the methods of processing or manufacturing nucleic acids in vitro involves the capping of messenger RNA (mRNA) for the manufacture of mRNA or a peptide encoded by the mRNA in industrial quantities. Two main strategies are currently used for the production of 5'-capped mRNA: co-transcriptional capping, whereby a synthetic oligonucleotide incorporating the cap structure is incorporated during transcription of the template DNA strand; and post-transcriptional capping, whereby the biosynthesis of the cap structure and associated reactions are enzymatically catalyzed.

SUMMARY

Efficient post-transcriptional mRNA capping requires prior processing of the reaction harvest obtained after in vitro transcription (IVT). The traditional method of pretreatment involves at least one purification operation between the TIV step and the enzymatic capping step to ensure sufficient capping efficiency. Such steps increase the time and complexity of the process and decrease the overall yield of capped RNA molecules. Accordingly, systems and methods for simple, inexpensive, and rapid processing of mRNA reaction harvesting to ensure effective downstream enzymatic capping are of interest. Disclosed herein, in certain aspects, is a method of producing at least one capped ribonucleic acid (RNA) molecule, comprising: providing a plurality of uncapped RNA molecules in a first solution; diluting the first solution by a factor of at least 4 to form a second solution containing the plurality of uncapped RNA molecules; contacting the second solution with a plurality of capping enzyme molecules; and adding a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule. In some embodiments, diluting the first solution includes adding a volume of a diluent to said first solution. In some embodiments, the first solution is diluted by a factor between about 4 and 1000. In some embodiments, the first solution is diluted by a factor of at least

2 BE2022/5035 about 200. In some embodiments, the first solution is diluted by a factor of at least about 50. In some embodiments, the first solution is diluted by a factor of at least about 10. In some embodiments, the method further includes removing an excess volume of the second solution. In some embodiments, excess volume removal includes ultrafiltration, microfiltration, tangential flow filtration, or a combination thereof. In some embodiments, the dilution of the first solution does not include an additional purification step.

In some embodiments, the additional purification step includes chromatography. In some embodiments, the dilution of the first solution occurs in the same vessel or reactor as the plurality of uncapped RNA molecules generated through an in vitro transcription (IVT) reaction. In some embodiments, the dilution of the first solution occurs in a different vessel or reactor from the plurality of uncapped RNA molecules generated through a TIV reaction. In some embodiments, the TIV reaction occurs in a continuous reactor or a batch reactor. In some embodiments, the plurality of capping enzymes are selected from the group consisting of cap-specific mRNA (nucleoside-2'-O-)-methyltransferase, Vaccinia capping enzyme (VCE), blue tongue disease virus capping enzyme, chlorella virus capping enzyme, S. cerevisiae virus capping enzyme, mimivirus capping enzyme, chlorella virus capping enzyme African swine fever, and avian reovirus capping enzyme. In some embodiments, the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 70%, at least 75%, at least at least 77%, at least 80%, at least 85%, at least 87%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. In some embodiments, the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at approximately 100% efficiency.

In some embodiments, the RNA includes a nucleic acid sequence encoding a peptide or protein. In some embodiments, the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, RNA polymerase I, RNA polymerase II , an RNA polymerase III, an RNA polymerase IV, an

RNA polymerase V, and a single subunit RNA polymerase. In some embodiments, the method further comprises synthesizing a peptide or

3 BE2022/5035 of a protein using at least one capped mRNA molecule. In some embodiments, diluting the first solution includes decreasing a concentration of a plurality of molecules that inhibits a capping reaction of adding a capping structure to a 5' end of an RNA molecule uncapped. In some embodiments, the concentration of the plurality of molecules is decreased to a level where the capping reaction is no longer inhibited. In some embodiments, diluting the first solution includes decreasing a concentration of the plurality of uncapped RNA molecules. In some embodiments, the concentration of the plurality of uncapped RNA molecules is decreased to a level where the capping reaction is no longer inhibited.

Disclosed herein, in certain aspects, is a pharmaceutical composition obtained using a method described herein. In some embodiments, the pharmaceutical composition is a vaccine and a pharmaceutically acceptable carrier.

Disclosed herein, in certain aspects, is a peptide or protein obtained using a method disclosed herein. In some embodiments, the peptide or protein is produced in vivo. In some embodiments, the peptide or protein is produced in vitro. In some embodiments, the peptide or protein is a prophylactic or therapeutic peptide or protein.

Disclosed herein, in certain aspects, is a composition comprising a plurality of 5'-capped RNA molecules, wherein said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping is produced post-transcriptionally, said composition comprises reagents for in vitro transcription, and wherein the concentration of RNA in said composition is less than 20 mg/ml.

Disclosed herein, in certain aspects, is a composition comprising a plurality of - 5' capped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping reaction is is produced post-transcriptionally, wherein the efficiency of the capping reaction is at least 75% without using chromatography.

Disclosed herein, in certain aspects, is a composition comprising a plurality of 5' capped and uncapped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said

4 BE2022/5035 capping has occurred post-transcriptionally, said composition comprises reagents for in vitro transcription wherein a ratio of the plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between 0.0001 to 1.

Disclosed herein is, in certain aspects, a system for producing at least one capped RNA molecule. The system includes a bioreactor configured to contain a first solution containing a plurality of uncapped RNA molecules, wherein the first solution is diluted by a factor of at least 4 to form a second solution, wherein the second solution comprises the plurality of uncapped RNA molecules; and the bioreactor or a second bioreactor configured to add a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule by contacting the second solution with a plurality of capping enzyme molecules.

BRIEF DESCRIPTION OF DRAWINGS

The present patent application contains at least one drawing executed in color. Copies of this patent or of this patent application with color drawing(s) will be furnished by the Office on request and after payment of the necessary costs.

Fig. 1 illustrates a non-limiting example of the system described herein for nucleic acid capping. The system includes a first batch reactor (101) comprising a first solution containing uncapped nucleic acid such as uncapped RNA molecules. The first solution can be passed through a continuous flow reactor (arrowed line; 104) where it is mixed with a suitable diluent through an inlet at a defined rate (q_dilution) to reach the target dilution. The dilute solution may also be passed through a filtration device (103) before arriving at a second batch reactor (102), from where it is passed to the enzyme capping reactor. Since the concentration of a plurality of molecules that negatively influence the capping reaction is decreased by diluting the first solution (to obtain a second solution), such dilution may increase the 5' capping of RNA molecules transcribed in vitro (VIT). The dilute first solution, i.e., the second solution containing the nucleic acid, may be followed by ultrafiltration or any other suitable method to form a third solution. Ultrafiltration or any other suitable method decreases the total volume of solution to be processed downstream and allows adjustment of the concentration of uncapped mRNA to a concentration that is optimal for the next downstream operation (e.g. the reaction of enzymatic capping). The capped nucleic acid can be processed further downstream, such as by chromatography, tangential flow filtration or other suitable purification methods.

Fig. 2 illustrates a non-limiting example of experimental protocols, where a concentration of uncapped RNA can be diluted 10-fold (10x) in some embodiments.

Fig. 3 illustrates a non-limiting example of an embodiment of the system, which comprises at least one or a first container (301) comprising a first solution (302) comprising at least one uncapped nucleic acid. The first solution (302) can be diluted directly in the first container (301) to obtain a second solution (304). The system may further include at least one additional container (eg, a second container, 303) for diluting the first solution (302) to obtain a second solution (304) in the second container (303). Due to the diminished plurality of molecules that negatively influence the capping reaction in the second solution (304), the capping agents, (e.g., capping enzyme(s)) then cap the nucleic acid (nucleic acid capped, 305). The capped nucleic acid (305) may be further processed, for example, to change the concentration (306) of the capped nucleic acid (305) or exchange buffer systems, before further downstream processing.

The new features of the disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments.

DETAILED DESCRIPTION

Overview

Efficient post-transcriptional capping of messenger RNA produced by in vitro transcription (IVT) is only possible after intermediate processing of the TIV reaction product. This intermediate treatment typically involves a purification operation (such as chromatography), usually in combination with a

6 BE2022/5035 tangential flow filtration stage. This suggests that the product of the in vitro transcription reaction contains substances that interfere with capping reagents. Enzyme capping immediately after TIV, without intermediate treatment of the reaction product, produces reduced or approaching 0% amounts of capped mRNA molecules. When capping reagents are added to TIV for “concomitant transcription and capping” (one-pot reaction), marginal capping can be achieved (less than 10%). This confirms that successful styling requires some intermediate processing of the TIV product. A conventional intermediate treatment, as mentioned above, involves one or more purification steps, which is accompanied by several disadvantages: inevitable loss of product (decreased yield of the process); increased complexity of the process requiring the development of additional steps; need for additional equipment; and increased costs due to product loss and the cost of additional operational steps. The need for additional equipment also leads to increased operational cost, increased system footprint, and extended process time.

Described herein are systems and methods for diluting the TIV product to decrease the concentration of interfering agents to a level where they no longer interfere with the enzymatic capping reaction of adding cap structures to uncapped RNA molecules. The decreased concentration of uncapped RNA molecules prior to performing the capping reaction can also help achieve an efficient capping reaction to add cap structure to an uncapped RNA molecule. As demonstrated herein, dilution of the TIV reaction product can provide a capping efficiency of at least about 90% to 99.9%. This is comparable or substantially similar to the capping efficiency that was achieved under a control condition, whereby the TIV was purified using a nucleic acid purification kit. By achieving substantially similar high mRNA capping efficiency compared to currently conventional methods used in the industry, current systems and methods also achieve significantly reduced operational cost and improved process yield. Furthermore, using current systems and methods, it is much simpler to scale up the production of capped mRNA molecules.

In some embodiments, the systems and methods described herein provide a dilution of a first solution comprising uncapped RNA molecules to form a second solution, which comprises a concentration decreased by a

7 BE2022/5035 plurality of molecules which negatively influence or inhibit the enzymatic capping reaction and the resulting capping efficiency. In some embodiments, the second solution is contacted with a plurality of capping enzymes and other reagents (e.g., —guanosine-5”-triphosphate, S-adenosylmethionine) to produce capped RNA molecules. In some cases, at least one type of capping enzyme is used. In some cases, more than one type of styling enzymes are used. In some embodiments, the dilution factor used to dilute the first solution to obtain the second solution is sufficient to decrease the inhibition of the capping reaction exerted by the plurality of molecules in the second solution. Fig. 1 provides a non-limiting example of a system described here. In this non-limiting example, the 5' capping reaction of an mRNA molecule can be inhibited by the presence of a plurality of molecules in a first solution. By diluting the first solution, a second solution is obtained, where the concentration of the plurality of molecules which negatively influences or inhibits the styling reaction is decreased. In some embodiments, the second solution further passes through a filtration step to remove bulk and refocus the plurality of uncapped RNA molecules. Such a decrease in concentration or removal of the plurality of molecules allows the capping reaction to occur efficiently. In some embodiments, the second solution may undergo buffer exchange by dialysis or other means of buffer exchange of the uncapped mRNA before proceeding to the capping reaction. In some embodiments, the solution containing the capped mRNA molecules can be further purified. In certain embodiments, capped mRNA molecules obtained from the systems and methods described herein can be used for purposes such as the manufacture of pharmaceutical or diagnostic compositions.

Systems

Disclosed herein are systems for processing nucleic acids with the methods described below, such as processing solutions containing uncapped RNA molecules derived from in vitro transcription (IVT) reactions to avoid inhibition of reactions capping procedures relating to the addition of a capping structure to an uncapped RNA molecule. The system may include an upstream portion intended to provide TIV reaction mixtures containing the uncapped RNA molecules. In some cases, the system may include a downstream portion for further processing of capped RNA molecules, such as purification to remove unwanted substances and flow-through filtration.

8 BE2022/5035 tangential to modify the composition and concentration of the solution of capped RNA molecules. In some embodiments, the system can further be configured to manufacture compounds, biomolecules, or pharmaceutical compositions using capped RNA as input. For example, the system described herein can synthesize or increase the yield of synthesis of an antigen encoded by capped RNA or capped mRNA, where the antigen can further be formulated into a vaccine. In some embodiments, the system includes components or devices for initiating or maintaining biological reactions. In some embodiments, the system can be configured to perform any sort of suitable process. Non-limiting examples of methods to which the systems disclosed herein may be suitable include the production of a biological compound; the production of a pharmaceutical or biopharmaceutical compound; RNA synthesis, including TIV, post-transcriptional processes and RNA purification; protein synthesis, including cell-dependent protein synthesis and cell-free protein synthesis (CFPS); or a combination thereof.

In some embodiments, the system described herein is modular, where each component of the system can be independently assembled or disassembled based on the functionality needed. In some embodiments, the system may be an open system or a closed system such that the system includes a continuous reactor or a batch reactor. In some cases, the system may include a continuous reactor. The system can be used in a continuous mode. In other cases, the system may include a batch reactor. The system can be employed in a non-continuous mode or a batch reaction mode. In other cases, the system may comprise a combination of a continuous reactor and a batch reactor and the system may be employed in a semi-continuous mode.

In some embodiments, the system as shown in FIG. 1 comprises at least two containers, where a first container 101 contains a first solution comprising the uncapped RNA molecules. In some embodiments, the first solution can be diluted to obtain a second solution. For example, a fraction of the first solution can be transferred to a second container to dilute the first solution in the second solution. In some cases, this transfer and dilution process is a continuous process. The transfer rate from the first solution to a second solution is regulated and controlled according to a speed of the dilution process. In some cases, after dilution of at least one

9 BE2022/5035 portion of the first solution containing the uncapped RNA molecules, the method further comprises mixing the dilute first solution before transferring the dilute first solution to a next step. In some cases, the transfer and dilution process is a non-continuous process or a batch process. In some cases, the transfer and dilution process is a combination of a non-continuous process and/or a continuous process.

In some cases, the mixing step is performed by a mixing system, which may include a magnetic object, a stirrer, a deflector, a ball, or any other suitable object that can be used to mix a solution.

After dilution or additional mixing, the concentration of the plurality of molecules that inhibit the capping reaction is decreased, leading to capping of uncapped RNA molecules in the second solution. In some embodiments, the dilution factor for diluting the first solution to form the second solution is at least 4, at least 5, at least 6, at least 7, at least 8 , at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, d at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1000, or all numerical number between the dilution factors mentioned above.

As shown in FIG. 1, the system may include a filtration device 103 that is configured to filter the dilute first solution to reconcentrate the plurality of uncapped RNA molecules prior to entry into the batch reactor 102. In some embodiments, the system does not does not include a filtration device 103 or the dilute first solution does not pass through the filtration device 103 before entering the batch reactor 102. The batch reactor 101, the container for diluting the first reaction to obtain the second solution, the mixing device, the filtration device 103, and the batch reactor 102 are in fluid communication with each other. In some embodiments, the capping reaction of adding a cap structure to an uncapped RNA molecule is performed in a batch reactor 102. Any necessary reagents, such as capping enzymes as described herein, guanosine -5'-triphosphate, buffers and salts, a methyl donor (e.g., S--adenosylmethionine), and other necessary reagents can be added to the batch reactor 102 via a valve or an opening .

10 BE2022/5035

In some embodiments, the system may include a filtration unit. Filtration units can include a dead-end filtration unit, a rotary filtration unit, a tangential flow filtration (TFF) unit, an alternating tangential flow filtration (ATF) unit, a microfiltration unit, a ultrafiltration or any other suitable filtration unit known in the art.

Solution can be transported from one part of the system to another (e.g., from one segment to another) or into or out of the system by the opening or closing of valves. The valves can be caused to open or close at certain times by the system. The system may further include pumps or other means, which are further driven by the system to transport the solution.

In some embodiments, the system may include a purification component or device to purify the biomolecule synthesized or present in solution (e.g., the capped RNA molecule or the encoded polypeptide from the RNA molecule capped) and prepare it for any suitable downstream reaction. A non-limiting example of the purification component or device can include chromatography or filtration.

Methods

Described herein are methods for capping RNA molecules (including mRNA molecules, for example) synthesized from a TIV reaction. In some embodiments, the method increases the capping efficiency of RNA molecules synthesized from a TIV reaction. In some embodiments, the method eliminates an inhibition of a capping reaction by adding a cap structure to an uncapped RNA molecule. In some embodiments, the method uses the systems described herein to dilute the plurality of molecules that inhibit the capping reaction. In some embodiments, the method includes diluting a first solution containing the uncapped RNA molecules or the uncapped mRNA molecules to form a second solution, wherein diluting the plurality of molecules that inhibits the capping reaction allows post-transcriptional capping of uncapped RNA or mRNA molecules in the second solution. In some embodiments, the capping reaction is initiated by contacting the second solution with a plurality of capping enzymes and other reagents to form at least one capped RNA molecule. In some embodiments, dilution of the first solution enables post-transcriptional capping efficiency to produce capped RNA molecules.

11 BE2022/5035 to achieve at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 77%, at least 80%, at at least 85%, at least 87%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of RNA molecules — capped, or any percentage between the percentages mentioned above.

In some embodiments, after an in vitro transcription (IVT) reaction to obtain a plurality of uncapped RNA molecules, the DNA template used in the IVT reaction may be removed. For example, the DNA template can be removed by DNase treatment. After treatment with DNase, the TIV reaction can be terminated by heating, cooling or contacting the solution containing the RNA molecules with a chelating agent. A non-limiting example of a chelating agent may include 8-hydroxyquinoline, carboplatin, VEDTA, EGTA, hexadecylpyridinum bromide or sodium tartrate. Prior to capping, uncapped RNA molecules can be denatured (e.g., by heating at 65°C for 5 minutes). The temperature of the denaturation and the duration of the denaturation can vary. In some embodiments, denaturation of uncapped RNA molecules is not performed prior to the capping reaction.

In some embodiments, the capping efficiency can be determined by dividing the amount of uncapped RNA molecules by the amount of capped RNA molecules to obtain a ratio of uncapped RNA/capped RNA. For example, but not limited to, liquid chromatography coupled with UV absorbance measurement and mass spectrometry (LC-UV-MS) can be used to assess concentrations of capped or uncapped RNA molecules . In some embodiments, the concentrations of uncapped RNA molecules and capped RNA molecules are calculated based on the read absorbance measurements of the eluted molecules as identified by on-line mass spectrometry.

The capping effectiveness is calculated based on the calculated concentrations. In some embodiments, the capping efficiency is calculated directly based on read measurements of absorbance of capped RNA molecules and uncapped RNA molecules.

In some embodiments, dilution of the first solution allows capping of uncapped RNA, where after the capping reaction the ratio of uncapped RNA/capped RNA is at most 1.0, at plus 0.8, max 0.6, max 0.4, max 0.2, max 0.1, max 0.09, max 0 .08, not more than 0.07, not more than 0.06, not more than 0.05, not more than 0.04, not more than 0.03, not more than 0.02 , at most 0.01, at

12 BE2022/5035 plus 0.001 or not more than 0.0001. In some embodiments, the ratio of a plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between about 0.001 to about 10. In some embodiments, the ratio of a plurality of uncapped RNA and the plurality of capped RNA molecules is between about 0.001 to about 0.002, about 0.001 to about 0.005, about 0.001 to about 0.01, about 0.001 to about 0.02, about 0.001 to about 0.05, about 0.001 to about 0.1, about 0.001 to about 0.2, about 0.001 to about 0.5, about 0.001 to about 1, about 0.001 to about 5, about 0.001 to about 10, about 0.002 to about 0.005, about 0.002 around

0.01, about 0.002 to about 0.02, about 0.002 to about 0.05, about 0.002 to about 0.1, about 0.002 to about 0.2, about 0.002 to about 0.5, about 0.002 to about 1, about 0.002 to about 5, about 0.002 to about 10, about 0.005 to about 0.01, about 0.005 to about 0.02, about 0.005 to about 0.05, about 0.005 to about 0.1, about 0.005 to about 0, 2, about 0.005 to about 0.5, about 0.005 to about 1, about 0.005 to about 5, about 0.005 to about 10, about 0.01 to about 0.02, about 0.01 to about 0.05, about 0 .01 to about 0.1, about 0.01 to about 0.2, about 0.01 to about 0.5, about 0.01 to about 1, about 0.01 to about 5, about 0.01 to about 10, about 0.02 to about 0.05, about 0.02 to about 0.1, about 0.02 to about 0.2, about 0.02 to about 0.5, about 0.02 to about 1, about 0.02 to about 5, about 0.02 to about 10, about 0.05 to about 0.1, about 0.05 to about 0.2, about 0.05 to about 0.5, about 0.05 to about 1, about 0.05 to about 5, about 0.05 to about 10, about 0.1 to about 0.2, about 0.1 to about 0.5, about 0.1 to about 1, about 0 .1 to about 5, about 0.1 to about 10, about 0.2 to about 0.5, about 0.2 to about 1, about 0.2 to about 5, about 0.2 to about 10, about 0 .5 to about 1, about 0.5 to about 5, about 0.5 to about 10, about 1 to about 5, about 1 to about 10, or about 5 to about 10. In some embodiments, the ratio of a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is about 0.001, about 0.002, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.2, about 0.5, about 1, about 5, or about 10. In some embodiments, the ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is at least about 0.001, about 0.002, about 0.005, about 0.01, about 0.02, about 0.05, about 0 .1, about 0.2, about 0.5, - about 1, or about 5. In some embodiments, the ratio of a plurality of uncapped RNA molecules to the plurality of capped RNA molecules

13 BE2022/5035 is at most about 0.002, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.2, about 0.5, about 1, about 5 , or about 10.

In some embodiments, the capping reaction can be performed at reduced or absent inhibition by diluting the first solution to form the second solution, where no additional purification or filtration is required.

For example, the capping reaction is performed by the method described here without the need to use chromatography to first purify uncapped RNA. In some embodiments, the TIV reaction mixture (eg, the first solution before dilution) is not purified or filtered (eg, by chromatography or conventional filtration) prior to the capping reaction. In some embodiments, the second solution before being contacted with the capping enzyme and the reagents for the capping reaction is filtered to reconcentrate the plurality of uncapped RNA molecules. In some embodiments, the second solution prior to performing the capping reaction is not filtered or purified. In some embodiments, the solution after the capping reaction containing a plurality of capped RNA molecules is not purified or filtered (e.g., by chromatography or conventional filtration) before being formulated into a composition or pharmaceutical composition described herein or to be used for any other application where a capped nucleic acid can be used. In some embodiments, the solution containing a plurality of capped RNA molecules after the capping reaction is purified or filtered.

In some embodiments, the first solution may be diluted by a factor of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 , at least 8, at least 9, at least 10, at least 12, at least 12, at least 14, at least 16, at least 18, d at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least least 100 or at least 1000 to form a second solution, wherein the capping reaction may occur in the second solution after contacting the uncapped RNA molecules with capping agents, for example capping enzymes , in the presence of, for example, enzyme substrates, capping reaction buffers, salts, and other reagents. In some embodiments, the first solution is diluted by a factor of about 1 to about 100.

In some embodiments, the first solution is diluted by a factor of about 1 to about 5, about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, from about 1 to about 50, from about 1 to

14 BE2022/5035 approximately 60, approximately 1 to approximately 70, approximately 1 to approximately 80, approximately 1 to approximately 100, approximately 2 to approximately 5, approximately 2 to approximately 10, approximately 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 60, about 2 to about 70, about 2 to about 80, about 2 to about 100, about 5 to about 10, about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50 , about 5 to about 60, about 5 to about 70, about 5 to about 80, about 5 to about 100, about 10 to about 20, about 10 to about 30, d about 10 to about 40, about 10 to about 50, about 10 to about 60, about 10 to about 70, about 10 to about 80, about 10 to about 100, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 70, about 20 to about 80, about 20 to about 100, about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 100 , about 40 to about 50, about 40 to about 60, about 40 to about 70, about 40 to about 80, about 40 to about 100, about 50 to about 60, d about 50 to about 70, about 50 to about 80, about 50 to about 100, about 60 to about 70, about 60 to about 80, about 60 to about 100, about 70 to about 80, about 70 to about 100, or about 80 to about 100. In some embodiments, the first solution is diluted by a factor of about 1, about 2, about 5 , about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 100. In some modes In some embodiments, the first solution is diluted by a factor of at least about 1, about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, or about 80. In some embodiments, the first solution is diluted by a factor of at most about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 100.

In some embodiments, the first solution may be diluted in the same vessel where the TIV and capping reaction occur. For example, one volume of diluent can be added to the first solution in the same container to form a second (diluted) solution. In some embodiments, any diluent that is inert and does not interfere with the capping reaction of uncapped RNA or mRNA molecules can be used. In some embodiments, the diluent is water.

15 BE2022/5035

In some embodiments, the first solution can be diluted by mixing a portion of the first solution with a diluent in a second container to form the second (diluted) solution. In some cases, the second solution may undergo buffer exchange for a suitable buffer or solution conditions for further processing to occur. In some embodiments, the second solution can be further purified (eg, by chromatography) or concentrated (eg, by filtration or ultrafiltration) prior to the capping reaction. In some embodiments, the volume of the second solution can be reduced prior to the capping reaction (eg, by filtration or ultrafiltration). For example, the second solution can be reconcentrated by contacting it with a membrane filter (eg, a membrane filter having a 10 kDa cutoff) to remove excess fluid. In some embodiments, the membrane may have a molecular weight cut-off (PMCO) of at least 1 kDa, 3 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa , 40 kda, 45 kda, 50 kda, 55 kda, 60 kda, 65 kda, 70 kda, 75 kda, 80 kda, 85 kda, 90 kda, 95 kda, 100 kda, 200 kda, 300 kda, 400 kda, 500 kDa, 600 kDa, 700 kDa, 750 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, or any PMCO between any two of the ranges described herein. In some embodiments, the PMCO is at least about 30 kDa to at least about 100 kDa. In some embodiments, the PMCO is at least about 30 kDa to at least about 500 kDa. In some embodiments, the PMCO is at least about 30 kDa to at least about 1000 kDa. In some embodiments, the second solution may be purified or reconcentrated prior to the capping reaction. In some embodiments, the second solution may be purified or reconcentrated prior to the capping reaction. In some embodiments, the second solution can only be purified prior to the capping reaction.

In some embodiments, the second solution can only be concentrated prior to the capping reaction.

In some embodiments, diluting the first solution to form the second solution creates a volume increase, where the second solution can be concentrated (eg, by filtration or ultrafiltration) to remove excess volume prior to the reaction of styling. In some embodiments, the dilution of the first solution to form the second solution does not create excess volume as required by downstream reactions and the capping reaction can be initiated directly in the second solution. In some embodiments, the capped RNA molecules can then be purified from the second

16 BE2022/5035 solution and further formulated (eg, into a pharmaceutical composition described herein) via various downstream processes. In certain embodiments, additional processes may be performed after the capping reaction to produce a final pharmaceutical composition. For example, capped RNA or mRNA molecules function as templates for the synthesis of peptides or proteins. Capped RNA molecules can also be encapsulated in or adsorbed onto lipid nanoparticles (LNPs) or other RNA delivery systems for vaccine production.

mRNA.

In some embodiments, the method first includes synthesizing the uncapped RNA or mRNA molecules. For example, uncapped RNA molecules can be synthesized from in vitro transcription (IVT). In some embodiments, the TIV reaction can be carried out in any of the vessels of the system described herein. In some embodiments, the TIV reaction occurs in a continuous reactor. In some embodiments, the TIV reaction occurs in a batch reactor. In some embodiments, the TIV reaction can be terminated by inactivation of RNA polymerase in the solution. RNA polymerase can be inactivated by heating, cooling, adding a chelator (e.g., EDTA), or a combination of these. Non-limiting examples of RNA polymerase that can be used to synthesize uncapped RNA molecules via TIV can include T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase. In some embodiments, the method includes template-DNA degradation after TIV.

In some embodiments, the method includes contacting the second solution (comprising the dilute plurality of molecules that inhibits the capping reaction), with a capping enzyme and other reagents to initiate the capping reaction in the second solution . Non-limiting examples of capping enzyme include mRNA (nucleoside-2'-O-)-specific cap-specific methyltransferase,

Vaccinia capping enzyme (VCE), blue tongue disease virus capping enzyme, chlorella virus capping enzyme, S. cerevisiae capping enzyme, capping enzyme mimivirus, African swine fever virus capping enzyme, or avian reovirus capping enzyme. Non-limiting examples of other reagents may include cap-specific mRNA (nucleoside-2"-O-)-methyltransferase, 2'-O-methyltransferase, a salt of

17 BE2022/5035 magnesium, guanosine-5'-triphosphate, S-adenosyImethionine, buffering agents, RNase inhibitor. Non-limiting examples of capping structures formed during the capping reaction include GpppN, m7GpppN (Cap 0), m7GpppméA, m7GpppmlA, m7GpppNm (Cap 1), m2.7GpppNm, m2.2.7GpppNm, m7Gpppm6Am, m7GpppmiAm, m7GpppNmpNm (Cap 2), m7GpppNmpNmpNm (Cap 3), m7GpppNmpNmpNmpNm (Cap 4), where N represents any nucleotide, A adenosine, G guanosine, m a methyl group and p a phosphate group. In some embodiments, the capping structure comprises a chemically modified nucleotide. In some embodiments, the capping reaction described herein yields a majority of one species of capped structure (eg, Cap 1).

In some embodiments, the capping reaction described herein yields other minor cap structures such as an unmethylated cap, Cap 0, Cap 2, or the like.

In some embodiments, the first solution comprises uncapped RNA molecules. In some embodiments, the uncapped RNA molecules can include long chain RNA, coding RNA, noncoding RNA, long noncoding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (ARNIin), circular RNA (circRNA), messenger RNA (mRNA), self-amplifying mRNA (SAM), trans-amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNAs (small RNAi), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immunostimulatory RNA (RNAis), ribozymes, aptamers, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a viral RNA (vRNA), a retroviral RNA or a replicon RNA, a small

nuclear RNA (small nRNA), small nucleolar RNA (small noRNA), microRNA (MIRNA), RNAs associated with the transcription start site (TSSa), upstream antisense RNAs (au), transcription products in upstream of the promoter (PROMPT), or a combination thereof. In some embodiments, the uncapped RNA molecules include at least one chemical modification including a backbone modification, a sugar modification, or a base modification. In this context, a modified RNA molecule includes nucleotide changes, e.g. backbone modifications, sugar modifications or base modifications. A sugar modification related to this disclosure is a chemical modification of the sugar of the nucleotides of the RNA molecule. Further, a base modification in connection with this disclosure is a chemical modification of the base group of the nucleotides of the RNA molecule. In this context, nucleotide modifications are

18 BE2022/5035 selected from nucleotide modifications which are applicable to transcription and/or translation. In additional embodiments, the modified RNA includes nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5- aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5- hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8- oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6- chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, and a combination thereof.

In some embodiments, the method includes diluting a TIV reaction mixture to perform an uninhibited capping reaction. In some embodiments, the TIV reaction mixture (eg, the first solution comprising a plurality of uncapped RNA molecules before dilution) is not purified (eg, by conventional chromatography or other purification methods adequate) before the capping reaction. In some embodiments, the solution before or after the capping reaction (e.g., the second solution before or after being contacted with the capping enzyme and other reagents for the capping reaction) is not purified (e.g., by conventional chromatography or other purification methods). In some embodiments, the second dilute solution does not go through a filtration step or any other adequate step to remove the increase in volume. In some embodiments, the efficiency of the second solution capping reaction is increased due to the dilution of the TIV reaction mixture and not due to the additional purification of the second solution. In some embodiments, diluting the TIV reaction mixture decreases the concentration of the plurality of molecules that inhibit the capping reaction, thereby increasing the efficiency of the capping reaction in the diluted TIV reaction mixture. In some embodiments, the dilute solution, after capping reaction, can be reconcentrated or purified before being formulated into a composition or pharmaceutical composition described herein or used for any other application where a capped RNA can be used. .

19 BE2022/5035

In some embodiments, the method includes diluting the TIV reaction mixture followed by removal of excess fluid volume by filtration or other suitable methods to concentrate the uncapped RNA molecule for downstream processing. In some embodiments, dilution or removal of excess fluid volume (via filtration or other suitable methods) lowers the concentration or removes the majority of the plurality of molecules that inhibits the reaction capping, thereby increasing the efficiency of the capping reaction. In some embodiments, the second diluted and then reconcentrated solution (e.g., via filtration), after capping reaction, may be further purified, concentrated, and/or filtered before passing through a series of downstream process steps to be formulated into a composition or pharmaceutical composition described herein or to be used for any other application where a capped nucleic acid may be used.

In some embodiments, the method includes diluting the TIV reaction mixture followed by removal of the increased volume of fluid by ultrafiltration or microfiltration to concentrate the uncapped nucleic acid molecule (e.g., uncapped RNA molecule). capped) for downstream processing. In some embodiments, the method includes diluting the TIV reaction mixture followed by removal of the increased fluid volume by tangential flow filtration to concentrate uncapped RNA molecules. In some embodiments, dilution or removal of excess fluid volume (via ultrafiltration using a membrane capable of retaining uncapped RNA molecules) decreases the concentration of the plurality of — molecules which inhibits the capping reaction, thereby increasing the efficiency of the capping reaction in the TIV reaction mixture. In some embodiments, the diluted and then reconcentrated TIV reaction mixture (e.g., via ultrafiltration), after capping reaction, may be further purified, concentrated, and/or filtered before being formulated into composition or pharmaceutical composition described herein or to be used for any other application where a capped RNA can be used.

Production of biomolecules

In some instances of this disclosure, the systems and methods described herein are designed to accommodate a reaction/process or part of a reaction/process taking place within the system. In some cases, the reaction involves processing a plurality of uncapped RNA molecules such that a reaction

20 BE2022/5035 capping of adding a cap structure to an uncapped RNA molecule can proceed without interference or inhibition. In some cases, the method also includes steps relating to the in vitro (cell-free) translation of RNA into protein. In some cases, the reaction is a combination of both processes, namely, DNA to RNA through transcription and RNA to protein through translation.

In some cases, in vitro transcription refers to a process in which RNA is synthesized in a cell-free (in vitro) system. In some cases, DNA cloning vectors, especially plasmid DNA vectors, are applied as a template for the generation of RNA transcripts following linearization of circular plasmid DNA. These cloning vectors are usually designed as a transcription vector. RNA can be obtained by DNA-dependent in vitro transcription of an appropriate DNA template. A promoter for controlling RNA transcription in vitro can be any promoter for any DNA-dependent RNA polymerase. In some embodiments, a viral RNA polymerase binds to a viral promoter that is at least one promoter selected from the list consisting of T7, T3, T7lac, SP6, pL, pR, CMV,

SV40 and CaMV35S. Alternatively or in combination, the nucleic acid fragment comprising the promoter sequence comprises a bacterial promoter. In some cases, a bacterial RNA polymerase binds to a bacterial promoter that is at least one promoter selected from the list consisting of araBAD, trp, lac and

Ptac. In some cases, the nucleic acid fragment comprising the promoter sequence includes a eukaryotic promoter. In some cases, eukaryotic RNA polymerase binds to a eukaryotic promoter which is at least one promoter selected from the list consisting of EFla, PGK1, Ubc, beta actin, CAG, TRE, UAS, Ac5,

Polyhedrin, CaMKIIa, ALB, GAL1, GAL10, TEF1, GDS, ADH1, Ubi, H1 and U6. In some cases, the eukaryotic promoter is at least one promoter selected from the list consisting of an RNA pol I promoter, an RNA pol II promoter and an RNA pol III promoter.

In some cases, the DNA-dependent RNA polymerases include at least one of a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase, an RNA polymerase I, an RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V, and single subunit RNA polymerase. The DNA template for in vitro RNA transcription can be obtained by cloning a nucleic acid, in particular a cDNA

21 BE2022/5035 corresponding to respective RNA to be transcribed in vitro, and introduction thereof into a suitable vector for transcription of RNA in vitro, for example into a circular plasmid DNA which is introduced into a host such as a bacterium.

cDNA can be obtained by reverse transcription of mRNA or chemical synthesis. In addition, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis or by amplification reactions, including polymerase chain reaction (PCR).

In some cases, the DNA template is a DNA molecule comprising a nucleic acid sequence encoding the RNA sequence. Template DNA is used as a template for in vitro RNA transcription to produce RNA encoded by template DNA. Therefore, the DNA template includes all the necessary elements for RNA transcription in vitro, in particular a promoter element for the binding of a DNA-dependent RNA polymerase like e.g. RNA polymerases T3, T7 and SP6 5' to the DNA sequence coding for the target RNA sequence. The poly(A) tail can either be encoded in the DNA template or enzymatically added to RNA in a separate step after in vitro transcription. In some cases, the template DNA includes primer binding sites 5' and/or 3' of the DNA sequence encoding the target RNA sequence to determine the identity of the DNA sequence encoding for the target RNA sequence e.g. by PCR or DNA sequencing. In some cases, the DNA template includes a 5' UTR or a 3' UTR. In some cases, the DNA template includes a DNA vector, such as plasmid DNA, which includes a nucleic acid sequence encoding the RNA sequence. In some cases, the DNA template comprises one molecule — linear or circular DNA.

In some instances of this disclosure, a DNA template encodes a different RNA molecular species. In some cases the DNA template contains a sub-genomic promoter and a large open reading frame encoding non-structural proteins which, following administration of the biopharmaceutical compound into the cytosol, are transcribed into four functional components (nsP1, nsP2 , nsP3 and nsp4) by encoded RNA-dependent RNA polymerase (RDRP). RDRP then produces a negative-sense copy of the genome that serves as a template for two positive-strand RNA molecules: genomic mRNA and a shorter subgenomic mRNA. This sub-genomic mRNA is transcribed at very high levels, allowing amplification of an mRNA encoding the antigen of choice. A different RNA molecular species can code for an antigen of different

22 BE2022/5035 serotypes or strains of a pathogen, a different allergen, a different autoimmune antigen, a different antigen of a pathogen, different adjuvant proteins, a different isoform or variant of a cancer or tumor, a tumor antigen different from a patient, an antibody from a group of antibodies that target different epitopes of a protein or group of proteins, different proteins of a metabolic pathway, a single protein from a group of proteins that are lacking in a subject, or a different isoform of a protein for molecular therapy.

In some embodiments, the RNA molecules capped by the method described herein comprise a coding region of a peptide or protein. In such a case, the capped RNA molecules serve as a template for the synthesis of peptides or proteins. For example, the capped RNA molecules can further be formulated into a composition or pharmaceutical composition for administration to a subject, where synthesis of the peptide or protein occurs in vivo. In some embodiments, the peptide or protein can be synthesized directly from capped RNA molecules in either the same or a different system vessel. The peptide or protein synthesized in vitro can then be formulated into a composition or a pharmaceutical composition. In certain embodiments, the peptide or protein encoded by the capped RNA molecules may be prophylactic. In certain embodiments, the peptide or protein encoded by the capped RNA molecules may be therapeutic. In some embodiments, the peptide or protein encoded by the capped RNA molecules has a molecular weight (MW) of from about 1 kDa to about 1000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules has a molecular weight (MW) of about 10 kDa to about 20 kDa, about 10 kDa to about 50 kDa, d about 10 kDa to about 100 kDa, about 10 kDa to about 200 kDa, about 10 kDa to about 300 kDa, about 10 kDa to about 400 kDa, about 10 kDa to about 500 kDa, d about 10 kDa to about 600 kDa, about 10 kDa to about 700 kDa, about 10 kDa to about 800 kDa, about 10 kDa to about 1000 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 100 kDa, from about 20 kDa to about 200 kDa, from about 20 kDa to about 300 kDa, from about 20 kDa to about 400 kDa, from about 20 kDa to about 500 kDa, from about about 20 kDa to — about 600 kDa, from about 20 kDa to about 700 kDa, from about 20 kDa to about 800 kDa, from about 20 kDa to about 1000 kDa, from about 50 kDa to about 100 kDa, from about 50 kDa to about 200 kDa, from about 50 kDa to about 300 kDa,

23 BE2022/5035 from about 50 kDa to about 400 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 600 kDa, from about 50 kDa to about 700 kDa, from about 50 kDa to about 800 kDa, about 50 kDa to about 1000 kDa, about 100 kDa to about 200 kDa, about 100 kDa to about 300 kDa, about 100 kDa to about 400 kDa, about 100 kDa to about 500 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 800 kDa, from about 100 kDa to about 1000 kDa, from about 200 kDa to about 300 kDa, from about 200 kDa to about 400 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 800 kDa, from about 200 kDa to about 1000 kDa, from about 300 kDa to about 400 kDa, from about 300 kDa to about 500 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 800 kDa, from about 300 kDa to about 1000 kDa, from about 400 kDa to about 500 kDa, from about 400 kDa to about 600 kDa, from about about 400 kDa to about 700 kDa, about 400 kDa to about 800 kDa, about 400 kDa to about 1000 kDa, about 500 kDa to about 600 kDa, about 500 kDa to about 700 kDa, d about 500 kDa to about 800 kDa, about 500 kDa to about 1000 kDa, about 600 kDa to about 700 kDa, about 600 kDa to about 800 kDa, about 600 kDa to about 1000 kDa , from about 700 kDa to about 800 kDa, from about 700 kDa to about 1000 kDa, or from about 800 kDa to about 1000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules has a molecular weight (MW) of about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, approximately 200 kDa, approximately 300 kDa, approximately 400 kDa, approximately 500 kDa, approximately 600 kDa, approximately 700 kDa, approximately 800 kDa or approximately 1000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules has a molecular weight (MW) of at least about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa or about 800 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules has a molecular weight (MW) of up to about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa or about 1000 kDa.

In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) of up to about

24 BE2022/5035

10 kDa to about 1000 kDa.

In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight

(PM) of at most about 10 kDa to about 20 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 200 kDa, about kDa to about 300 kDa, about 10 kDa to about 400 kDa, about 10 kDa to about 500 kDa, about 10 kDa to about 600 kDa, about 10 kDa to about

700 kDa, about 10 kDa to about 800 kDa, about 10 kDa to about 1000 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 100 kDa, about 20 kDa to about 200 kDa, about 20 kDa to about 300 kDa, about 20 kDa to about

10,400 kDa, approximately 20 kDa to approximately 500 kDa, approximately 20 kDa to approximately 600 kDa, approximately 20 kDa to approximately 700 kDa, approximately 20 kDa to approximately 800 kDa, approximately

20 kDa to about 1000 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 200 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about

400 kDa, about 50 kDa to about 500 kDa, about 50 kDa to about 600 kDa,

approximately 50 kDa to approximately 700 kDa approximately 50 kDa to approximately 800 kDa approximately 50 kDa to approximately 1000 kDa approximately 100 kDa to approximately 200 kDa approximately 100 kDa to approximately 300 kDa approximately 100 kDa to approximately 400 kDa approximately 100 kDa at approximately

500 kDa, approximately 100 kDa to approximately 600 kDa, approximately 100 kDa to approximately 700 kDa, approximately 100 kDa to approximately 800 kDa, approximately 100 kDa to approximately 1000 kDa, approximately

200 kDa to about 300 kDa, about 200 kDa to about 400 kDa, about 200 kDa to about 500 kDa, about 200 kDa to about 600 kDa, about 200 kDa to about

700 kDa, approximately 200 kDa to approximately 800 kDa, approximately 200 kDa to approximately 1000 kDa, approximately 300 kDa to approximately 400 kDa, approximately 300 kDa to approximately 500 kDa, approximately

300 kDa to about 600 kDa, about 300 kDa to about 700 kDa, about 300 kDa to

— approximately 800 kDa, approximately 300 kDa to approximately 1000 kDa, approximately 400 kDa to approximately 500 kDa, approximately 400 kDa to approximately 600 kDa, approximately 400 kDa to approximately 700 kDa, approximately 400 kDa to approximately 800 kDa, approximately 400 kDa to approximately about 1000 kDa, about

500 kDa to about 600 kDa, about 500 kDa to about 700 kDa, about 500 kDa to about 800 kDa, about 500 kDa to about 1000 kDa, about 600 kDa to about

700 kDa, about 600 kDa to about 800 kDa, about 600 kDa to about 1000 kDa, about 700 kDa to about 800 kDa, about 700 kDa to about 1000 kDa, or about 800 kDa to about 1000 kDa.

In some embodiments, the peptide or protein encoded by the capped RNA molecules has a molecular weight (MW) of up to about 10 kDa, about 20 kDa, about 50 kDa, about

100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa or about 1000 kDa.

In some embodiments, the peptide or protein encoded by the molecules

BE2022/5035 capped RNA comprises a molecular weight (MW) of at most at least about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa or about 800 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) of at most about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa , about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa or about 1000 kDa.

Compositions

Described herein is a composition comprising an agent or composition described herein (e.g., uncapped RNA molecules after treatment as described herein).

In some embodiments, the composition includes substances having a MW of 400 kDa or less at a concentration of less than 15% v/v. In some embodiments, the substances include a PM of about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa or about 1000 kDa. In some embodiments, the substances include a concentration of less than 30% v/v, less than 25% v/v, less than 20% v/v, less than 19% v/v, less than 18% v/v, less than 17% v/v, less than 16% v/v, less than 15% v/v, less than 14% v/v, less than 13% v/v , less than 12% v/v, less than 11% v/v, less than 10% v/v, less than 9% v/v, less than 8% v/v, less than 7 % v/v, less than 6% v/v, less than 5% v/v, less than 4% v/v, less than 3% v/v, less than 2% v/v or less than 1% v/v.

In some embodiments, the composition comprises a plurality of 5' uncapped RNA molecules, wherein said uncapped RNA molecules are obtained by means of an in vitro transcription reaction, said composition comprises reagents for in vitro transcription, and wherein the concentration of uncapped RNA in said composition is less than 20 mg/ml.

In some embodiments, the concentration of uncapped RNA in said composition comprises a range between about 0.01 mg/ml to about 20 mg/ml.

In some embodiments, the concentration of uncapped RNA in said composition comprises a range between about 0.01 mg/ml to about 0.02 mg/ml, about 0.01 mg/ml to about 0.05 mg /ml, about 0.01 mg/ml to about 0.1 mg/ml, about 0.01 mg/ml to about 0.2 mg/ml, about 0.01 mg/ml to about 0.5 mg/ml , about 0.01 mg/ml to about 1 mg/ml, about 0.01 mg/ml to about 2 mg/ml,

26 BE2022/5035 about 0.01 mg/ml to about 5 mg/ml, about 0.01 mg/ml to about 10 mg/ml, about 0.01 mg/ml to about 20 mg/ml, about 0.02 mg/ml to about 0.05 mg/ml, about 0.02 mg/ml to about 0.1 mg/ml, about 0.02 mg/ml to about 0.2 mg/ml, about 0.02 mg/ ml to approximately 0.5 mg/ml, approximately 0.02 mg/ml to approximately 1 mg/ml,

about 0.02 mg/ml to about 2 mg/ml, about 0.02 mg/ml to about 5 mg/ml, about 0.02 mg/ml to about 10 mg/ml, about 0.02 mg/ml to about 20 mg/ml, about 0.05 mg/ml to about 0.1 mg/ml, about 0.05 mg/ml to about 0.2 mg/ml, about 0.05 mg/ml to about 0.5 mg/ml, approximately 0.05 mg/ml to approximately 1 mg/ml, approximately 0.05 mg/ml to approximately 2 mg/ml, approximately 0.05 mg/ml to approximately 5 mg/ml,

about 0.05 mg/ml to about 10 mg/ml, about 0.05 mg/ml to about 20 mg/ml, about 0.1 mg/ml to about 0.2 mg/ml, about 0.1 mg/ ml to about 0.5 mg/ml, about 0.1 mg/ml to about 1 mg/ml, about 0.1 mg/ml to about 2 mg/ml, about 0.1 mg/ml to about 5 mg/ml ml, about 0.1 mg/ml to about 10 mg/ml, about 0.1 mg/ml to about 20 mg/ml, about 0.2 mg/ml to about 0.5 mg/ml, about

0.2 mg/ml to about 1 mg/ml, about 0.2 mg/ml to about 2 mg/ml, about 0.2 mg/ml to about 5 mg/ml, about 0.2 mg/ml to about 10 mg/ml, about 0.2 mg/ml to about 20 mg/ml, about 0.5 mg/ml to about 1 mg/ml, about 0.5 mg/ml to about 2 mg/ml, about 0, 5 mg/ml to about 5 mg/ml, about 0.5 mg/ml to about 10 mg/ml, about 0.5 mg/ml to about 20 mg/ml, about

1 mg/ml to about 2 mg/ml, about 1 mg/ml to about 5 mg/ml, about 1 mg/ml to about 10 mg/ml, about 1 mg/ml to about 20 mg/ml, about 2 mg /ml to about 5 mg/ml, about 2 mg/ml to about 10 mg/ml, about 2 mg/ml to about 20 mg/ml, about 5mg/ml to about 10 mg/ml, about 5 mg/ml to about 20 mg/ml, or about 10 mg/ml to about 20 mg/ml.

In certain embodiments, the concentration of uncapped RNA in said composition comprises a range between about 0.01 mg/ml, about 0.02 mg/ml, about 0.05 mg/ml, about 0.1 mg /ml, about 0.2 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 5 mg/ml, about 10 mg/ml or about 20 mg/ml.

In certain embodiments, the concentration of uncapped RNA in said composition comprises a range between at least about 0.01 mg/ml, about 0.02 mg/ml, about 0.05 mg/ml, about 0.1 mg /ml, about 0.2 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 5 mg/ml or about 10 mg/ml.

In certain embodiments, the concentration of uncapped VRNA in said composition comprises a range between at most about 0.02 mg/ml, about

0.05 mg/ml, about 0.1 mg/ml, about 0.2 mg/ml, about 0.5 mg/ml, about 1mg/ml, about 2 mg/ml, about 5mg/ml, about 10 mg /ml or about 20 mg/ml.

27 BE2022/5035

In some embodiments, the composition comprises a plurality of 5' capped RNA molecules, wherein a capping reaction occurs post-transcriptionally and wherein a ratio of a plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between 0.0001 and 1. In some embodiments, the composition comprises a plurality of 5' capped RNA molecules, wherein a capping reaction occurs post-transcriptionally and wherein a ratio of a plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between 0.01 and 0.5. In some embodiments, the composition comprises the capped RNA molecules or a peptide encoded by the capped RNA molecules.

In some embodiments, the capped RNA molecules or a peptide encoded by the capped RNA molecules can encode an antigen for a vaccine formulation. In some embodiments, the capped RNA molecules are further processed for formulation into a vaccine composition.

In some embodiments, the vaccine composition comprises at least one 5' capped RNA molecule, wherein the 5' capped RNA molecule is a 5' capped mRNA molecule. In some embodiments, at least one 5' capped mRNA molecule can be encapsulated in a nanoparticle to form a pharmaceutical composition. The nanoparticle can comprise lipids, carbohydrates, polypeptides, polymers formed from one or more monomers, or any combination of these (including molecular combinations of these substances). In some embodiments, at least one 5' capped RNA molecule is complexed with a charged polymer, e.g. by electrostatic interaction.

In some embodiments, the pharmaceutical composition comprising the at least one 5'-capped mRNA molecule may be formulated with a charged lipid or an amino lipid. In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule can be formulated by complexation with lipids, liposomes or lipoplexes. For example, complexation may include contacting the at least one 5'-capped mRNA molecule with a PEG-lipid or a zwitterionic lipid comprising a head group, where the positive charge is located near the region of the acyl chain and the negative charge is located at the distal end of the head group.

28 BE2022/5035

In some embodiments, the pharmaceutical composition comprising the at least one 5'-capped mRNA molecule may be formulated with a lipid bilayer carrier.

In some embodiments, the pharmaceutical composition comprising the at least one 5'-capped mRNA molecule may be formulated with a natural or synthetic polymer. Non-limiting examples of such polymers can include chitosan or cyclodextrin. In some embodiments, the pharmaceutical composition comprising the at least one 5'-capped mRNA molecule comprises may be formulated into a polymer formulation comprising a polymer such as polyethenes, polyethylene glycol (PEG), poly( l-lysine) (PLL), cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), polyamine derivative, modified poloxamer, biodegradable polymer, elastic biodegradable polymer, acrylic polymers, amine-containing polymer, dextran polymer, dextran polymer derivative, or a combination thereof. In some embodiments, the pharmaceutical composition comprising the at least one 5'-capped mRNA molecule may be formulated in a polyplex with one or more polymers commonly used in pharmaceutical formulation. In some embodiments, the polyplex comprises two or more cationic polymers such as a poly(ethylene imine) (PEI). In some embodiments, the pharmaceutical composition comprising the at least one 5'-capped mRNA molecule comprises may be formulated as a nanoparticle using a combination of polymers, lipids, or other biodegradable agents. . In some embodiments, the lipid nanoparticles may comprise a 5' capped mRNA core described herein and a polymer shell. The polymer shell can be any of the polymers known in pharmaceutical formulation.

In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier, excipient or diluent. In certain embodiments, the pharmaceutical composition described herein includes at least one additional active agent described herein. In certain embodiments, the at least one additional active agent is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an anti-hormonal agent, an anti-angiogenic agent or a checkpoint inhibitor. In certain embodiments, the at least one additional active agent is an adjuvant to increase the effectiveness of the vaccination.

29 BE2022/5035

In practicing the methods of treatment or use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease, disorder or condition to be treated.

In some embodiments, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. Therapeutic agents, and in some cases compositions described herein, can be used alone or in combination with one or more therapeutic agents as components of mixtures.

The pharmaceutical composition described herein can be administered to a subject by appropriate routes of administration, including, but not limited to, intravenous, intra-arterial, oral, parenteral, buccal, topical, transdermal, rectal , intramuscular, subcutaneous, intraosseous, transmucosal, by inhalation or intraperitoneal. The composition described herein may include, but is not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulse release formulations, multi-particulate formulations, and combination release formulations immediate and controlled.

The pharmaceutical composition including a therapeutic agent can be manufactured in a conventional manner, such as, by way of example only, using conventional mixing, chaotic mixing, laminar mixing, dissolution , encapsulation or other methods.

The pharmaceutical composition may include at least one exogenous therapeutic agent as the active ingredient in acid-free or base-free form, or in pharmaceutically acceptable salt form. Moreover, the methods and compositions described here include the use of N-oxides (if any), crystalline forms, amorphous phases, as well as active metabolites of these biomolecules having the same type of activity. In certain embodiments, the therapeutic agents exist in unsolvated form or in

30 BE2022/5035 solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and others. Solvated forms of the therapeutic agents are also considered to be disclosed herein.

In some embodiments, the pharmaceutical composition provided herein includes one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

The use of absolute or sequential terms, for example, "will", "will not", "should", "should not", "shall", "shall not", "first", "initially", "next", "next", "before", "after", "last" and "finally", are not intended to limit the scope of the present embodiments disclosed herein but by way of examples.

As used herein, the singular forms "un", "une", "le", and "la" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms "including", "includes", "having", "a", "comprising", or variations thereof, are used in the detailed description and/or claims, of such terms are intended to be inclusive in a manner similar to the term "comprising".

As used herein, the expressions "at least one", "one or more" and "and/or" are open expressions which are both conjunctive and disjunctive forms. For example, each of the expressions "at least one of A, Bet C", "at least one of A, B or C", "one or more of A, B and C", "one or more of A, B or C” and “A, B and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

As used herein, "or" may refer to "and", "or", or "and/or" and may be used both exclusively and inclusively. For example, the term "A or

B” can refer to “A or B”, “A but not B”, “B but not A”, and “A and

B”. In some cases, the context may dictate a particular meaning.

31 BE2022/5035

All systems, methods, software and platforms described here are modular. Accordingly, terms such as "first" and "second" do not necessarily imply priority, order of importance, or order of actions.

The term "approximately" when referring to a number or numeric range means that the number or numeric range referred to is an approximation within experimental variability (or within a statistical experimental error), and the number or numerical range may vary, for example, from 1% to 15% of the indicated number or numerical range.

In the examples, the term "approximately" refers to +10% of an indicated number or value.

The terms "increased", "increasing", or "increase" are used herein to generally refer to an increase by a statically significant amount. In some embodiments, the terms "increased" or "increase" refer to an increase of at least about 10% compared to a baseline, e.g., an increase of at least about 10%, at least about 20% , or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at at least about 80%, or at least about 90% or up to and including 100% or any other increase between 10 and 100% compared to a baseline, standard or control. Other examples of "increase" include an increase by a factor of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100 , at least 1,000 or more …—compared to a reference level.

The terms "decreased", "decreasing" or "decrease" are used herein to generally mean a decrease of a statistically significant amount.

In some embodiments, "decreased" or "decrease" means a reduction of at least 10% from a baseline, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least approximately 90% or up to and including 100% (eg, level absent or level not detectable — compared to a reference level), or any decrease between 10 and 100% compared to a reference level. In the context of a marker or symptom, these terms refer to a statistically significant decrease

32 BE2022/5035 of such a level. The decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably up to a level accepted as being within the normal range for a given disease-free individual.

The terms "RNA" or "RNA molecule" are used herein to generally refer to any type of RNA. Non-limiting example of RNA includes long chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (ARNIin), a

circular RNA (circRNA), messenger RNA (mRNA), self-amplifying mRNA (SAM), trans-amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (small RNA), small Hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immunostimulatory RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (small nRNA), small nucleolar RNA (small noRNA), microRNA (miRNA), start site associated RNAs transcription (TSSa), upstream antisense RNAs (au), promoter upstream transcripts (PROMPT), or a combination thereof.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of embodiments herein are not intended to be construed in a limiting sense. Many (many) variations, changes and substitutions will now occur to those skilled in the art without departing from the invention. Further, it is understood that not all aspects of the invention are limited to the specific representations, configurations or relative proportions set forth herein which depend on a variety of conditions and variables. It is understood that various variations to the embodiments of the invention described herein may be employed in the practice of the invention. It is therefore contemplated that the invention should also cover such variants, modifications, variations or equivalents. It is intended that the following claims

33 BE2022/5035 define the scope of the invention and that the methods and structures within the scope of these claims and their equivalents are covered by them.

EXAMPLES

The following illustrative examples are representative of embodiments of the systems and methods described herein and are not intended to be limiting in any way.

Example 1. Dilute Nucleic Acid Capping

Uncapped in vitro transcribed RNA can be diluted prior to the capping reaction. Such dilution decreases the concentration of a plurality of molecules which inhibits the capping reaction. The diluted uncapped RNA can then be contacted with capping reagents to initiate the capping reaction. Fig. 2 illustrates a non-limiting example for dilution and capping of in vitro transcribed RNA (IVT). The solution containing the uncapped RNA molecules can be diluted. TIV reagents and reactions can be used to synthesize uncapped TIV RNA. The solution containing uncapped TIV RNA can be diluted before adding capping reagents for capping reactions. The capping efficiency is then calculated as the proportion of capped RNA relative to the sum of uncapped and capped RNA. Capped RNA can also be purified for further downstream processing.

While the foregoing disclosure has been described in some detail for the purposes of clarity and understanding, it will be apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail may be made without straying from the true scope of the disclosure. For example, all of the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications and/or other documents cited in this application are incorporated by — reference in their entirety for all purposes to the same extent as if each publication, patent, application patent and/or other individual document was individually and separately indicated as incorporated by reference for all purposes.

All publications, patents and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application were

34 BE2022/5035 specifically and individually indicated as being incorporated by reference.

To the extent that the publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to take precedence and/or prevail over any such conflicting material.

Claims (32)

35 BE2022/5035 CLAIMS 1. A method of producing at least one capped ribonucleic acid (RNA) molecule, comprising: has. providing a plurality of uncapped RNA molecules in a first solution; b. diluting the first solution by a factor of at least 4 to form a second solution containing the plurality of uncapped RNA molecules; vs. contacting the second solution with a plurality of capping enzyme molecules; and D. the addition of a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule. 2. A method according to claim 1, wherein diluting the first solution comprises adding a volume of a diluent to said first solution. 3. A method according to claim 1 or claim 2, wherein the first solution is diluted by a factor between about 5 and 1000. 4. A method according to claim 3, wherein the first solution is diluted by a factor of at least about 200. 5. A method according to claim 3, wherein the first solution is diluted by a factor of at least about 50. 6. A method according to claim 3, wherein the first solution is diluted by a factor of at least about 10. 7. A method according to claims 1 to 6, further comprising removing an excess volume of the second solution. 8. A method according to claim 7, wherein the removal of excess volume comprises ultrafiltration, microfiltration, tangential flow filtration, or a combination thereof. 9. Method according to claim 7 or 8, in which the dilution of the first solution does not include an additional purification step. 10. A method according to claim 9, wherein the additional purification step comprises chromatography. 11. A method according to any one of claims 1 to 10, wherein the dilution of the first solution occurs in the same vessel or reactor as the plurality of uncapped RNA molecules generated via an in vitro transcription reaction (IVT). 12. Method according to any one of claims 1 to 10, in which the dilution of the first solution occurs in a (e) tank or a different reactor (e) 36 BE2022/5035 of the plurality of uncapped RNA molecules generated via a TIV reaction. 13. A method according to claim 11, wherein the TIV reaction occurs in a continuous reactor or a batch reactor. 14. A method according to any one of claims 1 to 13, wherein the plurality of capping enzymes is selected from the group consisting of cap-specific mRNA (nucleoside-2'-O-)-methyltransferase, Vaccinia capping enzyme (VCE), blue tongue disease virus capping enzyme, chlorella virus capping enzyme, S. cerevisiae capping enzyme, capping enzyme mimivirus, African swine fever virus capping enzyme, and avian reovirus capping enzyme. 15. A method according to any one of claims 1 to 14, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 87%, at least 90%, at least 95%, d at least 97%, at least 98%, at least 99% or 100%. 16. A method according to any one of claims 1 to 15, wherein the RNA comprises a nucleic acid sequence encoding a peptide or protein. 17. Method according to any one of claims 1 to 15, in which the RNA polymerase is selected from the group consisting of a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase, a RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V, and single subunit RNA polymerase. 18. A method according to any of claims 1 to 17, further comprising synthesizing a peptide or protein using at least one capped mRNA molecule. 19. A method according to any one of claims 1 to 18, wherein diluting the first solution comprises decreasing a concentration of a plurality of molecules which inhibits a capping reaction of adding a capping structure to a 5' end of an uncapped RNA molecule. 20. A method according to claim 19, wherein the concentration of the plurality of molecules is decreased to a level where the capping reaction is no longer inhibited. 21. A method according to any of claims 1 to 20, wherein diluting the first solution comprises decreasing a concentration of the plurality of uncapped RNA molecules. 37 BE2022/5035 22. A method according to claim 21, wherein the concentration of the plurality of uncapped RNA molecules is decreased to a level where the capping reaction is no longer inhibited. 23. Pharmaceutical composition obtained using the method according to any one of claims 1 to 22. 24. A pharmaceutical composition according to claim 23, wherein the pharmaceutical composition is a vaccine and a pharmaceutically acceptable carrier. 25. Peptide or protein obtained using the method according to any one of claims 1 to 24. 26. A peptide or protein according to claim 25, wherein the peptide or protein is produced in vivo. 27. A peptide or protein according to claim 25, wherein the peptide or protein is produced in vitro. 28. A peptide or protein according to claim 25, wherein (which) the peptide or protein is a prophylactic or therapeutic peptide or protein. 29. A composition comprising a plurality of 5' capped RNA molecules, wherein said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping has occurred post-transcriptionally, said composition comprises reagents for in vitro transcription, and wherein the concentration of RNA in said composition is less than 20 mg/ml. 30. A composition comprising a plurality of 5'-capped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping reaction has occurred post-transcriptionally , wherein the efficiency of the capping reaction is at least 75% without using chromatography. 31. A composition comprising a plurality of 5'-capped and uncapped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping has occurred post- transcriptional, said composition comprises reagents for in vitro transcription wherein a ratio of the plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between 0.0001 to 1. 32. A system for producing at least one capped ribonucleic acid (RNA) molecule, comprising: a bioreactor configured to contain a first solution containing a plurality of uncapped RNA molecules, wherein: 38 BE2022/5035 the first solution is diluted by a factor of at least 4 to form a second solution, wherein the second solution comprises the plurality of uncapped RNA molecules; and the bioreactor or a second bioreactor configured to add a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule by contacting the second solution with a plurality of capping enzyme molecules.