BE1030204B1 - 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 BE2022/5036

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; removing a plurality of molecules that has a molecular weight of at most about 1000 kDa in the first solution to form a second solution, such that a post-transcriptional capping efficiency of at least 75% is achievement ; 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 the at least one capped RNA molecule. In some embodiments, the plurality of molecules have a molecular weight of at most about

2 BE2022/5036

800 kDa.

In some embodiments, the plurality of molecules has a molecular weight of no more than about 600 kDa.

In some embodiments, the plurality of molecules has a molecular weight of no more than about 500 kDa.

In some embodiments, the plurality of molecules has a molecular weight of up to about 400 kDa.

In some embodiments, the plurality of molecules has a molecular weight of no more than about 200 kDa.

In some embodiments, the plurality of molecules have a molecular weight of at most about

100 kDa.

In some embodiments, the plurality of molecules have a molecular weight of up to about 30 kDa.

In some embodiments, the plurality of molecules have a molecular weight of no more than about 10 kDa.

In some embodiments, the plurality of molecules have a molecular weight of at most about

5 kDa.

In some embodiments, the plurality of molecules have a molecular weight of up to about 3 kDa.

In some embodiments the removal of the plurality of molecules includes filtering the first solution against a filter, wherein the filter includes a nominal pore size measured at a molecular weight cut-off (PMCO) of about 800 kDa, 600kDa, 500kDa,

400 kDa, 200 kDa, 100 kDa, 50 kDa 30 kDa, 10 kDa, 5 kDa, 3 kDa or 1 kDa.

In certain embodiments, the filtration is carried out by continuous or discontinuous diafiltration.

In some embodiments, the filtration includes tangential flow filtration.

In some embodiments, removing the plurality of molecules includes performing dialysis of the first solution in a suitable medium.

In some embodiments, the removal of the plurality of molecules does not include an additional purification step.

In some embodiments, the additional purification step is performed by chromatography.

In some embodiments, the plurality of uncapped RNA molecules are generated through an in vitro transcription (IVT) reaction. 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%. 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 75%. In some embodiments, adding the cap structure to

3 BE2022/5036 the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 77%. 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 80%. 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 85%. 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 90%. 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 95%. 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 97%. 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 98%. 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 99%. In some embodiments, the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at 100% efficiency.

In some embodiments, a concentration of the plurality of molecules is reduced by at least 50%, 60%, 70%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97 %, or 98%, 99%, or nearly 100% in the second solution compared to a concentration of the plurality of molecules in the first solution. In some embodiments, a concentration of the plurality of molecules is reduced by at least 99.9975% in the second solution compared to a concentration of the plurality of molecules in the first solution. In some embodiments, the concentration of the plurality of molecules in the second solution is less than or equal to 50%, or 40%, or %, or 20%, or 15%, or 10%, or 5%, or 4 %, or 3%, or 2%, or 1% of the concentration of the plurality of molecules in the first solution. In some embodiments, the method further comprises synthesizing a peptide or protein using the at least one capped RNA molecule.

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

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

4 BE2022/5036 the 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 has occurred from 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 capping is is produced post-transcriptionally, wherein a ratio of the plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between 0.0001 to 0.3.

Disclosed herein, in certain aspects, is a system comprising: at least one container containing the first solution or the second solution according to any preceding claim; and at least one filter for separating the plurality of molecules according to any preceding claim.

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 an experimental protocol including buffer exchange (e.g., diafiltration) to remove the plurality of molecules that inhibit the capping reaction from the first solution containing the uncapped RNA molecules.

Fig. 2 illustrates another non-limiting example of an experimental protocol including dialysis to remove the plurality of molecules that inhibit the capping reaction from the first solution containing the uncapped RNA molecules.

Fig. 3 illustrates a non-limiting example of a system described herein, where the system includes at least one container (301) including a first solution (302) including at least one uncapped nucleic acid. The first solution (302) can be brought into contact with a filter membrane (303). Contacting the filter membrane (303) removes a plurality of molecules capable of infiltrating the membrane which inhibits the capping reaction, thereby allowing the capping reaction to occur to generate capped RNA molecules ( 304). The capped nucleic acid (304) can be further purified or filtered to increase the concentration (305) of the capped RNA molecules (304) and to further remove unwanted substances from the solution.

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 tangential flow filtration step. 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, yields a small or near 0% amount 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%). That

6 BE2022/5036 confirms that successful capping 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.

Disclosed herein are systems and methods for removing (by filtration or dialysis) the plurality of molecules that inhibits the capping reaction based on size, wherein the plurality of molecules are smaller and can be filtered to a size of nominal pore smaller than the molecular size of RNA. In some embodiments, the systems and methods include direct filtration through a filter (eg, membrane filter) followed by resolubilization of the retentate in a suitable solvent (eg, water); diafiltration against an adequate number of diavolumes of an adequate solvent; or dialysis of the TIV harvest in a suitable medium to remove substances that inhibit the capping reaction of adding cap structures to uncapped RNA molecules. In some cases, systems and methods may include dilution of the TIV RNA molecules followed by ultrafiltration to remove a portion of the solvent. In some embodiments, the systems and methods lead to incomplete removal of solutes, depending on the chosen dilution factor. For example: a 50x dilution followed by a reconcentration in the original volume of solvent removes 98% of the solutes.

In some embodiments, the systems and methods described herein allow a capping reaction to occur without the need to first isolate or purify the uncapped RNA using conventional methods, such as chromatography. In certain embodiments, the systems and methods described herein achieve an in vitro mRNA 5' capping efficiency that is substantially similar to the capping efficiency achieved using conventional methods of purification of uncapped RNA molecules. In some embodiments, the systems and methods described herein include contacting a solution comprising uncapped RNA molecules with a filter.

In some cases, the filter material includes a ceramic. In some cases,

7 BE2022/5036 the filter material includes one or more minerals. In some cases, the filter material includes one or more metals. In some cases, the filter material includes a polymer. The filter includes pores having a pore size that: allows the plurality of molecules that inhibit the capping reaction to pass through, but retains uncapped RNA molecules. By performing such medium exchange, the plurality of molecules which inhibit the capping reaction of adding cap structures to uncapped RNA molecules are removed from the solution comprising the uncapped RNA molecules until the concentration of the plurality of molecules which inhibit the capping reaction is reduced to a level where it no longer inhibits the capping reaction. In some embodiments, the solution after buffer exchange is contacted with a plurality of capping enzymes and other reagents to form capped RNA molecules. In certain embodiments, capped RNA 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 capping reactions relating to the addition of a cap 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 the capped RNA molecules, such as purification to remove unwanted substances and tangential flow filtration to change the composition and concentration of the solution. capped RNA molecules. In some embodiments, the system can further be configured to make compounds, biomolecules, or pharmaceutical compositions — using the 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 system disclosed herein may be adapted include the production of a biological compound; there

8 BE2022/5036 production of a pharmaceutical or biopharmaceutical compound; RNA synthesis, including TIV and 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 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 instances, a system as disclosed herein may include more than one container. The more than one vessels may be in fluid communication with each other, some subset of the more than one vessels may be in fluid communication with the same or another subset of the more than one vessels, or more than one vessel may not be in fluid communication.

The system can be programmed to transport the medium from one container to another after a certain period of time. In some cases, the time period may be determined by an incubation or reaction time of a reagent or medium component, the length of the vessel, the volume of the vessel, the flow rate of the medium through the container, or some combination thereof. In some embodiments, the system described herein includes at least one filter described herein.

In some embodiments, the filter (eg, a membrane filter) can be positioned between two reactors to generate the second solution from the first solution.

In some embodiments, the system described herein includes at least one container. A first container contains a first solution comprising the uncapped RNA molecules. In some embodiments, the first solution may be filtered to obtain a second solution. Filtration units may include a dead-end filtration unit, rotary filtration unit, tangential flow filtration unit (TFF), alternating tangential flow filtration unit (ATF), or any other suitable filtration unit

9 BE2022/5036 known in the art. In some embodiments, the filtration unit is in fluid communication with the first container. In some embodiments, the filtration unit is not in fluid communication with the first container. After the filtration process, in some cases the systems may additionally include a mixing unit. The second filtered solution can pass through the mixing device. In some embodiments, the filtered second solution may not pass through the mixing device. In some embodiments, the mixing device is contained within the filtration unit.

In some embodiments, the second filtered solution may be transferred to a second container for the capping reaction to occur. In some cases, the capping reaction of adding a cap structure to an uncapped RNA molecule is performed in the second vessel. In some embodiments, the filtration unit is in fluid communication with the second container. In some embodiments, the filtration unit is not in fluid communication with the second container. Any necessary reagents, such as capping enzymes as described herein, capping enzyme substrates, buffers and salts, methyl donor, and other reagents, can be added to the second container via a valve or opening. In some embodiments, the second filtered solution may be returned to the first container for the capping reaction to occur. In some cases, the capping reaction of adding a cap structure to an uncapped RNA molecule is performed in the first vessel. Any necessary reagents, such as capping enzymes as described herein, capping enzyme substrates, buffers and salts, methyl donor, and other reagents, can be added to the first container via a valve or opening.

In some embodiments, the system described herein includes at least one container. A first container contains a first solution comprising the uncapped RNA molecules. In some embodiments, the first solution may undergo a dialysis process to obtain a second solution. In some embodiments, the system includes a dialysis device, which includes a filter suitable for performing a dialysis process. In some embodiments, the dialysis device is in fluid communication with the first container. In some embodiments, the dialysis device is not in fluid communication with the first container. After the process of

10 BE2022/5036 dialysis, in some cases the systems may additionally comprise a mixing unit. The treated second solution can pass through the mixing device.

In some embodiments, the treated second solution may not pass through the mixing device. In some embodiments, the mixing device is contained within the dialysis unit.

After dialysis or dialysis plus mixing, the treated second solution is transferred to a second vessel to perform a capping reaction. In some cases, the capping reaction of adding a cap structure to an uncapped RNA molecule is performed in the second vessel. In some embodiments, the dialysis unit is in fluid communication with the second container. In some embodiments, the dialysis unit is not in fluid communication with the second container. Any necessary reagents, such as capping enzymes as described herein, capping enzyme substrates, buffers and salts, methyl donor, and other reagents, can be added to the second container via a valve or opening. In some embodiments, the second filtered solution may be returned to the first container for the capping reaction to occur. In some cases, the capping reaction of adding a cap structure to an uncapped RNA molecule is performed in the first vessel. Any necessary reagents, such as capping enzymes as described herein, capping enzyme substrates, buffers and salts, methyl donor, and other reagents, can be added to the first container via a valve or opening.

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 includes a purification component or device to capture the compound or biomolecule synthesized or present in solution (e.g., capped RNA molecule or encoded polypeptide from the capped RNA molecule) to remove unwanted substances. A non-limiting example of the purification component or device includes chromatography or filtration.

Methods

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Described herein are methods for capping RNA molecules synthesized from a TIV reaction. In some embodiments, the method uses the systems described herein to remove and separate the plurality of molecules that inhibit the capping reaction from uncapped RNA molecules or uncapped mRNA molecules. In some embodiments, the method includes obtaining a first solution comprising the uncapped RNA molecules. In some embodiments, as shown in FIG. 1, the first solution is contacted with at least one filter (eg, a membrane filter) to form a second solution, where the plurality of molecules that inhibit the capping reaction are passed through the pore of the filter and separated uncapped RNA molecules or uncapped mRNA molecules. In some embodiments, filtration of the first solution by contacting the filter decreases a concentration of the plurality of molecules that inhibits the capping reaction in the second solution. In some embodiments, as shown in FIG.2, the first solution undergoes buffer exchange dialysis, where the plurality of molecules can pass through the pores of the dialysis filter (e.g., membrane filter of dialysis). In some embodiments, the dialysis filter includes the same material or pore size as the at least one filter membrane described herein. In some embodiments, the plurality of molecules in the second solution are eliminated or reduced by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, or any percentage between the percentages mentioned above by filtration or dialysis of the filter. In some embodiments, the plurality of molecules in the second solution is less than or equal to 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1 % or Less of the concentration of the — plurality of molecules in the first solution.

In some embodiments, the filter removes and separates a plurality of molecules that inhibits or interferes with a capping reaction of adding cap structures to uncapped RNA molecules in the first solution to obtain the second solution, where the filter separates the plurality of molecules comprising a molecular weight cut-off (PMCO) of at most approximately 1 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 1 000 kDa, or all — molecular weight between the molecular weight values mentioned above.

12 BE2022/5036

With respect to the filtration/diafiltration/ultrafiltration process, in certain embodiments, an optional step of diluting the reaction mixture of

TIV, i.e., the first solution containing a plurality of uncapped RNA molecules is performed before the filtration process. The diluent can be purified water or any other suitable solution that does not interfere with any downstream reactions. In some embodiments, the TIV reaction mixture is diluted by a factor of at least 1, 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, 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 100, at least 1,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000 or any numerical number between dilution factors mentioned above to form a second solution, where the capping effectiveness in the second solution is increased compared to the capping effectiveness of the undiluted solution. In some embodiments, the first dilution is diluted by a factor of about 1 to about 100. In some embodiments, the first dilution is diluted by a factor of about 1 to about 2, from 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, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 100, about 2 to about 5, about 2 to about 10, about 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 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, 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, d 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, about 50 to about 70, about 50 to about 80, about 50 to about 100, about 60 to about 70, about 60 to

13 BE2022/5036 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 dilution is diluted by a factor of approximately 1, approximately 2, approximately 5, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, 70, about 80, or about 100. In some embodiments, the first dilution 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 dilution 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, about 100, or any numerical number between the dilution factors mentioned above.

Further, as shown in FIG. 2, after the filtration/diafiltration/ultrafiltration process to remove at least a portion of the plurality of molecules which inhibits the capping reaction of adding cap structures to uncapped RNA molecules, an optional step of washing the plurality of uncapped RNA molecules retained by the filter. In some embodiments, RNAase-free purified water or another suitable washing agent can be used to perform this optional step. In some embodiments, this optional washing step can refocus the plurality of uncapped RNA molecules.

In some embodiments, removing the plurality of molecules that inhibits or interferes with a capping reaction increases the capping efficiency in the second solution or allows the capping reaction to achieve substantially similar capping efficiency compared using conventional DNA purification methods. 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 or at least one capped mRNA molecule . In some embodiments, filtration or dialysis of the first solution increases the capping efficiency of the second solution to 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 least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or any percentage between the percentages mentioned above. In some embodiments, filtration or dialysis of the first solution allows the capping reaction to occur at an efficiency of at least

14 BE2022/5036 approximately 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or any percentage between the percentages mentioned above -above.

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, filtration or dialysis of the first solution to obtain a second solution containing a plurality of uncapped RNA molecules and a reduced level of molecules that inhibit the capping reaction allows capping of the RNA molecules uncapped to begin and continue. After the capping reaction, the ratio of uncapped RNA/capped RNA is at most 1.0, at most 0.8, at most 0.6, at most 0.4, d 'at most 0.2, at most 0.1, at most 0.09, at most 0.08, at most 0.07, at most 0.06, at most no more than 0.05, no more than 0.04, no more than 0.03, no more than 0.02, no more than 0.01, no more than 0.001 or no 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 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 to about 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

15 BE2022/5036 0.02, approximately 0.005 to approximately 0.05, approximately 0.005 to approximately 0.1, approximately 0.005 to approximately 0.2, approximately 0.005 to approximately 0.5, approximately 0.005 to approximately 1, approximately 0.005 to approximately 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 to 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 of a plurality of uncapped RNA molecules to 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 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, during the step of filtration or dialysis of the first solution to form the second solution, no additional purification of uncapped RNA molecules is necessary. For example, the capping reaction can occur uninhibited by the method described here without the need to use chromatography or any other purification method used to increase the efficiency of DNA capping reactions by current industry standards.

For the filtration/diafiltration/ultrafiltration process, in some embodiments, the first solution is subjected to the process in the same vessel as

16 BE2022/5036 the RNA synthesis reaction by TIV. In some embodiments, the first solution is transferred to a different vessel to perform the filtration process after the TIV RNA synthesis reaction is complete. In some embodiments, filters (eg, membrane filters) having certain pore sizes as described herein are used to separate molecules based on their sizes. The pore sizes of the filters are selected to remove the plurality of molecules that inhibit the capping reaction of uncapped RNA molecules. Additionally, the pore sizes of the filters are selected to retain uncapped RNA molecules. In some cases, the filters are polymer filters (eg, polymer membranes). Filter materials (eg, membranes) can be any suitable material that can be used in the filtration process. In some embodiments, positive pressure is applied to the first solution against the filter during the filtration process to remove the plurality of molecules that inhibit the capping reaction of uncapped RNA molecules. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or near 100% of the plurality of molecules which inhibit the capping reaction are eliminated. In some embodiments, a forward flow filtration (DFF) process using at least one pumping or centrifugal device is used to collect the retentate, i.e., the second solution which includes a plurality of uncapped RNA molecules and comprises a sufficiently reduced level of a plurality of molecules which inhibits the capping reaction. In other embodiments, a tangential flow filtration (TFF) process is used to collect the retentate as described herein. During the TFF process, the first solution is passed — parallel to a membrane filter rather than being pushed through the filter perpendicularly.

In some embodiments, a diafiltration process is used to collect the retentate as described above. In some embodiments, the diafiltration process is a continuous diafiltration process. The diafiltration solution (water or other suitable buffer) is added to a container containing the first solution at the same rate that the filtrate (eg, the solution containing a plurality of molecules which inhibits the capping reaction) is generated. In this way, the volume in the container remains constant, but the smaller molecules (e.g., the plurality of molecules that inhibit the capping reaction and other salts; having a lower molecular weight and size than RNA molecules not capped) that can freely infiltrate the filter are evacuated. In some embodiments, the diafiltration process is batch diafiltration. In some cases, at

17 BE2022/5036 minus approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or nearly 100% by weight of the plurality of molecules which inhibit the capping reaction are removed.

For the dialysis process, in some embodiments, a dialysis device is provided to contain the TIV reaction mixture, i.e., the first solution.

The dialysis device may be made of a filter capable of retaining uncapped RNA molecules within the dialysis device and allowing the plurality of molecules which inhibit the capping reaction to diffuse out of the dialysis device .

In some embodiments, the filter includes a molecular weight cut-off of at most about 1 kDa to about 1000 kDa. In some embodiments, the filter includes a molecular weight cut-off of at most about 3 kDa to about 200 kDa. In some embodiments, the filter includes a molecular weight cut-off of at most about 5 kDa to about 400 kDa. In some embodiments, the filter includes a PMCO of up to about 10 kDa to about 750 kDa. In some embodiments, the filter includes a PMCO of at least about 3 kDa, 5 kDa, 10 kDa, or 30 kDa. In some embodiments, the filter includes a PMCO of at most about 200 kDa, 400 kDa, 500 kDa, 600 kDa, 750 kDa, or 800 kDa.

The dialysis step serves to remove at least a portion of the plurality of molecules which inhibits the capping reaction. The dialysis process may include changing the dialysis buffer and adding extemporaneous dialysis buffer to the TIV reaction mixture. This cycle of changing and adding can be repeated several times until the plurality of molecules which inhibit the capping reaction are removed to a level where they no longer inhibit the capping reaction. In some embodiments, the dialysis process does not require repeating the change-and-add cycle since adding dialysis buffer to the TIV reaction mixture once is sufficient to remove the plurality of molecules that— inhibits the capping reaction to the level where it no longer inhibits the capping reaction.

In some embodiments, the first solution (i.e., the reaction mixture from the TIV RNA synthesis process) is undiluted before proceeding to the filtration process to remove at least a portion of the plurality of = — molecules that inhibit the capping reaction. In certain other embodiments, the first solution is not diluted before passing to a dialysis process to reduce the concentration of the plurality of molecules which inhibits the

18 BE2022/5036 capping reaction. In some embodiments, the dialysis device can be of any shape, such as a tubular shape. The dialysis device containing the first solution is immersed in a suitable dialysis buffer (eg, water) to allow the plurality of molecules that inhibit the capping reaction to diffuse out of the dialysis buffer. The dialysis buffer can be agitated by a stirring device to facilitate the diffusion of the plurality of molecules which inhibits the capping reaction. In some embodiments, the dialysis buffer may be removed and replaced with extemporaneous dialysis buffer when an equilibrium of the concentrations of the plurality of molecules between the TIV reaction mixture contained in the dialysis device and the dialysis buffer is reached. The dialysis buffer may be replaced several times until a concentration of the plurality of molecules which inhibit the capping reaction is reduced to an adequate level such that the efficiency of the capping reaction is increased by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, close to 100% or any percentage between the percentages mentioned above. In some embodiments, electricity can be used to facilitate the dialysis process.

In some embodiments, the first solution may be diluted in the same vessel where the TIV and capping reaction occur. For example, additional solution can be added to the first solution in the same container to form a second (diluted) solution. In some embodiments, the first solution can be diluted by mixing a portion of the first solution with additional solution in a second container to form the second (diluted) solution. In some embodiments, any solution that is inert and does not interfere with the capping reaction of uncapped RNA or mRNA molecules can be used. In some embodiments, the diluting solution is water. 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). In some embodiments, the dilution of the first solution to form the second solution does not create excess volume and the capping reaction can be initiated directly in the second solution. In some embodiments, the capped RNA molecules or capped mRNA molecules can then be purified from the second solution and further formulated (e.g., into a composition

19 BE2022/5036 pharmaceutical described here). In some embodiments, an additional reaction can be performed in the vessel after the capping reaction, where the capped RNA molecules function as a template to synthesize the compounds or biomolecules described herein.

In some embodiments, the method further comprises purifying or filtering a solution after the second solution has undergone a capping reaction as shown in FIGS. 1 and 2. The solution contains a plurality of capped RNA molecules. The plurality of capped RNA molecules are purified or filtered to remove unwanted substances and prepared for any other suitable downstream reactions described herein to produce pharmaceutical compositions. In some embodiments, the plurality of capped RNA molecules are not further purified or filtered.

In some embodiments, the method first includes synthesizing the uncapped RNA 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 reaction of

TIV occurs in a continuous reactor. In some embodiments, the TIV reaction occurs in a batch reactor. In some embodiments, the TIV reaction occurs in a simulated semi-continuous/continuous mode, wherein the systems described herein include at least one batch reactor and at least one continuous reactor. In some embodiments, the reaction of

TIV can be terminated by inactivation of RNA polymerase in solution. RNA polymerase can be inactivated by heating, cooling, addition of a chelator (eg, 8-hydroxyquinoline, carboplatin, EDTA, EGTA, hexadecylpyridinum bromide, or sodium tartrate), or a combination of these. Non-limiting examples of RNA polymerase that can be used to synthesize uncapped RNA molecules via TIV can include a

T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase

IV, an RNA polymerase V and a single subunit RNA polymerase. In some embodiments, the polymerase is a T3 RNA polymerase, a T7

RNA polymerase or an SP6 RNA polymerase.

20 BE2022/5036

In some embodiments, the method includes contacting the second solution (with the plurality of molecules that inhibit the capping reaction removed by the filter membrane), with at least one capping enzyme and other reagents (eg. , enzyme substrates, buffering agents, magnesium salts) to initiate the capping reaction in the second solution. Non-limiting examples of capping enzyme include cap-specific mRNA (nucleoside-2'-O-)-methyltransferase, Vaccinia capping enzyme (VCE), tongue disease virus capping enzyme blue, the chlorella virus capping enzyme,

S. cerevisiae capping enzyme, mimivirus capping enzyme, 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, magnesium salt, guanosine-5'-triphosphate, S-adenosylmethionine, buffering agents, RNase inhibitor. Non-limiting examples of capping structure include GpppN, m7GpppN (Cap 0), m7Gpppm6A, m7Gpppm1A, m7GpppNm (Cap 1), m2.7GpppNm, m2.2.7GpppNm, m7Gpppm6Am, m7GpppmlAm, 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 (e.g., Cap 1). In some embodiments, the capping reaction described herein yields other minor cap structures such as 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), a small nucleolar RNA (small noRNA), a microRNA

21 BE2022/5036 (MIRNA), transcriptional start site associated RNAs (TSSa), upstream antisense RNAs (ua) and promoter upstream transcripts (PROMPT). 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 related to the present disclosure is a chemical modification of the base group of the nucleotides of the RNA molecule. In this context, nucleotide modifications are selected from nucleotide modifications which are applicable to transcription and/or translation. In additional embodiments, 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,

Vinosine, 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.

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 capping reaction of adding a capping structure to an uncapped RNA molecule can proceed without inhibition. In some cases, the reaction involves in vitro transcription (IVT) of RNA from a DNA template with ensuing post-transcriptional reactions, such as enzyme capping and/or tailing poly(A). In some cases, the reaction is related 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.

22 BE2022/5036

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 the circular plasmid DNA molecule. 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 and 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 and 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, the eukaryotic RNA polymerase binds to a eukaryotic promoter and 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 corresponding to respective RNA to be transcribed in vitro, and introducing it into a suitable vector for transcription of RNA in vitro, for example into circular plasmid DNA which is introduced into a host such as a bacterium.

cDNA can be obtained by reverse transcription of mRNA, chemical synthesis or by amplification (e.g. polymerase chain reaction). Also, the matrix

23 BE2022/5036 DNA for in vitro RNA synthesis can also be obtained by gene synthesis.

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 sub-genomic 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 may code for an antigen of different serotypes or strains of a pathogen, a different allergen, a different autoimmune antigen, a different antigen of a pathogen, different adjuvant proteins, an isoform or a different variant of a cancer or tumor antigen, a different tumor antigen from a patient, an antibody from a group of antibodies that target different epitopes of a protein or group of proteins,

24 BE2022/5036 different proteins of a metabolic pathway, a single protein among a group of proteins that are lacking in a subject, or a different isoform of a protein for a molecular therapy.

In some embodiments, the RNA molecules capped by the method described herein comprise a non-coding region of a peptide or protein. 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, from about 10 kDa to about 700 kDa, from about 10 kDa to about 800 kDa, from about 10 kDa to about 1000 kDa, from about 20 kDa to about 50 kDa, from 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 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, 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 kDa to about 700 kDa, from about 50 kDa to about 800 kDa, from about 50 kDa to about 1000 kDa, from about 100 kDa to about 200 kDa, from about 100 kDa to

25 BE2022/5036 approximately 300 kDa, from approximately 100 kDa to approximately 400 kDa, from approximately 100 kDa to approximately 500 kDa, from approximately 100 kDa to approximately 600 kDa, from approximately 100 kDa to approximately 700 kDa, from approximately about 100 kDa to about 800 kDa, about 100 kDa to about 1000 kDa, about 200 kDa to about 300 kDa, about 200 kDa to about 400 kDa, about 200 kDa to about 500 kDa, d 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, about 300 kDa to about 500 kDa, about 300 kDa to about 600 kDa, about 300 kDa to about 700 kDa, about 300 kDa to about 800 kDa, 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 400 kDa to about 700 kDa, from about 400 kDa to about 800 kDa, from about 400 kDa to about 1000 kDa, from about 500 kDa to about 600 kDa, from about 500 kDa to about 700 kDa, from about 500 kDa to about 800 kDa, from about 500 kDa to about 1000 kDa, from 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 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 has a molecular weight (MW) of up to about 10 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 to about 20 kDa, about 10 kDa to about 50 kDa,

26 BE2022/5036 approximately 10 kDa to approximately 100 kDa, approximately 10 kDa to approximately 200 kDa, approximately

10 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,

— approximately 20 kDa to approximately 50 kDa, approximately 20 kDa to approximately 100 kDa, approximately 20 kDa to approximately 200 kDa, approximately 20 kDa to approximately 300 kDa, approximately 20 kDa to approximately

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, about 50 kDa to about 700 kDa, 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, 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, about 200 kDa to about 800 kDa, about 200 kDa to about 1000 kDa,

approximately 300 kDa to approximately 400 kDa approximately 300 kDa to approximately 500 kDa approximately 300 kDa to approximately 600 kDa approximately 300 kDa to approximately 700 kDa approximately 300 kDa to approximately 800 kDa approximately 300 kDa to approximately 1000 kDa approximately 400 kDa at 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 1000 kDa, approximately

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 capped RNA molecules 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, approximately 400 kDa, approximately 500 kDa, approximately 600 kDa, approximately 700 kDa or

27 BE2022/5036 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, about 0.01 mg/ml to about 5 mg/ml, about 0.01 mg/ml to approximately 10 mg/ml, approximately 0.01 mg/ml to approximately 20 mg/ml, approximately 0.02 mg/ml to approximately 0.05 mg/ml,

28 BE2022/5036 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 about 0.5 mg/ml ml, about 0.02 mg/ml to about 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, about 0.02 mg/ml 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, about 0 0.05 mg/ml to about 1 mg/ml, about 0.05 mg/ml to about 2 mg/ml, about 0.05 mg/ml to about 5 mg/ml, about 0.05 mg/ml to about 10 mg/ml, approximately 0.05 mg/ml to approximately 20 mg/ml, approximately 0.1 mg/ml to approximately 0.2 mg/ml, approximately 0.1 mg/ml to approximately 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, about 0.1 mg/ml to approximately 10 mg/ml, approximately 0.1 mg/ml to approximately 20 mg/ml, approximately 0.2 mg/ml to approximately 0.5 mg/ml, approximately 0.2 mg/ml to approximately 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 approximately 10 mg/ml, approximately 0.5 mg/ml to approximately 20 mg/ml, approximately 1 mg/ml to approximately 2 mg/ml, approximately 1 mg/ml to approximately 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.

29 BE2022/5036

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, the 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 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.

In certain embodiments, the pharmaceutical composition comprising the au

30 BE2022/5036 minus a 5' capped mRNA molecule can be formulated with a lipid bilayer vector.

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.

31 BE2022/5036

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 suitable routes of administration, including, but not limited to, intravenous, intra-arterial, oral, parenteral, buccal, topical, - transdermal, rectally, intramuscularly, subcutaneously, intraosseously, transmucosally, by inhalation or intraperitoneally. 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 forms solvated with pharmaceutically acceptable solvents such as

32 BE2022/5036 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.

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.

33 BE2022/5036

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 1000 or any numerical number between the dilution factors mentioned above 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% (e.g., 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 a — symptom, by these terms is meant a statistically significant decrease in such 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

34 BE2022/5036 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 RNAi), 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 - 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.

35 BE2022/5036

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. Nucleic acid capping following buffer exchange

The capping efficiency of in vitro transcribed RNA was increased by diluting the solution containing the uncapped RNA molecules before the capping reaction. THE

Fig. 1 and Figs. 2 illustrate non-limiting examples of experiments to perform buffer exchange and dialysis for in vitro transcribed RNA (IVT) capping.

The solution containing the uncapped RNA molecules can be brought into contact with a filter comprising a molecular weight cutoff of 10 kDa (PMCO of 10 kDa). After removal of the solution by centrifugation, the retentate can be re-diluted in RNase-free water and the resulting solution again filtered through the 10 kDa PMCO filter. This process can be repeated once again.

The retentate can then be dissolved in RNase-free water, and the uncapped RNA molecules can be contacted with capping enzymes and other capping reagents (GTP, capping buffer concentrate, S-adenosylmethionine , MgCl2) to initiate the capping reaction.

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 that each publication, patent, patent application 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 specifically and individually indicated as being incorporated as

36 BE2022/5036 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 (45)

37 BE2022/5036 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. removing a plurality of molecules that has a molecular weight of at most about 1000 kDa in the first solution to form a second solution, such that a post-transcriptional capping efficiency of at least 75% is achievement ; C. contacting the second solution with a plurality of capping enzyme molecules; and D. adding a cap structure to a 5' end of an uncapped RNA molecule to form the at least one capped RNA molecule. 2. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 800 kDa. 3. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 600 kDa. 4. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 500 kDa. 5. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 400 kDa. 6. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 200 kDa. 7. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 100 kDa. 8. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 30 kDa. 9. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 10 kDa. 10. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 5 kDa. 11. A method according to claim 1, wherein the plurality of molecules have a molecular weight of at most about 3 kDa. 12. A method according to claim 1, wherein removing the plurality of molecules comprises filtering the first solution against a filter, wherein the filter comprises a nominal pore size measured at a cutoff of 38 BE2022/5036 molecular weight (PMCO) of about 800 kDa, 600 kDa, 500 kDa, 400 kDa, 200 kDa, or 100 kDa. 13. A method according to claim 1, wherein removing the plurality of molecules comprises diafiltering the first solution against a filter, wherein the filter comprises a nominal pore size measured in PMCO of about 50 kDa, 30 kDa , 10 kDa, 5 kDa, 3 kDa, or 1 kDa. 14. Method according to claim 13, in which the filtration is carried out by continuous or discontinuous diafiltration. 15. A method according to claim 13, wherein the filtration comprises tangential flow filtration. 16. Method according to claim 1, in which the elimination of the plurality of molecules comprises carrying out a dialysis of the first solution in a suitable medium. 17. Method according to any one of claims 15 or 16, in which the elimination of the plurality of molecules does not include an additional purification step. 18. A method according to claim 17, wherein the additional purification step is carried out by chromatography. 19. A method according to claim 1, wherein the plurality of uncapped RNA molecules is generated via an in vitro transcription (IVT) reaction. 20. A method according to any one of claims 1 to 19, wherein the plurality of capping enzymes is selected from the group consisting of cap-specific mRNA (nucleoside-2'-O-)-methyltransferase, — Vaccinia (VCE) capping enzyme, blue tongue disease virus capping enzyme, chlorella virus capping enzyme, S. cerevisiae capping enzyme, mimivirus capping, African swine fever virus capping enzyme, and avian reovirus capping enzyme. 21. A method according to any one of claims 1 to 20, wherein - addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 70% . 22. A method according to any one of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 75%. 23. A method according to any of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 77%. 39 BE2022/5036 24. A method according to any one of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 80%. 25. A method according to any one of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 85%. 26. A method according to any one of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 90%. 27. A method according to any of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 95%. 28. A method according to any of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 97%. 29. A method according to any one of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 98%. 30. A method according to any of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 99%. 31. A method according to any of claims 1 to 20, wherein the addition of the cap structure to the 5' end of the uncapped RNA molecule occurs at approximately 100% efficiency. 32. A method according to any of claims 1 to 31, wherein a concentration of the plurality of molecules is reduced by at least 50%, 60%, 70%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% in the second solution compared to a concentration of the plurality of molecules in the first solution. 33. A method according to any of claims 1 to 31, wherein a concentration of the plurality of molecules is reduced by at least 99.9975% in the second solution compared to a concentration of the plurality of molecules in the first solution. . 34, A method according to claim 33, wherein the concentration of the — plurality of molecules in the second solution is less than or equal to 50%, or %, or 30%, or 20%, or 15%, or 10%, or 5%, or 4%, or 3%, or 2%, or 1% of the concentration of the plurality of molecules in the first solution. 40 BE2022/5036 35. A method according to any of claims 1 to 34, further comprising synthesizing a peptide or protein using the at least one capped RNA molecule. 36. Pharmaceutical composition obtained using the method according to any one of claims 1 to 35. 37. A pharmaceutical composition according to claim 36, wherein the pharmaceutical composition is a vaccine. 38. Peptide or protein obtained using the method according to any one of claims 1 to 35. 39. A peptide or protein according to claim 38, wherein the peptide or protein is produced in vivo. 40. A peptide or protein according to claim 38, wherein the peptide or protein is produced in vitro. 41. A peptide or protein according to any one of claims 39 to 41, wherein (which) the peptide or protein is a prophylactic or therapeutic peptide or protein. 42. 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. 43. 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 has occurred post-transcriptionally, wherein the efficiency of the capping reaction is at least 75% without using chromatography. 44. 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, wherein a ratio of the plurality of uncapped RNA molecules to the plurality of capped RNA molecules is between 0.0001 to 0.3. 45, System comprising: has. at least one container containing the first solution or the second solution according to any one of the preceding claims; and B. at least one filter for separating the plurality of molecules according to any preceding claim.