Improved in vitro transcription of RNA using DNA beads

Abstract

The present invention relates, inter alia, to an RNA in vitro transcription composition comprising a DNA template (DNA beads) immobilized on beads contained in an RNA in vitro transcription (IVT) buffer wherein the composition is configured to reduce or prevent agglomeration of the DNA beads during the process of RNA in vitro transcription. The field has not recognized the problem of DNA bead agglomeration, which can be solved according to the invention by optimizing the components of the IVT buffer (e.g., adjusting the concentration of spermidine and / or Mg2 +). Further aspects relate in particular to a method for producing RNA using the RNA in vitro transcription composition, an RNA production device.

Description

Background Technology

[0001] This invention relates particularly to RNA in vitro transcription compositions comprising a DNA template (also referred to as DNA beads) immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the composition is configured to reduce or prevent, for example, the aggregation of the DNA beads during an RNA in vitro transcription process. The problem of DNA bead aggregation is not recognized in the art, and such problem can be addressed according to the invention by optimizing the composition of the IVT buffer. Further aspects relate to a method for producing RNA using the RNA in vitro transcription composition, an RNA manufacturing apparatus, and several uses.

[0002] Therapeutic RNAs represent an emerging class of drugs. RNA-based therapeutics include mRNA molecules encoding antigens for use as vaccines. Additionally, the use of RNA molecules in protein replacement therapy or cancer therapy is envisioned in this field. Furthermore, the therapeutic uses of non-coding immunostimulatory RNA molecules, other non-coding RNAs (such as microRNAs and long non-coding RNAs), or RNAs suitable for genome editing (e.g., CRISPR / Cas9 guide RNAs) have been explored. Therefore, RNA-based therapeutics belong to one of the most promising and fastest-growing therapeutic areas in modern medicine.

[0003] Established methods for manufacturing RNA molecules involve numerous individual manufacturing steps. A key step in RNA production is generating a suitable DNA template, which is a major cost factor on an industrial scale. Typically, the DNA template can only be used for a single RNA in vitro transcription reaction and subsequently needs to be destroyed by DNase digestion and ultimately purified to ensure the efficacy and safety of RNA-based therapeutics. Therefore, established manufacturing methods are time-consuming, costly, and require significant laboratory space and equipment.

[0004] Emerging RNA manufacturing methods utilize immobilized DNA templates. For example, methods for immobilizing PCR-amplified DNA templates (e.g., WO2020002598) or linearized plasmid DNA templates (e.g., WO2019122371) onto beads have recently been described. The advantages of DNA immobilization are that the resulting DNA beads can be reused for several in vitro RNA transcription cycles, can be used in automated production methods, and do not require a DNase digestion step, as the DNA beads can be separated from the obtained RNA product by, for example, magnetic separation or filtration.

[0005] However, in vitro RNA transcription using DNA beads, particularly automated in vitro RNA transcription using DNA beads, is not well established in the art and may require further optimization. Therefore, the object of this invention is to provide optimized in vitro RNA transcription compositions based on DNA templates immobilized on beads and optimized methods for RNA synthesis.

[0006] These optimized compositions and methods are particularly well-suited for the production of RNA drugs, especially for the automated production of therapeutic RNA. Summary of the Invention

[0007] In vitro transcription of RNA using DNA fixed on beads is not well established in this field and is therefore likely to be associated with several issues.

[0008] As outlined in the Examples section, the inventors have identified a problem, not yet described, related to undesirable DNA bead aggregation that can occur during in vitro RNA transcription (IVT) processes, particularly after more than one IVT cycle. Therefore, DNA bead aggregation can significantly reduce the yield of produced RNA, especially when more than one IVT cycle is performed, potentially interfering with automated processing steps and leading to contamination of the final RNA product. In the extensive studies described in the Examples section, the inventors have found that certain parameters of the in vitro RNA transcription buffer (IVT buffer) and / or DNA beads can be tuned to reduce or prevent undesirable DNA bead aggregation that strongly negatively impacts RNA yield. Furthermore, the inventors have developed an in vitro RNA transcription method using DNA beads that is particularly well-suited for reducing or preventing DNA bead aggregation.

[0009] Therefore, in a first aspect, the present invention provides an RNA in vitro transcription composition comprising a DNA template (hereinafter referred to as a DNA bead) immobilized on beads contained in an RNA in vitro transcription (IVT) buffer.

[0010] Suitable of the RNA in vitro transcription composition, the composition is configured to reduce or prevent the aggregation of the DNA beads during the RNA in vitro transcription process.

[0011] The RNA in vitro transcription composition comprises a DNA template immobilized on beads, wherein the beads are contained in an RNA in vitro transcription (IVT) buffer, the IVT buffer may contain... - Less than 2 mM spermidine; and / or - Mg molar ratio less than 1.4 2+ With NTP.

[0012] IVT buffer can be characterized by at least one of the following features F1 to F6, or a combination thereof. -F1: Less than 1 mM spermidine, preferably free of spermidine; -F2: Mg with a concentration ranging from 1 mM to 25 mM 2+ Preferably less than 20 mM Mg 2+ ; -F3: Spermine and Mg in the IVT buffer 2+ The total concentration of the additive was less than 25 mM; -F4: Mg 2+ The molar ratio to NTP ranges from 1.2 to 0.5, preferably from 0.8 to 0.6; -F5: Total NTP concentration of at least 3 mM, preferably in the range of 3 mM to 35 mM; -F6: Less than 10 mM DTT, preferably less than or about 1 mM DTT.

[0013] The DNA beads can be characterized by at least one of the following features B1 to B5, or a combination thereof. -B1: Contains a magnetizable material, preferably a (super)paramagnetic material; -B2: In the range of 1 µm to 10 µm, in the range of 4 µm to 5 µm, for example, a diameter of 4.5 µg; -B3: Contains polystyrene or polystyrene derivatives; -B4: The surface load of the beads contains approximately 1 ng DNA / mm². 2 Approximately 5 ng DNA / mm 2 ; -B5: The beads contain a biotin-streptavidin-fixed DNA template.

[0014] In a second aspect, the present invention provides the use of the RNA in vitro transcription composition of the first aspect in a method for manufacturing RNA.

[0015] In a third aspect, the present invention provides a method for generating RNA using an RNA in vitro transcription composition comprising a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer.

[0016] Appropriately, the characteristics of the RNA in vitro transcription composition are further characterized by any of the characteristics in the first aspect.

[0017] The fourth aspect of the invention relates to an RNA manufacturing apparatus comprising the RNA in vitro transcription composition of the first aspect, or configured to perform the method of the third aspect.

[0018] Preferably, the device includes a magnetic unit for mixing the DNA beads and / or for retaining the DNA beads.

[0019] The fifth aspect of the invention relates to the use of an IVT buffer for preventing or reducing the aggregation of DNA beads. Suitably, the IVT buffer for said use is characterized according to the first aspect.

[0020] Another aspect of the invention relates to the use of a buffer containing a chelating agent for preventing or reducing the aggregation of DNA beads during the in vitro transcription of RNA.

[0021] definition For clarity and readability, the following definitions are provided. Any technical features mentioned in these definitions may be read in relation to the various and specific embodiments of the invention. Further definitions and interpretations may be provided in the context of these embodiments.

[0022] about The term "about" is used when the determining factors or values ​​do not need to be exactly the same, i.e., 100% identical. In the context of this invention, "about" means that the determining factors or values ​​may deviate by no more than 20%, preferably no more than 10%, and particularly no more than 5%.

[0023] Reunion: In the context of beads, particularly DNA beads, the term "aggregation" as used herein refers to an assembly of beads that are co-aggregated or clustered. Aggregation is the loosely bound or tightly connected aggregation of beads. Typically, such aggregates can be broken up by mechanical force. Aggregation as used herein must be distinguished from bead trapping by a magnet, which can cause beads to temporarily accumulate in the vicinity of the magnet. However, upon release of the magnet, the temporary accumulation of beads will dissipate, and the beads will disperse within the composition.

[0024] Encoded sequence / encoded region: As used herein, the terms "coding sequence" or "coding region" and the abbreviation "cds" will be recognized and understood by those skilled in the art, and are intended, for example, to refer to a sequence of several nucleotide triplets that can be translated into a peptide or protein. In the context of this invention, cds can be an RNA sequence consisting of a plurality of nucleotides divisible by three, beginning with a start codon and preferably ending with a stop codon.

[0025] Heterogeneous:As used throughout this specification in the context of RNA or amino acid sequences, the term "heterologous" or "heterologous sequence" means (e.g., RNA, amino acid) a sequence that must be understood to originate from another gene, another allele, or, for example, another species or virus. Two sequences are generally considered "heterologous" if they are not derived from the same gene or the same allele. That is, although heterologous sequences may originate from the same organism or virus, they will not appear in the same nucleic acid or protein in nature.

[0026] RNA: The terms "RNA" and "mRNA," for example, refer to ribonucleic acid molecules, which are polymers composed of nucleotides. These nucleotides are typically adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (i.e., ribose) of the first monomer and the phosphate ester moiety of the second adjacent monomer. The specific sequence of monomers is called the RNA sequence. mRNA (messenger RNA) provides the nucleotide-coding sequence that can be translated into the amino acid sequence of a specific peptide or protein.

[0027] RNA in vitro transcription The term "in vitro RNA transcription" refers to a method in which RNA is synthesized in a cell-free system. RNA can be obtained through DNA-dependent RNA in vitro transcription of a suitable DNA template, which, according to the invention, can be a DNA template immobilized on beads. The promoter used to control the in vitro RNA transcription can be any promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5 RNA polymerases. The in vitro RNA transcription occurs in an in vitro transcription buffer (IVT buffer) containing the components required to transcribe the DNA template into RNA. A typical IVT buffer may contain: ribonucleotide triphosphates (NTPs) (e.g., adenine, cytosine, guanine, and uracil); optionally, cap analogs (e.g., Cap1 analogs); optionally, further modified nucleotides (e.g., N1-methylpseuuridine (m1ψ)); a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5 RNA polymerase) capable of binding to a promoter sequence within a DNA template; optionally, a ribonuclease (RNase) inhibitor for inactivating any potentially contaminating RNase; optionally, a pyrophosphatase for degrading pyrophosphate (an inhibitor of RNA synthesis); and MgCl2, which supplies Mg... 2+ Ions as cofactors of polymerase; buffer substances (e.g., TRIS or HEPES), which may also contain antioxidants (e.g., DTT); optional polyamines, such as spermidine. Detailed Implementation

[0028] When referring to the "SEQ ID NO" of other patent applications or patents, the sequence (e.g., amino acid sequence or nucleic acid sequence) is explicitly incorporated herein by reference. When referring to the "SEQ ID NO" in the context of an RNA sequence, a person skilled in the art can also deduce the RNA sequence from the referenced SEQ ID NO, provided that a DNA sequence is provided. When referring to the "SEQ ID NO" in the context of a DNA sequence, a person skilled in the art can also deduce the corresponding DNA sequence from the referenced SEQ ID NO, provided that an RNA sequence is provided.

[0029] RNA in vitro transcription composition: In a first aspect, the present invention provides an RNA in vitro transcription composition.

[0030] Preferably, the RNA in vitro transcription composition comprises a DNA template immobilized on beads, wherein the beads are contained in an RNA in vitro transcription (IVT) buffer.

[0031] It should be noted that the features and implementations described and defined in the context of the first aspect, particularly those related to IVT buffers or DNA beads, are equally applicable to any of the other aspects of the invention.

[0032] In the context of this invention, beads containing a fixed DNA template as detailed herein are also referred to as DNA beads. Suitably, the DNA beads are contained in an IVT buffer as dispersed DNA beads or particles. Preferably, the DNA beads are dispersed in the IVT buffer as free-floating DNA beads induced by mixing or rotating (e.g., by magnetic force or an oscillator) of the RNA in vitro transcription composition.

[0033] As described herein, the inventors encountered a problem of DNA bead aggregation, which has not yet been described, occurring during in vitro RNA transcription reactions using DNA beads as templates. Therefore, IVT buffer and DNA beads have been adapted to reduce or prevent DNA bead aggregation. Bead aggregation can be determined using a microscope at 500x or 1000x magnification.

[0034] In a preferred embodiment, the RNA in vitro transcription buffer is configured to reduce or prevent the aggregation of DNA beads during, for example, an RNA in vitro transcription process. In a preferred embodiment, the RNA in vitro transcription buffer is configured to reduce or prevent the aggregation of DNA beads (e.g., beads that are dispersed and / or free-floating) in the first, second, and / or third RNA in vitro transcription reactions (e.g., IVT cycles).

[0035] In a preferred embodiment, the RNA in vitro transcription composition comprises a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer contains ● Less than 2 mM spermidine; and / or ● Less than 25 mM Mg2+; and / or ●Molar ratio of Mg2+ to NTP less than 1.4.

[0036] In a preferred embodiment, the RNA in vitro transcription composition comprises a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer contains ● Less than 2 mM spermidine; and ●Molar ratio of Mg2+ to NTP less than 1.4.

[0037] In a preferred embodiment, the RNA in vitro transcription composition comprises a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer contains ● Less than 25 mM Mg2+; and ●Molar ratio of Mg2+ to NTP less than 1.4.

[0038] In a particularly preferred embodiment, the RNA in vitro transcription composition comprises a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer contains ● Less than 2 mM spermidine; and ● Less than 25 mM Mg2+; and ●Molar ratio of Mg2+ to NTP less than 1.4.

[0039] The preferred features of the IVT buffer will be described in further detail below.

[0040] Polyamines such as spermidine Typically, IVT buffers contain polyamine compounds, such as spermidine. Speridine is generally considered a key component of in vitro RNA transcription, especially because it is thought to improve the overall efficiency of RNA polymerase. However, as described herein, polyamines such as spermidine can cause or promote undesirable aggregation of DNA beads, an unexpected and undescribed negative effect that can, for example, lead to reduced RNA yield (especially when more than one IVT cycle) and / or reduced RNA quality. Therefore, reducing the amount of polyamine compounds (such as spermidine) in the IVT buffer is preferred.

[0041] In a preferred embodiment, the IVT buffer contains spermidine at concentrations of less than 2 mM, less than 1.5 mM, less than 1.0 mM, and preferably less than 500 µM. In a particularly preferred embodiment, the IVT buffer is substantially free of spermidine.

[0042] In a preferred embodiment, the IVT buffer contains a polyamine compound at a concentration of less than 2 mM, less than 1.5 mM, less than 1 mM, preferably less than 500 µM, and more preferably the IVT buffer is substantially free of polyamine compounds.

[0043] Surprisingly, the performance of in vitro RNA transcription using DNA beads (e.g., RNA yield) is not impaired by reduced concentrations of spermidine or polyamines in the IVT buffer (e.g., less than 2 mM). Even more surprisingly, the performance of in vitro RNA transcription using DNA beads is not impaired even when the reaction is carried out in an IVT buffer that is substantially free of spermidine or alternative polyamine compounds.

[0044] Mg2+ ions Typically, IVT buffers contain divalent cations, usually Mg2+. 2+ This is a cofactor required by RNA polymerase. Mg 2+ The ions are typically provided by magnesium salts such as MgCl₂ or MgOAc₂. However, as described herein, ions such as Mg 2+ Divalent cations can cause or promote undesirable aggregation of DNA beads, an unexpected and undescribed negative effect that can, for example, cause reduced RNA yield and / or RNA quality. In the preferred embodiment in this context, Mg 2+ Provided by MgCl2. In a preferred embodiment, Mg 2+ Keep it as low as possible to minimize the risk of DNA bead aggregation.

[0045] In a preferred embodiment, the IVT buffer contains less than 30 mM of divalent cations, preferably less than 25 mM or less than 20 mM of divalent cations.

[0046] In a preferred embodiment, the IVT buffer contains less than 25 mM, more preferably less than 20 mM of Mg. 2+ In the implementation method, Mg 2+ The concentration can be further reduced, particularly in embodiments where NTPs are fed during RNA in vitro transcription, as further described in the context of the third aspect. Therefore, the IVT buffer may contain less than 20 mM, less than 15 mM, less than 10 mM, or less than 5 mM of Mg. 2+ .

[0047] In a preferred embodiment, the IVT buffer contains Mg at a concentration of 1 mM to 25 mM, 2 mM to 25 mM, preferably in the range of 5 mM to 20 mM, more preferably in the range of 10 mM to 20 mM or 15 mM to 20 mM. 2+ Specifically, the IVT buffer contains Mg2+ at concentrations ranging from 18 mM to 20 mM. 2+ In a specific embodiment, the IVT buffer contains approximately 18 mM Mg. 2 + Approximately 19 mM Mg 2+ Or approximately 20 mM Mg 2+ .

[0048] In the implementation method, Mg 2+ The concentration can be further reduced, particularly in embodiments where NTPs are fed during RNA in vitro transcription, as further described in the context of the third aspect. Therefore, the IVT buffer can contain Mg in a concentration ranging from 1 mM to 20 mM, preferably from 1 mM to 15 mM. 2+ Specifically, the IVT buffer contains Mg2+ in concentrations ranging from 2 mM to 15 mM, 2 mM to 10 mM, and 2 mM to 5 mM. 2+ In a specific embodiment, the IVT buffer contains approximately 2 mM Mg. 2+ Approximately 3 mM Mg 2+ or approximately 4 mM Mg 2+ .

[0049] Surprisingly, the performance of in vitro RNA transcription using DNA beads (e.g., RNA yield) was not affected by the Mg in the IVT buffer. 2+ Concentration decreases (e.g., less than 25 mM Mg) 2+ And thus suffer damage.

[0050] Divalent or trivalent ions Typically, IVT buffers contain cationic components (e.g., Mg). 2+ Ions and / or polyamine compounds (such as spermidine)). However, as described herein, these cations can cause or promote undesirable aggregation of DNA beads, an unexpected and undescribed negative effect that can, for example, cause reduced RNA yield (especially in cases involving more than one IVT cycle) and / or reduced RNA quality. Therefore, reducing the total amount of cations in the IVT buffer is preferred.

[0051] In embodiments, the IVT buffer contains less than 30 mM of divalent or trivalent cations, less than 25 mM of divalent or trivalent cations, and preferably less than 20 mM of divalent or trivalent cations. In embodiments, the IVT buffer may contain less than 15 mM, less than 10 mM, or less than 5 mM of divalent or trivalent cations.

[0052] In a preferred embodiment, Mg in the IVT buffer 2+ The total concentration of added spermidine is less than 30 mM, preferably less than 25 mM, and more preferably less than 20 mM. In the embodiments, Mg 2+ The concentration of spermidine can be further reduced, especially in embodiments where NTPs are fed during RNA in vitro transcription. Therefore, the IVT buffer may contain added Mg2+ at total concentrations of less than 20 mM, less than 15 mM, less than 10 mM, or less than 5 mM. 2+ And spermidine.

[0053] In a preferred embodiment, the IVT buffer contains Mg2+ in a total concentration ranging from 1 mM to 30 mM, from 2 mM to 25 mM, preferably from 5 mM to 20 mM, and more preferably from 10 mM to 20 mM or 15 mM to 20 mM. 2+ And spermidine. Specifically, the IVT buffer contains Mg2+ with a total added concentration ranging from 18 mM to 20 mM. 2+ And spermidine. In a specific embodiment, the IVT buffer contains approximately 18 mM Mg. 2+ And spermidine, approximately 19 mM Mg 2+ And spermidine or about 20mM Mg 2+ And spermidine.

[0054] In the implementation method, Mg 2+The total concentration of spermidine added can be further reduced, especially in embodiments where NTPs are fed during RNA in vitro transcription. Therefore, the IVT buffer can contain a total added concentration ranging from 1 mM to 20 mM, preferably from 1 mM to 15 mM of Mg. 2+ And spermidine. Specifically, the IVT buffer contains added total concentrations ranging from 2 mM to 15 mM, 2 mM to 10 mM, and 2 mM to 5 mM of Mg. 2+ And spermidine. In a specific embodiment, the IVT buffer contains approximately 2 mM Mg. 2+ And spermidine, approximately 3 mM Mg 2+ and spermidine or about 4 mM Mg 2+ And spermidine.

[0055] NTP IVT buffer typically contains a mixture of ribonucleotide triphosphates (NTPs). To allow for efficient in vitro RNA transcription without causing DNA bead aggregation, the amount of NTPs can be adjusted to an appropriate value.

[0056] In a preferred embodiment, the NTP mixture comprises adenine, cytosine, guanine, and uracil.

[0057] In a preferred embodiment, the NTP mixture contains modified nucleotides.

[0058] In a preferred embodiment, the modified nucleotides in the NTP mixture are modified uracil nucleotides.

[0059] In some embodiments, the modified nucleotide is selected from pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, and 2'-O-methyluridine. Therefore, in a preferred embodiment, the IVT buffer of the present invention comprises a modified nucleotide selected from pseudouridine (ψ) and / or N1-methylpseuuridine (m1ψ). In the context of this invention, N1-methylpseuuridine (m1ψ) is particularly preferred.

[0060] In a preferred embodiment within this context, the IVT buffer comprises a mixture of NTPs containing G, C, A, and U nucleotides as defined herein. Therefore, in this embodiment, the IVT buffer does not contain modified nucleotides.

[0061] In other preferred embodiments within this context, the IVT buffer comprises an NTP mixture containing G, C, A, and m1ψ as defined herein.

[0062] In an alternative implementation within this context, the IVT buffer comprises an NTP mixture containing G, C, A, and ψ as defined herein.

[0063] In a preferred embodiment, the NTP mixture is optimized for the given RNA sequence to be generated (according to claims 1 to 35 of WO2015188933). Thus, in order to generate an RNA sequence with a G:C:A:U ratio of 1:2:3:2, the corresponding sequence-optimized NTP mixture contains G:C:A:U in a molar ratio of 1:2:3:2.

[0064] In a preferred embodiment, the IVT buffer contains NTPs at a total concentration of at least 1 mM, at least 2 mM, and at least 3 mM.

[0065] In a preferred embodiment, the IVT buffer contains NTPs in a total concentration ranging from 1 mM to 45 mM, 1 mM to 35 mM, 3 mM to 35 mM, preferably 7 mM to 30 mM, more preferably 15 mM to 30 mM, or 20 mM to 30 mM. In a particularly preferred embodiment, the IVT buffer contains NTPs in a total concentration ranging from 26 mM to 28 mM. In a specific embodiment, the IVT buffer contains approximately 26 mM NTP, approximately 27 mM NTP, and approximately 28 mM NTP.

[0066] In embodiments, the concentration of NTPs can be further reduced, particularly in embodiments where NTPs are fed during the in vitro transcription of RNA. Therefore, the IVT buffer can contain NTPs at concentrations ranging from 2 mM to 30 mM, preferably from 2 mM to 20 mM. Specifically, the IVT buffer can contain NTPs at concentrations ranging from 3 mM to 20 mM, 3 mM to 15 mM, or 3 mM to 7 mM. In specific embodiments, the IVT buffer contains approximately 3 mM NTP, approximately 4 mM NTP, or approximately 5 mM NTP.

[0067] It is worth noting that the term "total concentration" of NTP (NTP mixture) is related to the molar concentration of ribonucleotides (e.g., A, G, U, C) and modified ribonucleotides (e.g., m1ψ, ψ) that may be added to the NTP (NTP mixture) of the IVT buffer.

[0068] In a preferred embodiment within this context, the NTP (NTP mixture) may comprise a modified nucleotide as defined herein, preferably selected from pseudouridine (ψ) or N1-methylpseudouridine (m1ψ). Most preferably, the NTP comprises N1-methylpseudouridine (m1ψ).

[0069] In a particularly preferred embodiment, the IVT buffer contains a total concentration of NTPs as defined herein, wherein the NTPs include N1-methylpseudouridine. In a specific embodiment, the IVT buffer contains approximately 27 mM NTPs, wherein the NTPs include N1-methylpseudouridine.

[0070] The ratio of NTP to Mg2+ As described above, IVT buffer contains NTPs and Mg. 2+ Cations promote RNA synthesis. To allow for efficient in vitro RNA transcription without causing DNA bead aggregation, Mg can be used. 2+ The ratio of Mg to NTP (e.g., molar ratio) is adjusted to an appropriate value. In state-of-the-art IVT schemes, Mg... 2+ The ratio to NTP is usually greater than 1.5.

[0071] In a preferred embodiment, Mg in the IVT buffer 2+ The molar ratio to NTPs (as defined herein as total NTP concentration) is less than 1.4, preferably less than 1.2, and more preferably less than 1.0. For example, Mg 2+ The molar ratio to NTP is approximately 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4.

[0072] In a preferred embodiment, Mg in the IVT buffer 2+ The molar ratio to NTPs (as defined herein as total NTP concentration) ranges from 1.4 to 0.5, 1.3 to 0.5, 1.2 to 0.5, and 1.1 to 0.5, preferably from 1 to 0.5, and more preferably from 0.8 to 0.6. In a specific embodiment, Mg 2+ The molar ratio to NTPs (total NTP concentration as defined herein) is about 0.8, about 0.7, or about 0.6. In a particularly preferred embodiment, the Mg in the IVT buffer... 2+The molar ratio to NTPs (as defined herein as total NTP concentration) is approximately 0.7.

[0073] Surprisingly, the performance of in vitro RNA transcription using DNA beads (e.g., RNA yield) was not affected by Mg. 2+ The ratio to NTP (a ratio below 1.4) is impaired. Even more surprisingly, even Mg... 2+ Even with a ratio to NTPs below 1.2, the performance of in vitro RNA transcription using DNA beads is not compromised. Reducing Mg in in vitro RNA transcription... 2+ The ratio to NTPs has a beneficial effect, namely, it can reduce and / or prevent DNA bead aggregation.

[0074] In a preferred embodiment, Mg in the IVT buffer 2+ The molar ratio to NTP ranges from 1.5 to 0.5, 1.2 to 0.5, preferably from 1 to 0.5, more preferably from 0.8 to 0.6 (e.g., 0.7), and the IVT buffer contains Mg at concentrations of 1 mM to 25 mM, 2 mM to 25 mM, preferably from 5 mM to 20 mM, more preferably from 10 mM to 25 mM or 15 mM to 20 mM, particularly from 18 mM to 20 mM. 2+ In a specific implementation within this context, the Mg in the IVT buffer... 2+ The molar ratio to NTP ranges from 1.5 to 0.5, 1.2 to 0.5, preferably from 1 to 0.5, and more preferably from 0.8 to 0.6 (e.g., 0.7), and the IVT buffer contains approximately 18 mM Mg. 2+ Approximately 19 mM Mg 2+ Or approximately 20 mM Mg 2 + .

[0075] In other embodiments, Mg in the IVT buffer 2+ The molar ratio to NTP ranges from 1.5 to 0.5, 1.2 to 0.5, preferably from 1 to 0.5, and more preferably from 0.8 to 0.6 (e.g., 0.7), and the IVT buffer contains Mg in a concentration ranging from 1 mM to 20 mM, preferably from 1 mM to 15 mM, or from 2 mM to 15 mM, 2 mM to 10 mM, or 2 mM to 5 mM. 2+ In a specific embodiment within the aforementioned context, Mg in the IVT buffer... 2+The molar ratio to NTP ranges from 1.5 to 0.5, 1.2 to 0.5, preferably from 1 to 0.5, and more preferably from 0.8 to 0.6 (e.g., 0.7), and the IVT buffer contains approximately 2 mM Mg. 2+ Approximately 3 mM Mg 2+ or approximately 4 mM Mg 2+ .

[0076] In the preferred embodiment within this context, Mg in the IVT buffer 2+ The molar ratio to NTPs (as defined herein as total NTP concentration) ranged from 0.8 to 0.6, and the corresponding Mg... 2+ NTP concentration is selected from: -Mg concentration from 1 mM to 6 mM 2+ And NTPs at concentrations ranging from 1.3 mM to 10 mM; -Mg concentrations from 3 mM to 9 mM 2+ And NTPs at concentrations ranging from 3.8 mM to 15 mM; -Mg concentrations from 6 mM to 12 mM 2+ And NTPs at concentrations ranging from 7.5 mM to 20 mM; -Mg concentration of 9 mM to 15 mM 2+ And NTPs at concentrations ranging from 11.3 mM to 25 mM; -Mg concentrations of 12 mM to 18 mM 2+ and NTPs at concentrations of 15 mM to 30 mM; -Mg concentrations ranging from 15 mM to 21 mM 2+ And NTPs at concentrations ranging from 18.8 mM to 35 mM; -Mg concentrations ranging from 18 mM to 24 mM 2+ And NTPs at concentrations ranging from 22.5 mM to 40 mM; -Mg concentrations of 21 mM to 27 mM 2+ And NTP concentrations ranging from 26.25 mM to 45 mM.

[0077] In a more preferred embodiment within this context, Mg in the IVT buffer 2+ The molar ratio to NTPs (as defined herein as total NTP concentration) is approximately 0.7, and the corresponding Mg... 2+ NTP concentration is selected from: -Mg concentration from 1 mM to 6 mM 2+ And NTPs at concentrations ranging from 1.4 mM to 8.6 mM; -Mg concentrations from 3 mM to 9 mM2+ And NTPs at concentrations ranging from 4.3 mM to 12.9 mM; -Mg concentrations from 6 mM to 12 mM 2+ And NTPs at concentrations ranging from 7.5 mM to 17.1 mM; -Mg concentration of 9 mM to 15 mM 2+ And NTPs at concentrations ranging from 8.6 mM to 21.4 mM; -Mg concentrations of 12 mM to 18 mM 2+ And NTPs at concentrations ranging from 17.1 mM to 25.7 mM; -Mg concentrations ranging from 15 mM to 21 mM 2+ And NTPs at concentrations ranging from 21.4 mM to 30 mM; -Mg concentrations ranging from 18 mM to 24 mM 2+ And NTPs at concentrations ranging from 25.7 mM to 34.3 mM; -Mg concentrations of 21 mM to 27 mM 2+ And NTPs at concentrations ranging from 30 mM to 38.6 mM.

[0078] Hat analogues / hat structures In a preferred embodiment, the IVT buffer contains a cap analog, preferably a cap1 analog.

[0079] As used herein, the term "cap analogue" will be recognized and understood by those skilled in the art, and is intended, for example, to refer to a non-polymerizable dinucleotide or trinucleotide that has a cap function, as it facilitates translation or localization, and / or prevents degradation of RNA molecules upon incorporation at the 5' end of a nucleic acid molecule. "Non-polymerizable" means that the cap analogue will only be incorporated at the 5' end, as it lacks a 5' triphosphate and therefore cannot be extended in the 3' direction by template-dependent polymerases, particularly template-dependent RNA polymerases.

[0080] In implementations, the IVT buffer contains trinucleotide cap analogs disclosed in the following literature: WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, WO2017066797 and WO2023007019.

[0081] Preferably, the cap structure, which may be derived from the structure disclosed in claims 1-5 of WO2017053297, may be suitably contained in the IVT buffer.

[0082] Therefore, in a preferred embodiment, the cap1 analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG is contained in the NTP mixture of the IVT buffer. In this context, the particularly preferred cap1 analog is m7G(5')ppp(5')(2'OMeA)pG.

[0083] In other preferred embodiments within this context, the cap1 analogue is 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0084] Alternatively, a cap1 analogue as described in WO2023007019 may be used.

[0085] Suitable, the IVT buffer contains a cap analogue at a concentration of 1 mM to 10 mM, preferably 5 mM to 8 mM, preferably a cap1 analogue as defined herein. In the experiments provided herein, 5 mM to 8 mM cap1 analogue was used.

[0086] Antioxidants such as dithiothreitol (DTT) Typically, IVT buffer contains antioxidants such as dithiothreitol (DTT) or TCEP to prevent oxidation of RNA polymerase. However, as described herein, large amounts of DTT can cause undesirable aggregation of DNA beads and / or may affect the quality or integrity of the DNA beads. For example, when the beads contain iron (e.g., in the case of magnetic beads), DTT can potentially reduce the metal, which can affect bead quality and, consequently, the efficiency and / or quality of RNA synthesis.

[0087] In a preferred embodiment, the IVT buffer contains an antioxidant (e.g., DTT) at a concentration of less than 10 mM, preferably less than 5 mM, more preferably less than 2.5 mM, and most preferably less than or about 1 mM. In a preferred embodiment, the IVT buffer contains an antioxidant (e.g., DTT) at a concentration ranging from 0.5 mM to 10 mM, preferably from 0.5 mM to 5 mM, more preferably from 0.5 mM to 2.5 mM, and most preferably from 0.5 mM to 1.5 mM. In a specific embodiment, the IVT buffer contains about 1 mM of an antioxidant (e.g., DTT).

[0088] Surprisingly, the performance of in vitro RNA transcription using DNA beads (e.g., RNA yield) is not impaired by reduced antioxidant concentration in the IVT buffer (e.g., when DTT is present at approximately 1 mM).

[0089] RNA polymerase IVT buffer contains an enzyme that promotes transcription from a DNA template into RNA. DNA-dependent RNA polymerases are typically used. In the context of this invention, a suitable RNA polymerase may be selected from phage-derived RNA polymerases, such as T7, T3, SP6, or Syn5 RNA polymerases. These RNA polymerases can be engineered, for example, to improve the quality of the synthesized RNA (e.g., reduce dsRNA content, reduce short abortive byproducts, improve capping efficiency).

[0090] The appropriate amount of RNA polymerase in the RNA in vitro transcription composition of the present invention must be adjusted based on the fact that the DNA template is fixed on the beads. Since the proteins in the RNA in vitro transcription composition may promote the aggregation of DNA beads, the amount of RNA polymerase must be adjusted to allow sufficient RNA synthesis.

[0091] In a preferred embodiment, the IVT buffer contains at least 5 units / ml of RNA polymerase, preferably at least 10 units / ml of RNA polymerase. Suitably, the IVT buffer contains RNA polymerase in the range of 5 units / ml to 20 units / ml, preferably 10 units / ml to 20 units / ml, and most preferably 10 units / ml to 15 units / ml. In a specific embodiment, the IVT buffer contains about 12.5 units / ml of RNA polymerase. In the experiments provided herein, 10 units / ml to 15 units / ml of T7 RNA polymerase were used.

[0092] In a preferred embodiment, the RNA polymerase is selected from or derived from T7, T3, SP6, or Syn5 RNA polymerase. In a particularly preferred embodiment, the RNA polymerase is selected from or derived from T7 RNA polymerase.

[0093] In implementation, the IVT buffer may contain additional components to, for example, prevent or reduce RNA degradation (e.g., RNase), reduce or prevent the formation of pyrophosphate (e.g., pyrophosphatase), maintain a preferred pH (e.g., a buffer), and resolve secondary structures in DNA and / or RNA (e.g., betaine).

[0094] In this embodiment, the IVT buffer further comprises an RNase inhibitor, a pyrophosphatase (PP enzyme), and / or a buffer.

[0095] Preferably, the IVT buffer contains 0.1-0.3 units / µl of RNase inhibitor.

[0096] Preferably, the IVT buffer contains 0.003 - 0.01 units / µl of PP enzyme.

[0097] Preferably, the IVT buffer contains a buffer selected from the following: phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, HEPES buffer, and citrate buffer. In a preferred embodiment, the IVT buffer contains Tris (e.g., Tris-HCl) as a buffer.

[0098] Preferably, the IVT buffer contains Tris as a buffer at a concentration of about 30 mM to 150 mM, preferably 50 mM to 100 mM, and particularly at a concentration of about 80 mM (e.g., 80 mM Tris-HCl).

[0099] Appropriately, the pH of the IVT buffer is from about pH 7.5 to pH 8.5, especially about pH 8.0.

[0100] In the context of drug RNA production, reducing the number of components in the RNA in vitro transcription composition is preferred. Therefore, in a preferred embodiment, the RNA in vitro transcription composition does not contain PEG. In a preferred embodiment, the RNA in vitro transcription composition does not contain BSA. In a preferred embodiment, the RNA in vitro transcription composition does not contain Tween-20. In a preferred embodiment, the RNA in vitro transcription composition does not contain Triton. In a preferred embodiment, the RNA in vitro transcription composition does not contain NaCl.

[0101] According to a preferred embodiment, the RNA in vitro transcription composition containing a DNA template immobilized on beads is contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer is characterized by at least one, at least two, or a combination of the following features: F1 to F6. -F1: Less than 1 mM spermidine, Preferably Free of spermidine; -F2: Mg with a concentration ranging from 1 mM to 25 mM 2+ , Preferably Less than 20 mM Mg 2+ ; -F3: Spermine and Mg in the IVT buffer 2+ The total concentration of the additive was less than 25 mM; -F4: Mg 2+ The molar ratio to NTP ranges from 1.2 to 0.5, preferably from 0.8 to 0.6; -F5: Total NTP concentration of at least 3 mM, preferably in the range of 3 mM to 35 mM; -F6: Less than 10 mM DTT, preferably less than or about 1 mM DTT.

[0102] The IVT buffer is preferably configured to reduce or prevent the aggregation of DNA beads during processes such as in vitro RNA transcription.

[0103] In the preferred embodiment within this context, the IVT buffer of the RNA in vitro transcription composition is characterized by the following features (in ascending order of preference): -F1 and F2 -F1 and F2 and F3 -F1 and F2 and F4 -F3 and F4 -F1 and F2 and F3 and F4 -F3 and F5 or F2 and F5 -F1 and F2 and F3 and F4 and F5 -F1 and F2 and F3 and F4 and F5 and F6 DNA beads The RNA in vitro transcription compositions of the present invention comprise a DNA template immobilized on beads (DNA beads) or particles, wherein the DNA beads serve as a template for RNA in vitro transcription as defined herein. DNA templates immobilized on beads are particularly suitable in the context of automated pharmaceutical RNA production because the DNA beads can be reused for several IVT cycles, which improves the RNA yield of the RNA production method. Furthermore, the DNA beads can be easily removed (e.g., by filtration or magnetic force), and therefore the DNA used for RNA synthesis does not contaminate the final RNA product. As described above, the IVT buffer of the RNA in vitro transcription compositions is configured to reduce or prevent the aggregation of DNA beads. Additionally, the DNA beads used herein may also possess certain preferred properties to optimize RNA synthesis. These preferred properties are described in detail below.

[0104] In the context of this invention, the term "bead" or "granule" must be understood as a solid-phase carrier suitable for in vitro transcription of RNA. Therefore, the term "bead" itself does not limit size, shape, or composition. However, in the context of this invention, "bead" must be understood as a free-floating solid-phase carrier (e.g., free-floating during mixing, shaking, etc.).

[0105] In the context of this invention, DNA is used as a template for in vitro transcription (IVT) of RNA. Therefore, during IVT, a DNA sequence is transcribed into an RNA sequence. Thus, depending on the type of RNA to be produced, the DNA template may contain certain sequence elements. Preferably, the DNA template encodes (i.e., serves as its template) any type of therapeutic RNA, preferably mRNA, replicon RNA, or circular RNA.

[0106] DNA templates typically contain a 5' spacer sequence (a DNA sequence element that extends from the bead at the 5' end), an RNA polymerase promoter sequence (e.g., T7 promoter, SP6 promoter), and may contain additional elements selected from: Kozak sequence, IRES, UTR sequence (3' UTR and / or 5' UTR), coding sequence (cds), Poly(A / T) sequence (e.g., containing about 100 A / T nucleotides), or histone stem-loop.

[0107] In a preferred embodiment, the DNA template may include the following elements in the following order: The spacer subsequence at the -5' end; -RNA polymerase promoters, such as the T7 promoter; -5' UTR element; -Translate the initiation sequence (e.g., Kozak and / or IRES); - A coding sequence (cds) that encodes at least one peptide or protein. -3' UTR element; -Poly(A / T) sequence The amount of DNA template (or DNA beads) in the RNA in vitro transcription composition of the present invention may need to be adjusted to allow for efficient RNA synthesis without causing bead aggregation.

[0108] In a preferred embodiment, the RNA in vitro transcription composition comprises 10 µg / ml to 100 µg / ml of fixed DNA template, preferably 20 µg / ml to 80 µg / ml of fixed DNA template, and more preferably 50 µg / ml to 70 µg / ml of fixed DNA template. In a specific embodiment, the RNA in vitro transcription composition comprises about 60 µg / ml of fixed DNA template.

[0109] In the embodiments, the RNA in vitro transcription composition is substantially free of non-fixed DNA template, which means that the RNA in vitro transcription composition contains about 90% fixed DNA template and about 10% non-fixed DNA template, preferably about 95% fixed DNA template and about 5% non-fixed DNA template, more preferably about 99% or more than 99% fixed DNA template and about 1% or less than 1% non-fixed DNA template.

[0110] In a preferred embodiment, the RNA in vitro transcription composition is substantially free of non-fixed DNA or DNA template.

[0111] Particularly preferred and advantageous is that the RNA in vitro transcription composition is substantially free of non-fixed DNA template to avoid contamination by the resulting RNA. Therefore, it is also preferred to fix the DNA template onto beads after RNA in vitro transcription.

[0112] Therefore, in a preferred embodiment, after performing an in vitro RNA transcription reaction at about 37°C for at least 1 hour, the RNA in vitro transcription composition is substantially free of non-fixed DNA template.

[0113] “Non-fixed DNA templates” encompass all DNA species suitable for in vitro RNA transcription that are not fixed on beads as defined herein.

[0114] In a preferred embodiment, the RNA in vitro transcription composition is substantially free of other DNA species (including bacterial genomic DNA, bacterial plasmid DNA, viral genomic DNA, or eukaryotic DNA).

[0115] In a preferred embodiment, the RNA in vitro transcription composition comprises 1 mg / ml to 100 mg / ml DNA beads, preferably 10 mg / ml to 50 mg / ml DNA beads, more preferably 20 mg / ml to 40 mg / ml DNA beads. In a specific embodiment, the RNA in vitro transcription composition comprises about 35 mg / ml DNA beads.

[0116] DNA beads in RNA in vitro transcription compositions may possess certain properties that can be advantageous in the context of this invention. For example, the DNA density on the beads, the bead diameter, or the specific bead material can be adjusted to optimize efficient RNA synthesis and / or reduce or prevent DNA bead aggregation.

[0117] In a preferred embodiment, the DNA beads comprise about 1 mg DNA per 0.3 g to 0.8 g beads. In a specific embodiment, the DNA beads comprise about 1 mg DNA per 0.6 g beads.

[0118] In the context of this invention, a certain density of DNA template immobilized on the surface of the bead may be advantageous. Therefore, per mm 2 A certain amount of DNA on the surface of the bead can be suitable.

[0119] Therefore, in a preferred embodiment, the surface of the DNA beads contains approximately 0.5 ng DNA / mm². 2 Approximately 10 ng DNA / mm 2 Preferably about 1 ng DNA / mm 2 Approximately 5 DNA / mm 2The most preferred concentration is approximately 1.4 ng to 3.1 ng DNA / mm³. 2 .

[0120] The density of DNA on the surface of the beads is a factor affecting IVT yield and DNA bead aggregation. Increasing the density of DNA on the bead surface to, for example, more than 10 ng DNA / mm² is crucial. 2 It may have a negative impact on IVT performance and / or DNA bead aggregation.

[0121] The beads on which the DNA template is immobilized are preferably spherical and substantially uniform in diameter. The bead diameter can range from 500 nm to 50 µm. However, in the context of using the RNA in vitro transcription composition in a method for producing pharmaceutical RNA, a bead diameter within a certain preferred range may be advantageous to reduce the risk of bead contamination of the final RNA product (e.g., in the case of selecting beads with a diameter smaller than a certain size) or to reduce the risk of bead aggregation (e.g., in the case of selecting beads with a diameter larger than a certain size).

[0122] In a preferred embodiment, the beads have a (uniform) diameter in the range of 1 µm to 10 µm, preferably in the range of 1 µm to 5 µm, more preferably in the range of 4 µm to 5 µm, and even more preferably in the range of 4.3 µm to 4.7 µm. In a preferred embodiment, the beads have a (uniform) diameter of about 4.5 µm.

[0123] In other embodiments, the beads have a (uniform) diameter of about 2.8 µm. In other embodiments, the beads have a (uniform) diameter of about 1 µm.

[0124] To allow the removal of beads (e.g., after an in vitro RNA transcription reaction) by a commercially available filtration system (e.g., using a 0.2 µm filter), it may be preferable to use beads with a size greater than 1 µm, preferably greater than 2 µm.

[0125] Appropriately, the size distribution of the beads is uniform, meaning that the CV value of the size distribution is less than 5%. The size distribution can be measured using a multisizer device (Beckman Coulter).

[0126] The beads on which the DNA template is fixed may contain materials selected from or derived from the following: iron oxide, silica, cellulose, agarose gel, dextran gel, polystyrene, agarose, (poly)methacrylate, poly(meth)methacrylate or any derivative or combination thereof.

[0127] Preferably, the beads on which the DNA template is fixed are non-porous.

[0128] Preferably, the density of the beads on which the DNA template is immobilized is in the range of 1 g / cm³. 3 Up to 2 g / cm 3 1.3 g / cm 3 Up to 1.5 g / cm 3 Preferably, the density is about 1.4 g / cm³. 3 .

[0129] In a preferred embodiment, the beads on which the DNA template is immobilized contain polystyrene or a polystyrene derivative.

[0130] In various embodiments, the RNA in vitro transcription composition is configured and adapted for magnetic mixing and / or magnetic trapping. Thus, the beads on which the DNA template is immobilized comprise a magnetizable material, preferably a (super)paramagnetic material. Suitably, the beads comprise magnetic iron oxide, preferably about 10% to 50% magnetic iron oxide (e.g., γ-Fe₂O₃ and / or magnetite (Fe₃O₄)).

[0131] In a preferred embodiment, the beads on which the DNA template is immobilized are magnetic beads, preferably (super)paramagnetic beads. Therefore, the DNA beads of the RNA in vitro transcription composition are magnetic DNA beads, preferably (super)paramagnetic DNA beads. Advantageously, the magnetic DNA beads allow for mixing of the DNA beads during the IVT process and / or capture of the DNA beads after IVT cycles, which is important for automated RNA manufacturing methods. Mixing and / or capture are preferably induced by magnetic force, for example by a magnet.

[0132] In a preferred embodiment, the beads on which the DNA template is immobilized are functionalized with streptavidin (to bind to biotin). In embodiments within this context, the beads contain about 25 pmol to 400 pmol of biotin binding capacity per mg of beads, preferably about 50 to 300 binding capacity per mg of beads.

[0133] Preferred examples of beads suitable for the context of this invention are Dynabeads®, such as Dynabeads® M-270, Dynabeads® M-280, Dynabeads® M-450, or Dynabeads® MyOne.

[0134] In a specific embodiment, the beads on which the DNA template is fixed are Dynabeads® M-450, such as Dynabeads® M-450 E beads.

[0135] In the context of this invention, it is preferred that the linear DNA template is fixed to the bead via one DNA end, and the other end of the DNA template is free (meaning this free end is not fixed to the bead). Suitably, the DNA template is fixed to the DNA bead to allow run-off RNA transcription in vitro, wherein the free end of the DNA template defines the termination of the RNA transcription reaction (e.g., as in WO2019122371). Figure 1 (As shown in B).

[0136] In a preferred embodiment, the DNA beads contain a biotin-streptavidin-fixed DNA template, preferably a linear biotin-streptavidin-fixed DNA template. As described above, the DNA template is preferably fixed to allow run-off RNA transcription in vitro (e.g., as in WO2019122371). Figure 1 (As shown in B).

[0137] In a preferred embodiment, the DNA beads are produced by specifically immobilizing biotinylated PCR-amplified DNA template sites onto streptavidin-functionalized beads or by specifically immobilizing biotinylated linearized plasmid DNA template sites onto streptavidin-functionalized beads.

[0138] To immobilize the linearized plasmid DNA template, the method disclosed in WO2019122371 can be used, preferably as defined in claims 6 to 29 of WO2019122371. The biotinylated linearized plasmid DNA template can be suitably immobilized on streptavidin-functionalized beads.

[0139] To immobilize the DNA template for PCR amplification, biotinylated PCR primers can be used in PCR-based DNA template production. The biotinylated PCR amplified DNA template can then be appropriately immobilized on streptavidin-functionalized beads.

[0140] Preferably, the DNA beads of the present invention comprise a biotin-streptavidin-fixed DNA template. Preferably, these biotin-streptavidin-fixed DNA templates are generated by site-specific immobilization of biotinylated PCR-amplified DNA templates or biotinylated linearized plasmid DNA templates onto streptavidin-functionalized beads to allow run-off RNA transcription in vitro.

[0141] The DNA used for fixation is preferably purified DNA, such as DNA purified by RP-HPLC. Alternatively, the DNA can be purified by normal-phase chromatography, mixed-mode chromatography, anion-exchange chromatography, or size exclusion chromatography.

[0142] Suitable, the DNA beads of the present invention may comprise the following structural elements: X-(SB)-[spacer-RNAP-UTR-CDS-UTR-POLY(A)*] Where X is a bead, preferably a magnetic bead; SB is a streptavidin-biotin bond; the elements between square brackets [ ] represent DNA elements; the spacer is a DNA sequence element at the 5' end that elongates the separation from the bead; RNAP is the RNA polymerase promoter sequence; UTR is the untranslated region; CDS is the coding sequence; POLY(A) is the poly(A) sequence; and the asterisk indicates the free end of the DNA.

[0143] According to a preferred embodiment, DNA beads are characterized by at least one, at least two, or a combination of the following features B1 to B5: -B1: Contains a magnetizable material, preferably a (super)paramagnetic material; -B2: A diameter in the range of 1 µm to 10 µm, preferably in the range of 4 µm to 5 µm, for example, 4.5 µg; -B3: Contains polystyrene or polystyrene derivatives; -B4: The surface load of the beads contains approximately 1 ng DNA / mm². 2 Approximately 5 ng DNA / mm 2 ; -B5: The beads contain a biotin-streptavidin-fixed DNA template.

[0144] In the preferred embodiment within this context, the IVT buffer of the RNA in vitro transcription composition is characterized by the following features (in ascending order of preference): -B1 and B2; -B1, B2, and B3; -B1 and B2 and B3 and B4; -B1 and B2 and B3 and B4 and B5.

[0145] In one embodiment, the RNA in vitro transcription composition is configured to produce more than one RNA species, such as 2, 3, 4, 5, 6, 7, 8, 9, or even more RNA species. In such embodiments, the RNA in vitro transcription composition comprises more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, or even more different DNA templates immobilized on beads. These 2, 3, 4, 5, 6, 7, 8, 9, or even more different DNA templates contain CDs encoding different peptides or proteins (e.g., different tumor antigens, different viral antigens, etc.).

[0146] In a preferred embodiment, the composition is configured to reduce or prevent RNA from binding to DNA beads at an RNA concentration of more than 100 mg / L, preferably more than 500 mg / L, and more preferably more than 1 g / L.

[0147] The RNA in vitro transcription composition of the present invention particularly addresses the problem of DNA bead aggregation during RNA in vitro transcription, a problem not previously described. This problem is solved by adjusting the components of the RNA in vitro transcription as described herein. It is noteworthy that the inventors have observed that the DNA bead aggregation problem occurring during RNA in vitro transcription may depend on the RNA concentration (i.e., the RNA continuously generated during IVT), the duration of RNA in vitro transcription, or the fact that the DNA beads of the RNA in vitro transcription composition are intended for several IVT cycles.

[0148] In a preferred embodiment, the RNA in vitro transcription composition is configured to reduce or prevent DNA bead aggregation at RNA concentrations exceeding 0.1 g / L, preferably exceeding 1 g / L, and more preferably exceeding 5 g / L. In this context, it must be understood that the specified RNA concentration refers to the product of RNA synthesis occurring during RNA in vitro transcription. Aggregation can be determined and quantified using microscopy.

[0149] In a preferred embodiment, the RNA in vitro transcription composition is configured to reuse DNA beads, particularly for more than one IVT reaction or cycle, for example, two, three, four, five, six, or more IVT cycles. Preferably, one IVT cycle is performed at about 37°C for at least 1 hour. After each IVT cycle, IVT buffer is typically replenished while the DNA beads remain in the composition.

[0150] In a preferred embodiment, when DNA beads are incubated with more than one IVT reaction or cycle (e.g., 2, 3, 4, 5, 6 or more IVT cycles), each of the more than one IVT reaction or cycle (e.g., 2, 3, 4, 5, 6 or more IVT cycles) produces substantially the same amount of RNA (given that the conditions of each IVT cycle are substantially the same).

[0151] In a preferred embodiment, the RNA in vitro transcription composition is configured for incubation periods of more than 1 hour, more than 2 hours, more than 3 hours, or more than 4 hours, preferably at a temperature of about 37°C.

[0152] In a preferred embodiment, after incubating the RNA in vitro transcription composition for at least 1 hour, less than 10% of the DNA beads contained in the composition are aggregated, preferably less than 5% of the DNA beads contained in the composition are aggregated, and more preferably less than 1% of the DNA beads contained in the composition are aggregated. In a preferred embodiment, the incubation temperature is about 37°C. Aggregation can be determined and quantified by microscopy.

[0153] In a preferred embodiment, the RNA in vitro transcription composition is substantially free of pyrogens, bacteria or fragments thereof, viruses or fragments thereof, and / or bacteriophages or fragments thereof. Preferably, the RNA in vitro transcription composition is suitable for pharmaceutical manufacturing, for example, for GMP (Good Manufacturing Practice) manufacturing.

[0154] In a preferred embodiment, the RNA in vitro transcription composition is used to generate any type of RNA as defined herein, preferably any type of therapeutic RNA as defined herein. In a preferred embodiment, the RNA in vitro transcription composition is used to generate (therapeutic) mRNA.

[0155] Uses of RNA in vitro transcription compositions: In a second aspect, the present invention provides the use of an RNA in vitro transcription composition as defined in the context of the first aspect in a method of RNA manufacturing.

[0156] It should be noted that the features and embodiments described in the context of the RNA in vitro transcription composition of the first aspect are also applicable to the use of the second aspect. Similarly, the features and embodiments described in the context of the use of the second aspect are also applicable to the RNA in vitro transcription composition of the first aspect.

[0157] In a preferred embodiment within this context, the manufacturing method is an automated RNA manufacturing method. In a preferred embodiment within this context, the manufacturing method is a GMP (Good Manufacturing Practice) compliant RNA manufacturing method. In a preferred embodiment within this context, the manufacturing method is a method for producing more than 1 mg of RNA, preferably more than 1 g of RNA.

[0158] In a preferred embodiment within this context, the RNA in vitro transcription composition as defined in the context of the first aspect can be used in methods for manufacturing any type of RNA as defined herein. Preferably, the RNA in vitro transcription composition as defined in the context of the first aspect can be used in methods for manufacturing any type of therapeutic RNA as defined herein. Most preferably, the RNA in vitro transcription composition as defined in the context of the first aspect can be used in methods for manufacturing any type of therapeutic coding RNA (preferably mRNA, such as mRNA for infectious disease vaccines or tumor vaccines) as defined herein.

[0159] Methods for producing RNA: In a third aspect, the present invention provides a method for generating RNA using an RNA in vitro transcription composition comprising a DNA template immobilized on beads.

[0160] It should be noted that the features and embodiments described in the context of the RNA in vitro transcription composition of the first aspect are also applicable to the RNA production method of the third aspect. Similarly, the features and embodiments described in the context of the RNA production method of the third aspect are also applicable to the RNA in vitro transcription composition of the first aspect.

[0161] In various embodiments, the RNA production method includes the step of incubating an RNA in vitro transcription composition under conditions that allow RNA transcription, wherein the RNA in vitro transcription composition comprises a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer. Preferably, the IVT buffer is configured to reduce or prevent the aggregation of DNA beads during incubation (i.e., during the RNA transcription process).

[0162] The method is suitable for producing any type of RNA.

[0163] In this embodiment, the RNA is selected from single-stranded RNA or double-stranded RNA, and / or coding RNA or non-coding RNA, and / or linear RNA or circular RNA. Therefore, the RNA can be a circular double-stranded non-coding RNA, or a linear single-stranded non-coding RNA, or a linear double-stranded coding RNA. Suitably, the RNA is a linear or circular single-stranded coding RNA.

[0164] In the implementation, the RNA is selected from viral RNA, retroviral RNA, replicon RNA, small interfering RNA (siRNA), antisense RNA, saRNA (small activating RNA), CRISPR RNA (small guide RNA, sgRNA), ribozymes, aptamers, riboswitch, immune-stimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), self-replicating RNA, circular RNA, or messenger RNA (mRNA).

[0165] In a preferred embodiment, the RNA is coding RNA. In a preferred embodiment, the RNA contains at least one coding sequence.

[0166] The coding RNA can be any type of RNA, characterized in that the RNA contains at least one coding sequence (cds) that translates into at least one amino acid sequence (when applied to, for example, a cell).

[0167] In a preferred embodiment, the RNA is selected from mRNA, (encoding) circular RNA, (encoding) self-replicating RNA, (encoding) viral RNA, or (encoding) replicon RNA.

[0168] In the embodiments, the length of the RNA ranges from about 500 nucleotides to about 10,000 nucleotides, from about 1,000 nucleotides to about 10,000 nucleotides, and from about 1,500 nucleotides to about 5,000 nucleotides.

[0169] The problem of DNA bead aggregation may be more pronounced when producing RNA of a certain length. In a preferred embodiment, the method for producing RNA is a method for producing RNA with a length of at least 1000 nucleotides, preferably at least 1500 nucleotides, and more preferably at least 2000 nucleotides. Specifically, the length of the RNA ranges from 1500 nucleotides to about 5000 nucleotides.

[0170] In a particularly preferred embodiment, the RNA is mRNA. Therefore, the method is a method for generating RNA, preferably mRNA. In a preferred embodiment, the RNA is therapeutic RNA.

[0171] The term "therapeutic RNA" refers to RNA that provides a therapeutic effect. In this context, the term "therapeutic" must be understood as "providing a therapeutic function" or "suitable for therapy or administration." Furthermore, "therapeutic RNA" is generally produced using methods and compositions suitable for pharmaceutical manufacturing. However, in this context, "therapeutic" should not be understood as limited to a particular mode of treatment. Typically, the term "therapeutic RNA" does not include natural RNA extracts or RNA preparations (e.g., obtained from bacteria or plants) that are unsuitable for administration to subjects (e.g., animals, humans).

[0172] In a preferred embodiment, the RNA is artificial RNA.

[0173] As used herein, the term "artificial RNA" refers to RNA that is not naturally occurring. In other words, artificial RNA can be understood as a non-natural RNA molecule. Such RNA molecules can be non-natural due to their respective sequences (e.g., coding sequences modified with G / C content, UTRs) and / or due to other modifications (e.g., structural modifications of nucleotides). Typically, artificial RNA can be genetically engineered and / or produced to correspond to an artificial sequence of desired nucleotides. In this context, artificial RNA is a sequence that can be non-naturally occurring, i.e., a sequence that differs from the wild-type or reference / naturally occurring sequence by at least one nucleotide (through codon modifications, as further detailed below). The term "artificial RNA" is not limited to meaning "a single molecule," but is understood to include a group or number of substantially identical RNA molecules.

[0174] It has been shown that the problem of DNA bead aggregation may be more pronounced when producing RNA with a certain G / C content or coding sequences containing increased or maximized GC content. In this context, the disclosure of WO2002098443 is included in the present invention in its entirety. G / C-optimized coding sequences are indicated by the abbreviation "gc". In embodiments, the G / C content of the RNA sequence or coding sequence is at least about 50%, 55%, or 60%. In specific embodiments, the G / C content of the RNA or coding sequence is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%. Therefore, in a preferred embodiment, the method for producing RNA is a method for producing RNA sequences or coding sequences with a G / C content of at least about 55%.

[0175] In a preferred embodiment, the resulting RNA comprises at least one 3' UTR and / or at least one 5' UTR sequence. Suitable UTR sequences and combinations may be selected from published PCT patent application WO2019077001, preferably from the UTR sequences and combinations disclosed in claims 1 to 10 of WO2019077001. Preferably, the 5' UTR is selected from or derived from HSD17B4, and the 3' UTR is selected from or derived from PSMB3. In a preferred embodiment, the resulting RNA comprises at least one histone stem-loop sequence (hSL). Suitable hSL sequences that may be used within the present invention may be derived from formula (I) or (II) of WO2012019780. In a preferred embodiment, the resulting RNA comprises at least one poly(A) sequence or at least two poly(A) sequences (see, for example, WO2016091391). In a preferred embodiment, the poly(A) sequence represents the 3' end of the RNA (see, for example, WO2022162027). Suitably, the poly(A) sequence is about 100 Å in length. In some implementations, the length of the Poly(A) sequence is approximately 64A.

[0176] In a preferred embodiment, the method for generating RNA is a method for generating therapeutic coding RNA (e.g., therapeutic mRNA).

[0177] Appropriately, the therapeutic coding RNA (preferably mRNA) contains at least one coding sequence encoding at least one therapeutic peptide or protein.

[0178] In embodiments described in this context, the at least one therapeutic peptide or protein is selected from or derived from antibodies, intracellular antibodies, receptors, receptor agonists, receptor antagonists, binding proteins, CRISPR-associated endonucleases, molecular chaperones, transport proteins, ion channels, membrane proteins, secretory proteins, transcription factors, enzymes, peptide or protein hormones, growth factors, structural proteins, cytoplasmic proteins, cytoskeletal proteins, viral antigens or epitopes, bacterial antigens or epitopes, protozoan antigens or epitopes, allergens, tumor antigens or epitopes, or fragments, variants, or combinations thereof.

[0179] In embodiments described in this context, the at least one therapeutic peptide or protein is selected from or derived from antigens or epitopes of pathogens (e.g., viral antigens or epitopes, bacterial antigens or epitopes, protozoan antigens or epitopes) or antigens or epitopes of tumors.

[0180] In a preferred embodiment of the third aspect, the method for generating RNA includes the following steps: (A) Provides an RNA in vitro transcription composition comprising DNA beads in an IVT buffer contained in a reaction vessel; and (B) A first or initial IVT cycle is performed by mixing the DNA beads with the IVT buffer to produce an RNA batch.

[0181] According to a preferred embodiment, the RNA in vitro transcription composition of the method is characterized by any of the features in the first aspect.

[0182] Suitable, the RNA in vitro transcription composition comprises a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer preferably contains less than 2 mM spermidine and less than 25 mM Mg 2+ ; and Mg with a molar ratio below 1.4 2+ With NTP.

[0183] Preferably, the RNA in vitro transcription composition of the method comprises a DNA template (DNA bead) immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer is characterized by at least one, at least two, or a combination of the following features: -F1: Less than 1 mM spermidine, preferably free of spermidine; -F2: Mg with a concentration ranging from 1 mM to 25 mM 2+ Preferably less than 20 mM Mg 2+ ; -F3: Spermine and Mg in the IVT buffer 2+ The total concentration of the additive is less than 30 mM; -F4: Mg 2+ The molar ratio to NTP ranges from 1.2 to 0.5, preferably from 0.8 to 0.6; -F5: Total NTP concentration of at least 3 mM, preferably in the range of 3 mM to 35 mM; -F6: Less than 10 mM DTT, preferably less than or about 1 mM DTT.

[0184] The IVT buffer is preferably configured to reduce or prevent DNA bead aggregation, for example, during in vitro RNA transcription or at repeated IVT cycles, at high RNA concentrations.

[0185] In a preferred embodiment within this context, the IVT buffer for the RNA in vitro transcription composition used in the method is characterized by the following features (in ascending order of preference): -F1 and F2 -F1 and F2 and F3 -F1 and F2 and F4 -F3 and F4 -F1 and F2 and F3 and F4 -F3 and F5 or F2 and F5 -F1 and F2 and F3 and F4 and F5 -F1 and F2 and F3 and F4 and F5 and F6 In a preferred embodiment of the method, the RNA in vitro transcription composition comprises 10 µg / ml to 100 µg / ml of fixed DNA template, preferably 20 µg / ml to 80 µg / ml of fixed DNA template, and more preferably 35 µg / ml to 65 µg / ml of fixed DNA template. In a specific embodiment, the RNA in vitro transcription composition comprises about 50 µg / ml of fixed DNA template.

[0186] In a preferred embodiment of the method, the RNA in vitro transcription composition is substantially free of non-fixed DNA template. In a preferred embodiment of the method, the RNA in vitro transcription composition is substantially free of other DNA species (including bacterial genomic DNA, viral genomic DNA, or eukaryotic DNA).

[0187] In a preferred embodiment of the method, the RNA in vitro transcription composition comprises 1 mg / ml to 100 mg / ml DNA beads, preferably 10 mg / ml to 50 mg / ml DNA beads, more preferably 20 mg / ml to 40 mg / ml DNA beads. In a specific embodiment, the RNA in vitro transcription composition comprises about 35 mg / ml DNA beads.

[0188] The methods in this area have been optimized and adapted to allow for the efficient use of DNA templates (DNA beads) immobilized on the beads, thereby particularly reducing or preventing unwanted DNA bead aggregation and / or unwanted DNA bead escape.

[0189] Preferred DNA beads particularly suitable for the method are described in the context of the first aspect. It is worth noting that the features related to DNA beads provided in the context of the first aspect can also be applied to DNA beads for the method of the third aspect.

[0190] Particularly preferred embodiments of the DNA beads are summarized below.

[0191] According to a preferred embodiment of the method, DNA beads are characterized by at least one, at least two, or a combination of the following features B1 to B5: -B1: Contains a magnetizable material, preferably a (super)paramagnetic material; -B2: A diameter in the range of 1 µm to 10 µm, preferably in the range of 4 µm to 5 µm, for example, 4.5 µg; -B3: Contains polystyrene or polystyrene derivatives; -B4: The surface of the bead contains approximately 1 ng DNA / mm². 2 Approximately 5 ng DNA / mm 2 ; -B5: The beads contain a biotin-streptavidin-fixed DNA template.

[0192] In a preferred embodiment within this context, the DNA beads used in the method are characterized by the following features (in ascending order of preference): -B1 and B2; -B1, B2, and B3; -B1 and B2 and B3 and B4; -B1 and B2 and B3 and B4 and B5.

[0193] In a preferred embodiment of the method, the beads on which the DNA template is immobilized are magnetic beads, preferably (super)paramagnetic beads. Advantageously, magnetic DNA beads allow for mixing of DNA beads during the IVT process and / or capture of DNA beads after IVT cycles, which is important for automated RNA manufacturing methods. Mixing and / or capture are preferably induced by magnetic force, for example by a magnet, as further detailed herein.

[0194] Preferred examples of beads suitable for the context of this invention are Dynabeads®, such as Dynabeads® M-270, Dynabeads® M-280, Dynabeads® M-450, or Dynabeads® MyOne.

[0195] In a specific embodiment of the method, the beads on which the DNA template is immobilized are Dynabeads® M-450, such as Dynabeads® M-450 E beads.

[0196] In a preferred embodiment of the method, the DNA beads contain a biotin-streptavidin-fixed DNA template, preferably a linear biotin-streptavidin-fixed DNA template. As described above, the DNA template is preferably fixed to allow run-off RNA transcription in vitro (e.g., as in WO2019122371). Figure 1 (As shown in B).

[0197] In a preferred embodiment of the method, the DNA beads are produced by specifically immobilizing the biotinylated PCR-amplified DNA template site onto streptavidin-functionalized beads or by specifically immobilizing the biotinylated linearized plasmid DNA template site onto streptavidin-functionalized beads.

[0198] Therefore, the DNA beads produced by this method contain a biotin-streptavidin-fixed DNA template.

[0199] Appropriately, the DNA beads of the method may contain the following structural elements: X-(SB)-[spacer-RNAP-UTR-CDS-UTR-POLY(A)*] Where X is a bead, preferably a magnetic bead; SB is a streptavidin-biotin bond; the elements between square brackets [ ] represent DNA elements; the spacer is a DNA sequence element at the 5' end that elongates the separation from the bead; RNAP is the RNA polymerase promoter sequence; UTR is the untranslated region; CDS is the coding sequence; POLY(A) is the poly(A) sequence; and the asterisk indicates the free end of the DNA.

[0200] In the context of this invention, the reaction vessel can be a microchannel, tube, vial, container, or bioreactor. Suitably, the reaction vessel is one that can be used for drug production (e.g., in compliance with GMP). The reaction vessel can be composed of materials selected from glass, metal, ceramic, or polymers (e.g., plastic materials). Preferred materials are metals and ceramics. The volume of the reaction vessel (e.g., the volume containing the RNA in vitro transcription composition) can be from 1 µl to 1000 l, preferably from 1 ml to 10 l, more preferably from 10 ml to 1 l, and even more preferably from 10 ml to 500 ml.

[0201] In a preferred embodiment, the reaction vessel is a bioreactor made of metal or ceramic, and has a volume of at least 1 ml, preferably from 10 ml to 500 ml. In one specific embodiment, the reaction vessel is a bioreactor made of ceramic and is configured to contain at least 50 ml.

[0202] In the context of this invention, an RNA batch must be understood as RNA produced during the IVT process. An RNA batch may contain more than 1 mg of RNA, for example, from 1 mg to 1000 g of RNA, preferably from 1 mg to 100 g of RNA, more preferably from 10 mg to 10 g of RNA. The RNA batch preferably does not contain DNA beads or fragments thereof.

[0203] In a preferred embodiment, the method for generating RNA is configured to achieve a high RNA concentration during IVT (or IVT cycling). Suitably, a concentration exceeding 100 mg / L, preferably exceeding 1 g / L, and more preferably exceeding 5 g / L, is achieved. Preferably, DNA bead aggregation is reduced or prevented during the process of said method at an RNA concentration exceeding 100 mg / L, preferably exceeding 1 g / L, and more preferably exceeding 5 g / L.

[0204] In the context of this invention, an IVT cycle must be understood as one in vitro RNA reaction cycle required for in vitro transcription of RNA at the desired yield. Typically, an IVT cycle is performed over a period of at least one hour (e.g., at least one hour at about 37°C). Typically, after an IVT cycle, free NTPs are substantially consumed and integrated into the resulting RNA molecules. In the context of this invention, methods for producing RNA may include more than one IVT cycle (preferably using the same DNA beads as templates and by feeding fresh IVT buffer into the reaction vessel, as further outlined below).

[0205] In a preferred embodiment, each IVT cycle of the method is performed under controlled temperature conditions.

[0206] Suitable, the IVT cycle of the method is performed at 20-40°C (e.g., 30°C to 40°C, preferably 36°C to 38°C). In some embodiments, the IVT cycle is performed at approximately 37°C.

[0207] In a preferred embodiment, the IVT cycle is performed for at least 1 hour, 2 hours, 3 hours, or 4 hours, for example, from 90 minutes to 180 minutes. In some embodiments, each IVT cycle is performed for approximately 2 hours.

[0208] After performing steps (A) and (B), RNA batches can be collected.

[0209] In a preferred embodiment, the method for generating RNA further includes the following steps: (C) Retain the DNA beads in the reaction vessel and collect the RNA batch.

[0210] Therefore, in a preferred embodiment, the DNA beads are preferably retained in the reaction vessel and the generated RNA batch is collected after steps (A) and (B). The step of retaining the DNA beads can be performed, for example, by using magnetism, centrifugation, or filtration.

[0211] The retention step facilitates the physical separation of DNA beads and the resulting RNA batches. Physical separation may be necessary, especially for reusing DNA beads for additional IVT cycles (e.g., by feeding fresh IVT buffer into the DNA beads) and / or removing DNA beads from the obtained RNA product.

[0212] RNA batches are preferably collected and transferred to an RNA product container or bag via an outlet. In an embodiment, the outlet may be a pipe. In an embodiment, the collection step may be automated and is preferably controlled by a pump. In a preferred embodiment, RNA batches are collected via a pipe connected to a product container or bag, wherein the transfer of RNA batches to the product container or bag is controlled by a pump.

[0213] It is important that DNA beads do not aggregate during the retention and / or collection steps, as DNA bead aggregation can cause DNA bead escape. Such DNA bead escape can lead to contamination of downstream equipment (e.g., purification equipment) and may contaminate the RNA product. By optimizing the RNA in vitro transcription composition as defined herein, DNA bead aggregation and / or DNA bead escape can be prevented or reduced.

[0214] In a preferred embodiment of the third aspect, the method for generating RNA includes the following steps: (A) Provides an RNA in vitro transcription composition comprising DNA beads in an IVT buffer contained in a reaction vessel; (B) A first or initial IVT cycle is performed by mixing the DNA beads with the IVT buffer to produce an RNA batch; (C) Retain the DNA beads in the reaction vessel and collect the RNA batch.

[0215] In a preferred embodiment, the method for generating RNA includes at least one additional IVT cycle, which is preferably performed after step (C).

[0216] To perform at least one additional IVT cycle, fresh IVT buffer must be added to the reaction vessel, preferably to the DNA beads retained in the reaction vessel. After adding fresh IVT buffer, a new IVT cycle can be performed to produce additional RNA batches by mixing the DNA beads with the newly added IVT buffer (e.g., using magnetic force).

[0217] In a preferred embodiment, the method for generating RNA further includes the following steps: (E) Add fresh IVT buffer to the reaction vessel containing the preserved DNA beads.

[0218] In this context, the IVT buffer added in step (E) contains the components required for in vitro RNA transcription on the retained DNA beads. The newly added IVT buffer is preferably configured to reduce or prevent DNA bead aggregation, for example, as defined in the context of the first aspect (e.g., characterized by any of the features F1 to F6). The newly added IVT buffer does not contain a DNA template or DNA beads. After adding fresh IVT buffer to the reaction vessel, further IVT cycles can be performed as defined herein.

[0219] Therefore, in a preferred embodiment, the method for generating RNA further includes at least one additional IVT cycle, said at least one additional IVT cycle comprising the following steps: (E) Add fresh IVT buffer to the reaction vessel containing the retained DNA beads; (F) Perform additional IVT cycles by mixing the DNA beads with newly added IVT buffer to produce additional RNA batches; (G) The DNA beads are retained in the reaction vessel and the additional RNA batches are collected.

[0220] Step (F) is preferably performed using similar or substantially the same conditions selected for step (B). Step (G) preferably involves collecting the RNA batch through an outlet and transferring it into an RNA product container or bag.

[0221] Appropriately, methods for producing RNA, as defined herein, may include more than one additional IVT cycle as defined herein, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional IVT cycles.

[0222] Therefore, in a preferred embodiment, the method further includes at least one additional IVT cycle as defined herein, wherein the at least one additional IVT cycle is performed 1 to 20 times (e.g., 3 to 10 times), or in other words, wherein steps (E) to (G) are repeated 1 to 20 times (e.g., 3 to 10 times).

[0223] Suitable methods for generating RNA may include a first IVT cycle (or a first round of in vitro RNA transcription) as defined herein, preferably including steps (A), (B) and (C), and at least one additional IVT cycle (or at least one additional round of in vitro RNA transcription) including steps (E), (F) and (G).

[0224] In the context of this invention, the RNA in vitro transcription composition of the first aspect particularly addresses the problem of DNA bead aggregation, which has not yet been described. Specifically, the inventors have observed this problem in RNA production methods that include more than one IVT cycle using (the same) DNA beads. As shown by the inventors, the risk of DNA bead aggregation in such methods can be further reduced by implementing a DNA bead washing step or a DNA bead recovery step. Without being bound by theory, such washing steps can remove molecules such as RNA, spermidine, or Mg that may attach to the DNA beads. 2+ These linked molecules can be removed through a washing step under certain conditions, which can prevent or reduce the risk of DNA bead aggregation.

[0225] Therefore, in a preferred embodiment, the method includes the following steps: (D) Wash the retained DNA beads in washing buffer, wherein step (D) is preferably performed after step (C) and / or after step (G).

[0226] Preferably, the DNA beads are mixed during step (D) (to keep them free-floating, for example). Suitablely, the mixing is performed using magnetic force.

[0227] In an implementation, step (D) includes at least two washing steps, wherein in each of the at least two washing steps, fresh washing buffer is added to the DNA beads.

[0228] In a preferred embodiment, the washing buffer used in step (D) contains at least one chelating agent. In the context of this invention, a cationic chelating agent is preferred, for example, one that can be used with Mg... 2+ Ion-binding chelating agents.

[0229] In a preferred embodiment within this context, the chelating agent is EDTA or an EDTA derivative.

[0230] Preferably, the washing buffer contains a chelating agent, preferably EDTA, in a concentration range of 0.1 mM to 50 mM, more preferably 1 mM to 10 mM, and even more preferably 1 mM to 5 mM.

[0231] In other embodiments, the wash buffer contains urea, preferably 0.5 M to 5 M urea. In this context, the wash buffer may contain a chelating agent as defined herein and urea as defined herein.

[0232] In a preferred embodiment within this context, the wash buffer is adjusted to a pH between 7.5 and 8.5. In a preferred embodiment, the pH of the wash buffer is approximately 8.0.

[0233] In a preferred embodiment within this context, the wash buffer contains at least one buffer. In this context, the preferred buffer is Tris.

[0234] In embodiments described in this context, the wash buffer contains 1 mM to 500 mM of buffer, preferably Tris. In a preferred embodiment, the wash buffer contains 1 mM to 50 mM of buffer, preferably Tris.

[0235] In a preferred embodiment within this context, the wash buffer comprises 1 mM EDTA and 10 mM Tris (pH 8.0).

[0236] In a preferred embodiment within this context, step (D) is performed subsequently in 1-5 reaction volumes. Preferably, step (D) is performed in at least 2 subsequent reaction volumes. DNA beads are preferably mixed during the washing step. In an embodiment, the washing step is performed for at least 15 minutes. In an embodiment, the washing step is performed at a temperature of approximately 37°C. After the washing step, the DNA beads are retained (e.g., by magnetic force, filtration, centrifugation) and the washing buffer is removed from the reaction vessel (e.g., through the waste port).

[0237] In a preferred embodiment, step (D) reduces or prevents the aggregation of DNA beads and / or reduces or prevents the attachment of RNA to DNA beads (during the in vitro transcription of RNA). Furthermore, the implementation of step (D) stabilizes the amount of RNA produced in each IVT cycle (e.g., the RNA yield does not decrease from cycle to cycle).

[0238] Therefore, in the preferred embodiment, the RNA yield generated in each IVT cycle is substantially stable; in other words, the RNA yield does not decrease from IVT cycle to IVT cycle.

[0239] In this implementation, IVT cycles are performed until an RNA concentration of at least 5 mg / ml is achieved.

[0240] Therefore, in a preferred embodiment, the method for generating RNA includes the following steps: (A) Provides an RNA in vitro transcription composition as defined herein, said RNA in vitro transcription composition comprising DNA beads in an IVT buffer contained in a reaction vessel as defined herein; (B) IVT cycling is performed by mixing the DNA beads with the IVT buffer to produce RNA batches; (C) Retain the DNA beads in the reaction vessel and collect the RNA batch; (D) Wash the retained DNA beads in a wash buffer as defined herein; (E) Add fresh IVT buffer to the reaction vessel containing the retained DNA beads; (F) Perform additional IVT cycles by mixing the DNA beads with newly added IVT buffer to produce additional RNA batches; (G) The DNA beads are retained in the reaction vessel and the additional RNA batches are collected; And optionally, n additional IVT cycles are then performed, wherein the additional IVT cycles include steps (D) to (G).

[0241] The method is performed in the following appropriate order: A, B, C, [D, E, F, G]n, where n Related to the number of other IVT cycles (usually) n = 1 to 20, preferably 3 to 10).

[0242] In a preferred embodiment, the method may include at least one NTP feeding step, which is preferably performed during the first IVT cycle (step (B)) and / or during any additional IVT cycle (step (F)).

[0243] In the context of this invention, the NTP feeding step includes adding an NTP feed mixture to a reaction vessel.

[0244] In a preferred embodiment, NTP feeding is performed 1 to 10 times during each IVT cycle, preferably 2 to 8 times, more preferably 3 to 5 times. In a preferred embodiment, the NTP feed mixture does not contain cap analogues. Preferably, NTP feeding is performed at predefined intervals or at time points when the NTP concentration in the reaction is below a certain predefined threshold (e.g., by measuring the in vitro transcription of RNA).

[0245] In a preferred embodiment within this context, the NTP feed mixture comprises an NTP mixture containing G, C, A, and U nucleotides. In other preferred embodiments within this context, the NTP feed comprises an NTP mixture containing G, C, A, and m1ψ. In alternative embodiments within this context, the NTP feed mixture comprises an NTP mixture containing G, C, A, and ψ. In a preferred embodiment, the NTP feed is optimized for a given RNA sequence to be produced (according to claims 1 to 35 of WO2015188933). Therefore, to produce an RNA sequence with a G:C:A:U ratio of 1:2:3:2, the corresponding sequence-optimized NTP mixture comprises a G:C:A:U molar ratio of 1:2:3:2.

[0246] Implementing an NTP feeding step during the IVT process can increase the overall RNA yield of an RNA batch. Furthermore, implementing an NTP feeding step may be suitable for ensuring a more efficient IVT reaction. Additionally, by implementing an NTP feeding step, Mg... 2+ The initial concentration can be kept low (e.g., below 20 mM, below 10 mM as defined herein), which has the beneficial effect of reducing and / or preventing the aggregation of DNA beads. Furthermore, by implementing the NTP feeding step, the formation of byproducts such as dsRNA can be reduced or prevented.

[0247] In one embodiment, the method includes additional optional steps for modifying the resulting RNA. Preferably, these additional optional steps to modify the resulting RNA are performed after the collection step. In one embodiment, the resulting RNA is capped in an enzymatic capping step (e.g., using a capping enzyme as described in WO2016193226 or a fixed capping enzyme). In one embodiment, the resulting RNA is polyadenylated in an enzymatic polyadenylation step (e.g., using a poly(A) polymerase as described in WO2016174271 or a fixed poly(A) polymerase).

[0248] In a preferred embodiment, the collected RNA batch contains less than 1 µg / ml of DNA beads, preferably less than 100 ng / ml of DNA beads, more preferably less than 1 ng / ml of DNA beads, and most preferably the collected RNA batch is substantially free of DNA beads. In this context, "collected RNA batch" refers to RNA obtained directly through in vitro transcription of RNA without the need for RNA purification or DNA digestion steps.

[0249] In a preferred embodiment, the collected RNA batch contains less than 1 µg / ml of DNA template, preferably less than 100 ng / ml of DNA template, more preferably less than 1 ng / ml of DNA template, and most preferably the collected RNA batch is substantially free of DNA template. In this context, "collected RNA batch" refers to RNA obtained directly through in vitro transcription of RNA without the need for RNA purification or DNA digestion steps.

[0250] In a particularly preferred embodiment, the collected RNA is substantially free of DNA beads or fragments thereof.

[0251] In a preferred embodiment, the collected RNA batch contains RNA with a concentration of more than 500 mg / L, preferably more than 1 g / L, and more preferably more than 5 g / L.

[0252] In a preferred embodiment, during any of steps A, B, C, D, E, F, and G, less than 10% of the DNA beads in the composition are aggregated, preferably less than 5% of the DNA beads in the composition are aggregated, and more preferably less than 1% of the DNA beads in the composition are aggregated. Bead aggregation can be determined and quantified using a microscope.

[0253] A key feature of this invention is the performance of in vitro transcription of RNA onto DNA immobilized on beads (referred to herein as "DNA beads"). As detailed herein, the beads on which the DNA template is immobilized typically contain magnetic or (super)paramagnetic materials. In the context of this invention, the use of magnetic or (super)paramagnetic beads is preferred because the mixing and / or trapping of DNA beads can be readily achieved by magnetic force (e.g., using magnets or magnetic units).

[0254] The magnetic unit can be configured to capture or introduce the movement of DNA magnetic beads contained in the reaction vessel. Through such movement, the magnetic unit can induce mixing or agitation of the DNA magnetic beads and the IVT composition. When the DNA magnetic beads and the IVT composition are mixed or agitated due to the movement of the DNA magnetic beads induced by the magnetic unit, the resulting homogeneous mixture of DNA magnetic beads and the IVT composition supports in vitro RNA transcription from template DNA to RNA. Furthermore, after in vitro RNA transcription is complete, the DNA magnetic beads can be captured or retained by the magnetic unit.

[0255] In one embodiment, the movement of the DNA magnetic beads during the IVT process (in step B or F) is configured to prevent bead sedimentation within the reaction vessel. Alternatively, the movement of the DNA magnetic beads during the IVT process (in step B or F) is configured to keep the DNA beads contained in the reaction vessel free-floating, thereby preventing sedimentation at the bottom of the reaction vessel. Furthermore, keeping the DNA beads free-floating in the container and / or preventing or reducing bead aggregation during the IVT process (in step B or F) improves the mixing or spinning method. Advantageously, keeping the DNA beads free-floating and / or preventing DNA bead sedimentation reduces the risk of DNA bead aggregation.

[0256] In this embodiment, the magnetic unit is configured to rotate about the longitudinal axis of the reaction vessel, wherein the direction of rotation of the magnetic unit is switchable during mixing. The magnetic unit can induce movement of the DNA magnetic beads in the radial direction relative to the longitudinal axis of the reaction vessel. The magnetic force can be static or dynamically generated by rotating the magnetic unit about the reaction vessel to induce rotation, thus mixing the DNA magnetic beads. The direction of rotation of the magnetic unit can be changed clockwise or counterclockwise and / or alternately relative to the longitudinal axis of the reaction vessel. Therefore, the DNA magnetic beads can remain free-floating in a non-contact manner, thereby improving the mixing of the components. Once the rotation of the magnetic unit stops, the DNA magnetic beads are trapped at the inner surface of the reaction vessel and no longer rotate. Thus, the magnetic unit is configured to (i) rotate about the longitudinal axis of the reaction vessel to induce movement of the DNA magnetic beads as explained above, and is configured to (ii) trap the DNA magnetic beads upon cessation of rotation (e.g., in step B or G).

[0257] Therefore, in a preferred embodiment, the mixing of DNA beads in steps (B) and / or (F) and / or (D) is performed by a magnetic unit as defined herein. Appropriately, the mixing allows the DNA magnetic beads to remain freely floating during the IVT process.

[0258] In a preferred embodiment, the retention of the DNA beads in steps (C) and / or (G) and / or (D) is performed using a magnetic unit as defined herein. Suitablely, the retention step keeps the DNA magnetic beads connected to the reaction vessel, preferably to the inner surface of the reaction vessel.

[0259] In this embodiment, the magnetic unit can be an array of electromagnets, a permanent magnet, an electromagnet, or an induction coil. Preferably, the magnetic unit is positioned near the outer surface of the reaction vessel. In a preferred embodiment, the magnetic unit does not come into direct contact with the RNA in vitro transcription composition. In one embodiment, the magnet unit includes a magnetic ring, which is designed to surround the reaction vessel. To facilitate the assembly and rotation of the magnet unit around the reaction vessel, the magnet unit can be formed as a ring. In other words, the reaction vessel can be positioned at the center of the ring-shaped magnet unit such that the magnet unit surrounds the reaction vessel.

[0260] Therefore, in a preferred embodiment, the magnet unit is a magnet ring surrounding the reaction vessel.

[0261] To effectively capture or retain DNA magnetic beads, the rotation of the magnetic rings can be stopped after the components are mixed in the reaction vessel (e.g., in step B or G).

[0262] In one embodiment, the reaction vessel used in the method is paramagnetic, such that DNA magnetic beads can be retained on the inner wall of the reaction vessel through the cooperation of the paramagnetic vessel and a magnetic unit positioned at the reaction vessel. Thus, the entire reaction vessel can be paramagnetic, or the inner surface of the reaction vessel can be paramagnetic, for example, by comprising a paramagnetic or magnetically conductive material. The term "magnetizable" throughout the invention indicates that the reaction vessel or its inner surface can be temporarily magnetized, such that magnetic beads can be attracted and retained at the reaction vessel wall. However, the magnetization of the reaction vessel or its inner surface can be reversed, allowing the DNA magnetic beads retained at the reaction vessel wall to be released. Therefore, it is important that the material of the reaction vessel (e.g., a bioreactor) and / or the inner surface of the reaction vessel (e.g., a bioreactor) is not permanently magnetized (i.e., non-ferromagnetic) by activating the magnetic unit.

[0263] Therefore, in a preferred embodiment, the reaction vessel is paramagnetic. In other embodiments, the reaction vessel is configured to allow magnetic fields to pass through without being magnetized.

[0264] Suitably, the method for producing RNA is carried out in a bioreactor, as described in WO2020002598, preferably as described in claims 1 to 59 of WO2020002598. Figures 1 to 1 Figure 1 illustrates a particularly suitable bioreactor in the context of the present invention.

[0265] In a preferred embodiment, step (A) includes a DNA production step (A1) and a DNA fixation step (A2) as well as an optional DNA trimming step on the beads (A3).

[0266] In a preferred embodiment, the DNA production (A1) step is a polymerase chain reaction (PCR). Suitablely, primers are used to amplify the DNA template to obtain PCR-amplified template DNA. Suitablely, biotinylated primers are used for PCR amplification to allow the resulting DNA template to be immobilized on streptavidin-functionalized beads. Preferably, the resulting PCR-amplified biotinylated DNA template is purified.

[0267] Alternatively, the DNA production (A1) step is bacterial plasmid DNA (pDNA) amplification. Suitablely, bacterial fermentation is used to amplify the DNA template. Suitablely, the resulting pDNA is linearized and functionalized with biotin to allow the resulting DNA template to be immobilized on streptavidin-functionalized beads. Preferably, the resulting biotinylated DNA template is purified.

[0268] In a preferred embodiment, the DNA fixation step (A2) includes binding biotinylated DNA (e.g., PCR template or linear pDNA) to streptavidin-functionalized beads (e.g., magnetic beads) in a fixation buffer. Step A2 is performed in a reaction vessel used in an RNA production method. After fixation, the obtained DNA beads can be further modified.

[0269] Preferred modifications to DNA beads include a DNA trimming step (A3) on the beads, for example, when the DNA is generated by PCR amplification.

[0270] DNA templates used for in vitro transcription of RNA can contain a Poly(A) box at the end of the DNA.

[0271] The poly(A) box can be introduced using PCR primers or amplified using flanking primers. The advantage of amplifying the poly(A) box using flanking primers is that the PCR reaction is more uniform and stable (e.g., the PCR product size is constant). The disadvantage is that RNA molecules generated using such DNA templates will not terminate with a poly(A) box. However, it has been shown that having a terminal poly(A) sequence is important for the biological activity of, for example, mRNA (that is, the poly(A) box is the end of the RNA; see, for example, WO2022162027).

[0272] Therefore, in the embodiment where PCR is used in step A1 to amplify a DNA template containing poly(A) via flanking primers, the immobilized DNA template (obtained after step A2) can be further trimmed using a restriction endonuclease that binds to the flanking region of the DNA and cuts at or within the poly(A) sequence to produce a poly(A) 3' end. Suitable, the DNA on the beads is digested using the TypIIS restriction enzyme in step A3 to obtain the poly(A) 3' end of the DNA template. The result of DNA trimming step A3 is beads containing a immobilized DNA template with a free, non-immobilized poly(A) 3' end.

[0273] In a preferred embodiment, the method further includes an RNA purification step of the collected RNA to obtain purified RNA.

[0274] As used herein, the terms "purified RNA" or "purified mRNA" must be understood as RNA with a higher purity than the starting material (e.g., the collected in vitro transcribed RNA) after certain purification steps (e.g., HPLC, TFF, oligo(dT) purification, precipitation steps). Typical impurities that are essentially absent in purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, such as RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double-stranded RNA (dsRNA)), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), DNA beads, etc. Other potential impurities that may originate from, for example, fermentation processes include bacterial impurities (bioload, bacterial DNA) or impurities derived from the purification process (organic solvents, etc.). Therefore, at this point, it is desirable for "RNA purity" to be as close to 100% as possible. Therefore, as used herein, the purity of “purified RNA” exceeds 75%, 80%, 85%, particularly preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably 99% or higher. Purity is determined, for example, by analytical HPLC, wherein the percentages provided above correspond to the ratio between the area of ​​the target RNA peak and the total area of ​​all peaks, including those representing byproducts. Alternatively, purity may be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0275] Appropriately, the collected RNA is transferred to an RNA purification module and subjected to at least one purification step.

[0276] In a preferred embodiment, the RNA purification step (H) includes at least one step selected from the following: RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, nuclear bead flow chromatography, oligomer (dT) purification, cellulose-based purification, or any combination thereof.

[0277] In a more preferred embodiment, the RNA purification step (H) includes at least a step selected from RP-HPLC (preferably performed as described in WO2008077592) and TFF (preferably performed as described in WO2016193206). In a particularly preferred embodiment, the RNA purification step (H) includes at least one oligomer (dT) purification step, and optionally at least one TFF and / or RP-HPLC step. In other preferred embodiments, the RNA purification step (H) includes at least one oligomer (dT) purification step and at least one cellulose purification step (preferably performed as described in WO2022162027).

[0278] In a preferred embodiment, the method for producing RNA does not include a DNase treatment step. Suitablely, the DNA beads of the method are effectively captured and retained, thereby preventing DNA contamination of the produced RNA. This DNA bead capture allows for the reuse of the DNA beads and eliminates the need for the DNase treatment step. Since DNase treatment can introduce additional contaminants (e.g., DNA fragments, DNase) into the produced RNA batch, the use of DNA beads improves the purity of the produced RNA.

[0279] In a preferred embodiment, the method further includes step (I) of preparing RNA (preferably preparing purified RNA obtained from step (H)).

[0280] In a preferred embodiment, step (I) is an LNP preparation step. For example, LNPs can be prepared by mixing the lipid composition with the aqueous RNA composition using a T-shaped or Y-shaped mixing element. Alternatively, LNPs can be prepared using microfluidic mixing. After LNP preparation, the LNPs can be purified or reburied using filtration and / or TFF.

[0281] In a preferred embodiment, the method further includes a filling and finishing step (J) for, for example, obtaining a formulated RNA drug. Suitably, the formulated RNA drug (preferably an LNP-formulated RNA drug) is aseptically filled in liquid or lyophilized form. Therefore, a lyophilization step (preferably according to WO2016165831), a spray freeze-drying step (preferably according to WO2016184576), or a spray drying step (preferably according to WO2016184575) can be performed in step (J). Suitably, the drug is filled into vials or syringes (preferably syringes compatible with LNP-formulated RNA drugs (suitably selected from syringes described in WO2022207862)).

[0282] It is worth noting that, in the context of this invention, it is advantageous and particularly preferred to strongly reduce or prevent DNA bead aggregation during the IVT process to ensure that downstream method steps (e.g., (H), (I), (J)) are not impaired, for example, by contamination caused by DNA beads.

[0283] Preferably, the method for generating RNA produces more than 1 mg of RNA, more than 1 g of RNA, more than 10 g of RNA, or more than 100 g of RNA. For example, from 1 mg to 1000 g of RNA, preferably from 1 mg to 100 g of RNA, more preferably from 1 g to 100 g of RNA. The RNA is optionally formulated in a lipid-based vector (e.g., in an LNP as defined herein).

[0284] Preferably, the RNA produced has an RNA purity of at least 85%, more preferably at least 90%, and even more preferably at least 95%, 96%, 97%, 98%, or 99%. Preferably, the RNA produced has an RNA integrity of at least 75%, more preferably at least 80%, and even more preferably at least 85%. RNA integrity is determined using IP-RP-HPLC.

[0285] Preferably, the method is an RNA production method that can be operated under GMP. Preferably, the method is an RNA production method configured and optimized for use in automated RNA manufacturing apparatus (see fourth aspect).

[0286] RNA manufacturing apparatus: In a fourth aspect, the present invention provides an RNA manufacturing apparatus comprising an RNA in vitro transcription composition of the first aspect, or configured to perform an RNA production method of the third aspect.

[0287] It should be noted that the features and embodiments described in the context of the RNA in vitro transcription composition of the first aspect or the method of the third aspect are also applicable to the RNA manufacturing apparatus of the fourth aspect. Similarly, the features and embodiments described in the context of the RNA manufacturing apparatus of the fourth aspect are also applicable to the RNA in vitro transcription composition of the first aspect or the method of the third aspect.

[0288] In a preferred embodiment, the RNA manufacturing apparatus includes a bioreactor for in vitro transcription of RNA, the bioreactor comprising: (a) A reaction vessel adapted to contain the RNA in vitro transcription composition of the first aspect. The DNA beads mentioned therein are DNA templates immobilized on magnetic beads, and (b) A magnet unit, said magnet unit being located at the reaction vessel. The magnetic unit is configured to capture DNA beads or introduce the movement of DNA beads.

[0289] Preferably, the magnet unit is a permanent magnet or an electromagnet that is movable in the longitudinal direction along at least one of the longitudinal axes of the reaction vessel, such that the magnet unit is configured to capture DNA beads or introduce movement into DNA beads.

[0290] In a preferred embodiment of the RNA manufacturing apparatus, the magnet unit is a magnet ring surrounding the reaction vessel.

[0291] In a particularly preferred embodiment, the RNA manufacturing apparatus comprises a bioreactor as described in WO2020002598, preferably as described in claims 1 to 59 of WO2020002598. (WO2020002598) Figures 1 to 1 Figure 1 illustrates a particularly suitable bioreactor in the context of the present invention.

[0292] In embodiments, the RNA manufacturing apparatus further includes a module for DNA production (e.g., for performing PCR), suitably as described in WO2022112498, specifically as described in claims 1 to 28 of WO2022112498, or as described in WO2022112498. Figures 1 to 1 The modules shown in 1.

[0293] In an embodiment, the RNA manufacturing apparatus further includes a purification module, preferably configured to purify RNA using the following methods: RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, nuclear bead flow chromatography, oligomer (dT) purification, cellulose-based purification, or any combination thereof.

[0294] In an embodiment, the RNA manufacturing apparatus further includes a dispensing module, preferably an LNP dispensing module, and / or a filling and finishing module suitable for the aseptic filling of RNA drugs.

[0295] In a preferred embodiment, the bioreactor for in vitro RNA transcription and other optional modules are integrated into a GMP manufacturing apparatus, module, or system. Preferably, the GMP manufacturing apparatus, module, or system is as described in WO2022049093, specifically as described in claims 1 to 72 of WO2022049093, or as described in WO2022049093. Figures 1 to 6 The devices, modules or systems shown in the document.

[0296] Its use in IVT buffer is to prevent or reduce the aggregation of DNA beads. In another aspect, the present invention relates to the use of IVT buffer for preventing or reducing the aggregation of DNA beads (and thus also contributing to RNA yield), preferably for preventing or reducing the aggregation of DNA beads in RNA manufacturing methods during the process of in vitro transcription of RNA.

[0297] It should be noted that the features and embodiments described in the context of the RNA in vitro transcription composition of the first aspect or the method of the third aspect are also applicable to this aspect.

[0298] Therefore, the IVT buffer for the stated purpose is characterized by any of the characteristics associated with suitable IVT buffers as disclosed herein (e.g., as disclosed in the first aspect). For example, characteristics related to spermidine, DTT, NTP, Mg... 2+ Mg 2 + The characteristics associated with the NTP ratio, the preferred amount of RNA polymerase, or other additives as defined herein also apply to IVT buffers used in the purposes described herein.

[0299] Particularly preferred embodiments of the IVT buffer in this regard are summarized below.

[0300] Appropriately, the IVT buffer contains less than 2 mM spermidine and Mg2+ in a molar ratio of less than 1.4. 2+ With NTP. Preferably, the IVT buffer contains less than 25 mM Mg. 2+ .

[0301] Preferably, the IVT buffer for the intended use is characterized by at least one, at least two, or a combination of the following features F1 to F6: -F1: Less than 1 mM spermidine, preferably free of spermidine; -F2: Mg with a concentration ranging from 1 mM to 25 mM 2+ Preferably less than 20 mM Mg 2+ ; -F3: Spermine and Mg in the IVT buffer 2+ The total concentration of the additive is less than 30 mM; -F4: Mg 2+ The molar ratio to NTP ranges from 1.2 to 0.5, preferably from 0.8 to 0.6; -F5: Total NTP concentration of at least 3 mM, preferably in the range of 3 mM to 35 mM; -F6: Less than 10 mM DTT, preferably less than or about 1 mM DTT.

[0302] In a preferred embodiment within this context, the IVT buffer for the intended use is characterized by the following features (in ascending order of preference): -F1 and F2 -F1 and F2 and F3 -F1 and F2 and F4 -F3 and F4 -F1 and F2 and F3 and F4 -F3 and F5 or F2 and F5 -F1 and F2 and F3 and F4 and F5 -F1 and F2 and F3 and F4 and F5 and F6 In a preferred embodiment of the described use, DNA beads can be characterized by any of the features disclosed in the context of the first or third aspect.

[0303] According to a preferred embodiment, the DNA beads for the intended use are characterized by at least one, at least two, or a combination of the following features B1 to B5: -B1: Contains a magnetizable material, preferably a (super)paramagnetic material; -B2: A diameter in the range of 1 µm to 10 µm, preferably in the range of 4 µm to 5 µm, for example, 4.5 µg; -B3: Contains polystyrene or polystyrene derivatives; -B4: The surface of the bead contains approximately 1 ng DNA / mm². 2 Approximately 5 ng DNA / mm 2 ; -B5: The beads contain a biotin-streptavidin-fixed DNA template.

[0304] In a preferred embodiment within this context, the DNA beads for the intended use are characterized by the following features (in ascending order of preference): -B1 and B2; -B1, B2, and B3; -B1 and B2 and B3 and B4; -B1 and B2 and B3 and B4 and B5.

[0305] Preferred examples of beads suitable for the context of this invention are Dynabeads®, such as Dynabeads® M-270, Dynabeads® M-280, Dynabeads® M-450, or Dynabeads® MyOne. In a specific embodiment of the method, the beads on which the DNA template is immobilized are Dynabeads® M-450, such as Dynabeads® M-450 E beads.

[0306] In a preferred embodiment of the application, DNA bead aggregation is reduced or prevented at RNA concentrations exceeding 100 mg / L, preferably exceeding 1 g / L, and more preferably exceeding 5 g / L.

[0307] In a preferred embodiment of the described use, if the DNA beads are used for more than one in vitro RNA transcription cycle, aggregation of the DNA beads is reduced or prevented. For example, if the DNA beads are used for 1 to 20 IVT cycles, preferably 3 to 10 IVT cycles, aggregation of the DNA beads is reduced or prevented.

[0308] The use of wash buffers containing chelating agents to prevent or reduce the aggregation of DNA beads. In another aspect, the present invention relates to the use of a buffer containing a chelating agent (wash buffer) for preventing or reducing the aggregation of DNA beads, preferably for preventing or reducing the aggregation of DNA beads in RNA manufacturing methods (e.g., during the process of in vitro transcription of RNA).

[0309] As demonstrated by the inventors, the risk of DNA bead aggregation during in vitro RNA transcription can be further reduced by implementing at least one DNA bead washing step or at least one DNA bead recovery step. This washing step is preferably performed between IVT cycles. Without being bound by theory, such washing steps can remove molecules such as RNA, spermidine, or Mg that may be attached to DNA beads. 2+ These linked molecules can be removed through a washing step under certain conditions, which can prevent or reduce the risk of DNA bead aggregation.

[0310] In a preferred embodiment of the described use, the washing buffer contains at least one chelating agent. In the context of this invention, a cationic chelating agent is preferred, for example, one that can be used with Mg... 2+ Ion-binding chelating agents.

[0311] In a preferred embodiment within this context, the chelating agent is EDTA or a derivative of EDTA. Preferably, the wash buffer for the intended use contains a chelating agent, preferably EDTA, at a concentration ranging from 0.1 mM to 50 mM, from 1 mM to 10 mM, and preferably from 1 mM to 5 mM.

[0312] In other embodiments, the wash buffer contains urea, preferably 0.5 M to 5 M urea. In this context, the wash buffer may contain a chelating agent as defined herein and urea as defined herein.

[0313] In a preferred embodiment of the application, the washing buffer is adjusted to a pH between 7.5 and 8.5. In a preferred embodiment, the pH of the washing buffer is approximately 8.0.

[0314] In a preferred embodiment of the described use, the wash buffer contains at least one buffer. In this context, the preferred buffer is Tris.

[0315] In embodiments described above, the wash buffer contains 1 mM to 500 mM of buffer, preferably Tris. In a preferred embodiment, the wash buffer contains 1 mM to 50 mM of buffer, preferably Tris.

[0316] In a preferred embodiment of the application, the washing buffer contains 1 mM EDTA and 10 mM Tris (pH 8.0).

[0317] In a preferred embodiment of the described use, the wash buffer is used between two RNA in vitro transcription cycles as defined herein to reduce DNA bead aggregation or to recover DNA beads for additional IVT cycles.

[0318] In a preferred embodiment of the described use, the wash buffer prevents or reduces the aggregation of DNA beads having certain characteristics. Suitably, the DNA beads, as defined in the context of the first aspect, are preferably characterized by at least one or all of the characteristics B1 to B5. Preferred examples of beads suitable for this context are Dynabeads®, such as Dynabeads® M-270, Dynabeads® M-280, Dynabeads® M-450, or Dynabeads® MyOne. In a specific embodiment, the beads are Dynabeads® M-450, such as M-450 E. Attached Figure Description

[0319] Figure 1 A schematic view of an exemplary bioreactor and magnet unit as used herein is shown (image adapted from WO2020002598). A magnet unit 3 surrounds a reaction vessel 2 containing the IVT composition 1, such that the magnet unit 3 can rotate around the reaction vessel 2 (e.g., to introduce mixing of DNA magnetic beads) or can be stopped (e.g., to capture DNA magnetic beads). The magnet unit 3 is connected to a member 4 that allows the magnet unit 3 to move. Thus, uniform mixing of the IVT composition 1 in the reaction vessel 2 is achieved by introducing movement into the DNA magnetic beads. IVT reagents can be added through an inlet port 5, and RNA products can be discharged through an outlet port 6.

[0320] Figure 2 A superposition of HPLC chromatograms from a cycle of DNA beads in an in vitro transcription reaction of RNA is shown. RNA yield decreases from cycle to cycle, as evidenced by a reduction in the area under the curve for each chromatogram. Numbered arrows indicate the chromatograms for the corresponding IVT cycles 1 through 8. Further details are provided in Example 1.

[0321] Figure 3 Endoscopic photographs of the interior of the bioreactor (initial IVT and IVT cycles C1 to C11) are shown. Dashed circles indicate the location of the outlet port opening. Bead aggregates or particles (black) are indicated by "P". White arrows in images C9 to C11 indicate that magnetic beads escaped from the bioreactor and accumulated in large quantities at the bottom (dark black area). Further details are provided in Example 2.

[0322] Figure 4 A microscopic image of the DNA magnetic beads that escaped from the bioreactor after cycle 9 of Example 2 is shown. The corresponding treatment is indicated. The scale bar indicates 50 µm. Further details are provided in Example 3.

[0323] Figure 5A micrograph of the DNA magnetic beads that escaped from the bioreactor after cycle 9 of Example 2 is shown. The corresponding treatment is indicated. The scale bar indicates 50 µm. Further details are provided in Example 3.

[0324] Figure 6 This image shows an endoscopic photograph of the interior of a bioreactor used for in vitro transcription of RNA in the presence of 2 mM spermidine. Image 1 shows the end of the initial IVT cycle; Image 2 shows the end of cycle 1; Image 3 shows intercycle bead washing with TE after cycle 1; Image 4 shows the end of cycle 2; Image 5 shows the end of cycle 3. Dashed circles indicate the location of the outlet port opening. Bead aggregates or bead particles (black) are indicated by “P”. Figure 3 The white arrows indicate that the magnetic beads escaped from the bioreactor and accumulated in large numbers at the bottom (dark black area). Further details are provided in Example 5.

[0325] Figure 7 Endoscopic images of the interior of a bioreactor used for in vitro transcription of RNA are shown in the absence of spermidine. Image 1 shows the end of the initial IVT cycle; Image 2 shows the end of cycle 1; Image 3 shows intercycle bead washing with TE after cycle 1; Image 4 shows the end of cycle 2; Image 5 shows the end of cycle 3. Dashed circles indicate the location of the outlet port opening. Bead aggregates or bead particles are not visible. No magnetic beads escape from the bioreactor or accumulate in large quantities at the bottom (as opposed to...). Figure 6 (In comparison). Further details are provided in Example 5.

[0326] Figure 8 RNA yields obtained from cyclic RNA IVT reactions (to produce RNA R1) in the presence of different concentrations of spermidine (0 mM to 6 mM) are shown. Further details are provided in Example 6.

[0327] Example: The following description presents embodiments illustrating various aspects and embodiments of the invention. However, the scope of the invention should not be limited to the specific embodiments presented herein and should be understood to be applicable to other compositions and / or methods, such as those defined in the specification. Therefore, to enable those skilled in the art to more clearly understand and practice the invention, the following preparations and embodiments are provided. The scope of the invention is not limited to the illustrated embodiments, which are intended only to illustrate a single aspect of the invention, and functionally equivalent methods are also within the scope of the invention. In fact, various modifications to the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description, the accompanying drawings, and the following embodiments.

[0328] Example A: Experimental RNA construct Table 1: RNA constructs generated in this embodiment The table below describes the characteristics of the mRNA constructs used for RNA R1, which encodes multiple tumor epitopes, RNA R2, which encodes viral antigens, and RNA R3, which encodes tumor antigens.

[0329] Table 1: RNA constructs generated in this embodiment

[0330] Example 1: Decreased RNA yield observed in in vitro RNA transcription using DNA beads. PCR-amplified biotinylated linear template DNA was immobilized onto streptavidin-functionalized magnetic beads (Dynabeads® M-450 E; Thermo Fisher Scientific) to produce DNA beads. The resulting DNA magnetic beads were used in an IVT bioreactor (exemplarily set up in...) Figure 1 The method for in vitro transcription of cyclic RNA (shown in Table 1) was used. The resulting RNA was 1514 nucleotides in length (R1; see Table 1).

[0331] For the in vitro RNA transcription in this experiment, the following RNA in vitro transcription composition was used: 34 mg / mL DNA beads (60 ng / µL DNA), 27 mM NTP (sequence optimized), 19 mM MgCl2, 80 mM Tris pH 8.0, 2 mM spermidine, and 1 mM DTT. Additionally, the RNA in vitro transcription composition contained T7 RNA polymerase, a cap analog (CleanCap AG), pyrophosphatase, and an RNase inhibitor. The IVT reaction was incubated at 37°C for approximately 90 minutes.

[0332] During the IVT process, a magnet was used to introduce the mixing of the RNA in vitro transcription composition. The IVT step was stopped by capturing DNA beads and emptying the reactor to obtain the product RNA. Subsequently, the captured DNA beads were washed twice in the reactor with fresh IVT buffer (containing 80 mM Triss pH 8.0, 2 mM spermidine, and 1 mM DTT). Fresh RNA in vitro transcription composition was added to the reactor to initiate the next RNA IVT cycle by reusing the DNA beads. This method was repeated 8 times. The RNA product obtained from each IVT cycle was purified using RP-HPLC. An exemplary HPLC chromatogram is shown below. Figure 2 As shown in the superposition.

[0333] result: During RNA R1 production, a decrease in RNA yield can be observed from the initial cycle (the first RNA in vitro transcription; indicated by "1" in the chromatogram) to any subsequent cycle (see [link to relevant documentation]). Figure 2 RNA yield decreased continuously from cycle to cycle, resulting in a sharp decrease in RNA yield for example, cycle 8 (indicated by "8" in the chromatogram). For IVT cycle 9, RNA product could not be eluted from the HPLC column at all. Another important observation was the loss of DNA beads during the RNA manufacturing process, caused by DNA bead aggregation (see examples below).

[0334] Example 2: Decreased RNA yield observed in in vitro transcription of circulating RNA PCR-amplified biotinylated linear template DNA was immobilized onto streptavidin-functionalized magnetic beads (Dynabeads® M-450 E; Thermo Fisher Scientific) to generate DNA beads. The resulting magnetic DNA beads were used in an IVT bioreactor (exemplarily set up in...). Figure 1 The method for in vitro transcription of cyclic RNA is shown in Table 1. The resulting RNA is 4009 nucleotides in length (R2; see Table 1).

[0335] For the in vitro RNA transcription in this experiment, the following RNA in vitro transcription composition was used: 34 mg / mL DNA beads (60 ng / µL DNA), 27 mM NTP (sequence optimized), 19 mM MgCl2, 80 mM Tris pH 8.0, 2 mM spermidine, and 1 mM DTT. Additionally, the RNA in vitro transcription composition contained typical concentrations of T7 RNA polymerase, a cap analogue (CleanCapAG), pyrophosphatase, and an RNase inhibitor. The IVT reaction was incubated at 37°C for approximately 90 minutes.

[0336] During the IVT process, a magnet was used to introduce the mixing of the RNA in vitro transcription composition. The IVT step was stopped by capturing DNA beads and emptying the reactor to obtain the product RNA. Subsequently, the captured DNA beads were washed once with buffer A (80 mM Tris pH 8.0, 2 mM spermidine, 1 mM DTT). Fresh RNA in vitro transcription composition was added to the reactor to initiate the next RNA IVT cycle by reusing the DNA beads. A total of 12 IVT cycles were performed.

[0337] To analyze the behavior of DNA beads during the IVT process in a bioreactor, an endoscope was placed inside the bioreactor to observe the opening of the bioreactor's outlet port when bead separation occurs at the end of the IVT cycle. Figure 3 (The dashed circle in the middle).

[0338] result: Using endoscopic imaging, proper mixing of DNA beads was observed after the initial IVT. Following the initial IVT, the DNA beads were well-distributed and did not accumulate around the bioreactor opening. With each subsequent IVT cycle, bead aggregates or particles (composed of…) were observed. Figure 3 The “P” in the diagram indicates that the magnetic beads begin to form and expand, and the magnetic beads begin to approach the opening at the bottom of the bioreactor (the opening is located at...). Figure 3 (In the center of the circle). Additionally, starting from cycle 5, larger bead aggregates can be detected in the endoscopic images. For IVT cycle 9, magnetic beads escape from the bioreactor (by... Figure 3 (As indicated by the arrow in the diagram) and accumulate in large quantities at the bottom. After cycle 9, the DNA beads that escaped from the bioreactor were recovered from a depth filter located after the outlet of the bioreactor, and the DNA beads were further analyzed (see Example 3).

[0339] The results clearly demonstrate that in vitro transcription of RNA using DNA beads under these conditions is associated with fundamental problems, and the decrease in RNA yield observed in Example 1 was caused by DNA bead aggregation. Therefore, further adaptations are needed to optimize in vitro transcription of RNA using DNA beads.

[0340] Example 3: Microscopic evaluation of DNA bead aggregates Magnetic beads that escaped from the bioreactor after cycle 9 of Example 2 were recovered from the depth filter by pushing 1x IVT buffer through the filter using a syringe. The aggregates of magnetic beads were analyzed by phase contrast microscopy at 1000x magnification.

[0341] To identify the cause of DNA bead aggregation, cycle 9 aggregated DNA magnetic beads were treated with RNase digestion of RNA, DNase digestion of DNA, or proteinase K digestion of proteins (see [link to study]). Figure 4 In addition, the DNA magnetic beads were treated with different buffers, such as 1x TE buffer (10 mM Tris pH 8.0, 1 mM EDTA), 100 mM Tris, and 100 mM Tris + 20 mM MgCl2 (see [link to buffer]). Figure 5 ).

[0342] result: like Figure 4 As shown, incubation of DNA beads with RNase leads to the disintegration of DNA bead aggregates, while RNase and proteinase K have no effect. Figure 5 As shown, incubation in 1x TE at 37°C for 15 minutes dissolved DNA bead aggregates. Conversely, 20 mM MgCl2 stabilized DNA bead aggregates at 37°C in 100 mM Tris pH 9, while the DNA bead aggregates dissolved in the absence of MgCl2. Therefore, the aggregation of DNA magnetic beads depends on RNA nonspecifically bound to the magnetic beads. Furthermore, Mg... 2+ The presence of ions makes the aggregation of DNA beads highly stable.

[0343] In summary, Mg in in vitro RNA transcription 2+ The reduction of ions can further reduce unwanted DNA bead aggregation. In addition, the washing step in a buffer containing a chelating agent (such as EDTA in Tris) allows for the separation of DNA bead aggregates (potentially RNA-promoted) formed in the bioreactor during IVT cycles.

[0344] Example 4: In the model system, Mg 2+ Stabilize DNA bead aggregates To further evaluate Mg 2+ The study investigated the effects of different washing buffers on bead aggregation and identified potential washing buffers to address this aggregation. Bead aggregates were artificially induced in a model system, and the effects of different washing buffers were analyzed.

[0345] 30 ml of MyOne streptavidin magnetic beads (Thermo Fisher Scientific; 1 µM diameter) or 34 ml of M-450 streptavidin beads (Thermo Fisher Scientific; 4.5 µm diameter) were incubated in 0.6 g / L R2 RNA and induced to aggregate in tubes in the presence of 1 M NaCl and 10% Peg-8000. To assess whether the washing solutions could recover R2 RNA from the surface of the beads, the aggregated beads were washed three times for 5 minutes each with the following: (i) 1x TE buffer (10 mM Tris pH 8.0, 1 mM EDTA), (ii) 1x buffer A (80 mM Tris pH 8.0, 2 mM spermidine, 1 mM DTT), or (iii) 1x buffer A containing 19 mM MgCl2 (80 mM Tris pH 8.0, 2 mM spermidine, 1 mM DTT). The results are summarized in Tables 2 and 3. The concentration of RNA in the washing solution was quantified using the NanodropOne device via UV absorbance.

[0346] Table 2: RNA bound to different bead types. The binding ratio is relative to the amount used for passing through 1 M NaCl and 10% The concentration of RNA precipitated by Peg-8000 was calculated.

[0347]

[0348] Table 3: RNA recovery from M-450 E streptavidin beads using different washing solutions. (Compared to recovery via 1x...) Normalization of RNA concentration recovered by TE

[0349] result: As shown in Table 2, in the absence of DNA, 1 M NaCl and Peg-8000, acting as molecular crowders, readily facilitated RNA deposition on the surfaces of various magnetic beads. Therefore, the data indicate that linked RNA can play a role in bead aggregation. This is consistent with the findings of Example 3, where RNase treatment led to the dissociation of DNA bead aggregates, while DNase treatment was ineffective. At comparable concentrations, RNA binding was more efficient for the smaller diameter MyOne streptavidin beads.

[0350] As shown in Table 3, RNA bound to M-450 E streptavidin beads was completely dissolved (recovered) upon washing in TE buffer or buffer A. However, in the presence of Mg... 2+ In the presence of ions (1x buffer A + 19 mM MgCl2), the bound RNA was stable and insoluble (recovery rate 1.1%).

[0351] In summary, the data support the role of Mg in in vitro RNA transcription. 2+Reducing ions can decrease unwanted DNA bead aggregation caused by RNA binding to the surface. Additionally, a washing step using a buffer containing a chelating agent (e.g., Tris containing EDTA) can be implemented to reduce or prevent bead aggregation.

[0352] Example 5: Identification of spermidine as another cause of bead aggregation PCR-amplified biotinylated linear template DNA was immobilized on streptavidin-functionalized magnetic beads (Dynabeads® M-450 E; Thermo Fisher Scientific) to produce DNA beads. The resulting magnetic DNA beads, with or without spermidine, were used in an IVT bioreactor (exemplarily set up in...) Figure 1 The method for in vitro transcription of circulating RNA (shown in Table 1) was employed. The resulting RNA encoded a tumor antigen and was 1928 nucleotides in length (R3 RNA; see Table 1).

[0353] For the in vitro RNA transcription in this experiment, the following RNA in vitro transcription composition was used: 34 mg / mL DNA beads (60 ng / µL DNA), 27 mM NTP (sequence optimized), 19 mM MgCl2, 80 mM Tris pH 8.0, optionally 2 mM spermidine, and 1 mM DTT. Additionally, the RNA in vitro transcription composition contained typical concentrations of T7 RNA polymerase, a cap analog (CleanCap AG), pyrophosphatase, and an RNase inhibitor. The IVT reaction was incubated at 37°C for approximately 90 minutes.

[0354] During the IVT process, a magnet was used to introduce the mixing of the RNA in vitro transcription composition. The IVT step was stopped by capturing DNA beads and emptying the reactor to obtain the product RNA. Subsequently, the captured DNA beads were washed twice in 1x TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Fresh RNA in vitro transcription composition was added to the reactor to initiate the next RNA IVT cycle by reusing the DNA beads. A total of 4 IVT cycles were performed.

[0355] To analyze the behavior of DNA beads during the IVT process in a bioreactor, an endoscope was placed inside the bioreactor in the presence of 2 mM spermidine (see [link to IVT process]). Figure 6 ) and spermidine is absent (see Figure 7 Observe the outlet port opening of the bioreactor (dashed circle) under the condition of ).

[0356] result: Endoscopic imaging was used to analyze the results after the initial IVT cycle ( Figure 6 and 7Image 1 in the image), after the first loop ( Figure 6 and 7 Image 1), after cyclic bead washing with TE ( Figure 6 and 7 Image 2 in the image), at the end of loop 2 Figure 6 and 7 Image 4 in the middle) and at the end of loop 3 ( Figure 6 and 7 Image 5) shows the behavior of DNA beads.

[0357] like Figure 6 As shown, DNA bead aggregation or particle formation was observed in the presence of 2 mM spermidine (indicated by "P"). Furthermore, after inter-cycle magnetic bead washing with 1x TE (Image 3), magnetic beads escaped from the bioreactor (indicated by arrows). Further bead aggregation or particle formation was observed in subsequent cycles (Images 4 and 5). Figure 7 As shown, no bead aggregation or particle formation was observed in the absence of spermidine. Furthermore, DNA magnetic beads did not escape from the bioreactor in the absence of spermidine.

[0358] These images clearly demonstrate that RNA production using a cycle-based in vitro transcription reaction of DNA beads can be carried out without bead escape in the absence of spermidine, and DNA bead aggregation can be prevented.

[0359] Example 6: Spermine has a negative impact on RNA yield As shown in the previous examples, spermidine induces bead aggregation in a cyclic IVT method using DNA beads as templates. To assess the effect of spermidine concentration on RNA yield, cyclic IVT reactions were performed in the presence of different concentrations of spermidine (0–6 mM) to generate RNA R1.

[0360] Biotinylated linear template DNA amplified by PCR was immobilized on streptavidin-functionalized magnetic beads (Dynabeads® M-450 E; Thermo Fisher Scientific) to generate DNA beads. The resulting magnetic DNA beads were used in a circulating in vitro RNA transcription method in reaction tubes in the presence of different spermidine concentrations.

[0361] For the in vitro RNA transcription in this experiment, the following RNA in vitro transcription composition was used: 34 mg / mL DNA beads (60 ng / µL DNA), 27 mM NTP (sequence optimized), and 19 mM MgCl₂. 2,The composition contains 80 mM Tris at pH 8.0 and 1 mM DTT, with spermidine concentrations ranging from 0 mM to 6 mM. Additionally, the RNA in vitro transcription composition includes typical concentrations of T7 RNA polymerase, a cap analog (CleanCap AG), pyrophosphatase, and an RNase inhibitor.

[0362] IVT was performed in 1.5 mL reaction tubes and incubated at 37°C for approximately 90 minutes. Two IVT cycles were performed, and the RNA concentration of the second IVT cycle was determined by capillary gel electrophoresis. Results are shown in... Figure 8 middle.

[0363] result: like Figure 8 As shown, increasing spermidine concentration negatively impacts RNA yield in in vitro RNA transcription reactions using DNA beads. At concentrations of 2 mM spermidine or higher, RNA yield decreases sharply, most likely due to the DNA beads beginning to aggregate (as observed in previous experiments). Concentrations of spermidine below approximately 1.5 mM result in the highest RNA yield.

[0364] Example 7: Effect of reducing spermidine concentration on IVT using DNA beads As demonstrated in previous examples, spermidine induces bead aggregation in cyclic IVT methods using DNA beads as templates, and thus reduces RNA yield. To further evaluate the effect of reducing spermidine on RNA integrity and capping efficiency, IVT reactions were performed in the presence of different concentrations of spermidine (0–2 mM) to produce different RNA constructs (R1 and R3).

[0365] Biotinylated linear template DNA amplified by PCR was immobilized on streptavidin-functionalized magnetic beads (Dynabeads® M-450 E; Thermo Fisher Scientific) to generate DNA beads. The resulting magnetic DNA beads were used in a circulating in vitro RNA transcription method in reaction tubes in the presence of different spermidine concentrations.

[0366] For the in vitro RNA transcription in this experiment, the following RNA in vitro transcription composition was used: 34 mg / mL DNA beads (60 ng / µL DNA), 27 mM NTP (sequence optimized), and 19 mM MgCl₂. 2, The composition contains 80 mM Tris at pH 8.0 and 1 mM DTT, with spermidine concentrations ranging from 0 mM to 2 mM. Additionally, the RNA in vitro transcription composition includes typical concentrations of T7 RNA polymerase, a cap analog (CleanCap AG), pyrophosphatase, and an RNase inhibitor.

[0367] IVT was performed in 1.5 mL reaction tubes and incubated at 37°C for approximately 90 minutes. Two IVT cycles were performed to determine the RNA yield (determined by Qubit BR RNA assay) and RNA integrity (determined by analytical IP-RP-HPLC) of the unpurified RNA. Additionally, the degree of capping of the purified RNA (AgencourtAMPure XP purified) was analyzed using a ribozyme-based capping assay. The results are summarized in Table 4.

[0368] Table 4: Effects of decreased spermidine concentration on RNA quality properties

[0369] result: As shown in Table 4, RNA yield and capping efficiency were improved in the absence of spermidine. With increasing spermidine concentration, both RNA yield and capping efficiency decreased. For RNA construct R3, a negative impact of spermidine on RNA integrity was observed.

[0370] In summary, the reduction or absence of spermidine has no negative impact on the physicochemical properties of RNA. Therefore, in vitro transcription of RNA using DNA beads should be performed at the lowest possible spermidine concentration (e.g., less than 2 mM or less than 1 mM), or in the absence of spermidine, to avoid problems associated with DNA bead aggregation.

[0371] Example 8: Mg 2+ Evaluation of the beneficial molar ratio with NTP As demonstrated in previous examples, MgCl2 has the effect of inducing and / or stabilizing DNA bead aggregation in in vitro RNA transcription reactions. This effect is potentially mediated by free Mg in the IVT transcription composition. 2+ Anion-induced factors may cause bead aggregation and thus reduce RNA yield. To further evaluate Mg... 2+ The effects of these factors were investigated, and IVT reactions were performed in the presence of different concentrations of MgCl2, NTP, and spermidine to produce RNA R3.

[0372] Biotinylated linear template DNA amplified by PCR was immobilized on streptavidin-functionalized magnetic beads (Dynabeads® M-450 E; Thermo Fisher Scientific) to generate DNA beads. The resulting magnetic DNA beads were used in a circulating in vitro RNA transcription method in reaction tubes in the presence of different spermidine concentrations.

[0373] For the in vitro RNA transcription in this experiment, the following RNA in vitro transcription composition was used: 34 mg / mL DNA beads (60 ng / µL DNA), 80 mM Tris pH 8.0, and 1 mM DTT, with varying concentrations of spermidine, NTP, and MgCl2 (see Table 5, conditions A to E). Additionally, the RNA in vitro transcription composition contained typical concentrations of T7 RNA polymerase, a cap analog (CleanCap AG), pyrophosphatase, and an RNase inhibitor.

[0374] The IVT reaction was performed in 1.5 mL reaction tubes and incubated at 37°C for approximately 90 minutes. Two IVT cycles were performed and RNA yield was determined (using a Qubit BR RNA assay). The relative RNA yield (compared to 0 mM spermidine) was calculated. The results are summarized in Table 5.

[0375] Table 5: Effect of the Mg2+ to NTP ratio on RNA in vitro transcription during the second IVT cycle

[0376] result: As shown in Table 5, the highest RNA yield is typically achieved with 0 mM spermidine. Interestingly, when Mg... 2+ When the NTP is above 1.0, RNA yield decreases sharply with increasing spermidine concentration (see condition E, where the relative RNA yield decreases from 100 to 11 in the presence of 2 mM spermidine). By using Mg... 2+ Setting the / NTP ratio to a value below 1.4 stabilizes RNA yield and prevents yield reduction induced by spermidine (see condition D, where the relative RNA yield decreases from 100 to 90 in the presence of 1 mM spermidine). By using Mg... 2+ Setting the / NTP ratio to a value of approximately 0.7 (see conditions A through C) can further stabilize RNA yield, even in the presence of larger amounts of spermidine.

[0377] In summary, by using Mg below 1.4 2+ Compared to NTPs, the in vitro RNA transcription yield using DNA beads was significantly increased. Consequently, the free Mg in the reaction... 2+ The level of Mg is reduced, for example, by forming complexes with NTPs, which reduces or prevents DNA bead aggregation and thus reduces RNA yield. Furthermore, Mg levels should be reduced. 2+ The total concentration (e.g., below 25 mM) is used to avoid unwanted DNA bead aggregation.

[0378] Example 9: Antioxidant compounds have a negative impact on DNA beads In stability studies, DNA beads (Dynabeads® M-450 E; Thermo Fisher Scientific) used in this paper were observed to darken during the in vitro transcription of RNA. Since a negative impact on RNA yield and / or quality could not be ruled out (particularly in the production of therapeutic RNA), a route cause analysis was initiated, identifying the antioxidant DTT as a potential cause.

[0379] Therefore, the DNA beads used in this study were incubated in IVT buffer (80 mM Tris pH 8.0, 2 mM spermidine) with gradually increasing DTT concentrations (0 mM to 40 mM) and stored at 37°C for 65 hours. The color was visually examined and compared to a control sample incubated in 1x TE buffer. Darkening of the beads was recorded and visually assessed on a scale of 0 (same color as control, red / orange) to 10 (black). The results are presented in Table 6. Furthermore, IVT reactions were tested to assess the effect of reducing DTT on in vitro RNA transcription yield (see Table 7).

[0380] Table 6: DTT has a negative impact on the physicochemical properties of DNA beads.

[0381] Table 7: The effect of DTT on DNA beads when the IVT mixture is incubated at 25°C for 48 hours before transcription with DNA beads. Its physicochemical properties have negative effects

[0382] result: As shown in Table 6, the color began to darken when the DTT concentration was above 1 mM, indicating that the DTT concentration affected the physicochemical properties of the beads. At the highest tested concentration (40 mM), the beads had a dark color. As shown in Table 7, the complete absence of DTT in the RNA in vitro transcription composition resulted in a lower RNA yield (5555 ng / µl) compared to reaction solutions containing DTT (RNA yield of approximately 7500 ng / µl from 1 mM DTT to 40 mM DTT).

[0383] Overall, data suggests that when using DNA beads (e.g., Dynabeads) for in vitro RNA transcription, the DTT concentration in the reaction solution should be reduced to avoid bead integrity issues. However, since DTT increases RNA yield, the DTT concentration should ideally be set at at least 1 mM.

Claims

1. An RNA in vitro transcription composition comprising a DNA template immobilized on beads contained in an RNA in vitro transcription (IVT) buffer, wherein the IVT buffer contains • Less than 2 mM spermidine; and • Mg molar ratio less than 1.4 2+ With NTP.

2. The RNA in vitro transcription composition of claim 1, wherein the IVT buffer is configured to reduce or prevent the aggregation of the DNA beads during RNA in vitro transcription.

3. The RNA in vitro transcription composition according to claim 1 or 2, wherein the IVT buffer contains Mg at a concentration of less than about 25 mM. 2+ .

4. The RNA in vitro transcription composition according to claims 1 to 3, wherein the IVT buffer contains Mg in a concentration ranging from 2 mM to 20 mM. 2+ .

5. The RNA in vitro transcription composition according to claims 1 to 4, wherein the IVT buffer contains less than 1.5 mM spermidine, preferably less than 1 mM spermidine.

6. The RNA in vitro transcription composition according to claims 1 to 5, wherein the IVT buffer is substantially free of spermidine.

7. The RNA in vitro transcription composition according to claims 1 to 6, wherein the IVT buffer contains NTPs in a total concentration of at least about 3 mM.

8. The RNA in vitro transcription composition according to claims 1 to 7, wherein the IVT buffer contains NTPs in a total concentration ranging from 3 mM to 35 mM.

9. The RNA in vitro transcription composition according to claim 7 or 8, wherein the NTP comprises modified nucleotides. Preferably Selected from pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

10. The RNA in vitro transcription composition according to claims 1 to 9, wherein the Mg in the IVT buffer... 2+ The molar ratio with NTP is less than 1.2, preferably less than 1.

0.

11. The RNA in vitro transcription composition according to claims 1 to 10, wherein the Mg in the IVT buffer... 2+ The molar ratio to NTP ranges from 1.2 to 0.5, 1.0 to 0.5, and preferably from 0.8 to 0.

6.

12. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the IVT buffer comprises a cap analogue. Preferably cap1 analogues.

13. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the IVT buffer contains an antioxidant at a concentration of less than 5 mM, preferably 1 mM.

14. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the IVT buffer contains at least 5 units / ml of RNA polymerase. Optional The RNA polymerase mentioned therein is T7 RNA polymerase.

15. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the composition comprises 10 μg / ml to 100 μg / ml of fixed DNA template, preferably 50 μg / ml to 70 μg / ml of fixed DNA template.

16. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the composition is substantially free of non-fixed DNA template.

17. An RNA in vitro transcription composition according to any one of the preceding claims, wherein the composition comprises 1 mg / ml to 100 mg / ml DNA beads, preferably 20 mg / ml to 40 mg / ml DNA beads.

18. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the surface of the beads contains about 0.5 ng DNA / mm². 2 Approximately 10 ng DNA / mm 2 Preferably about 1 ng DNA / mm 2 Approximately 5 ng DNA / mm 2 .

19. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the diameter of the DNA beads is in the range of 1 μm to 10 μm, preferably in the range of 4 μm to 5 μm.

20. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the DNA beads comprise polystyrene or a polystyrene derivative.

21. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the DNA beads are magnetic DNA beads. Preferably (Super)paramagnetic DNA beads.

22. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the DNA beads comprise a biotin-streptavidin-fixed DNA template.

23. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the DNA beads are produced by specifically immobilizing biotinylated PCR-amplified DNA template sites onto streptavidin-functionalized beads or by specifically immobilizing biotinylated linearized plasmid DNA template sites onto streptavidin-functionalized beads.

24. The RNA in vitro transcription composition according to any one of the preceding claims, wherein the IVT buffer is configured to reduce or prevent DNA bead aggregation at an RNA concentration of more than 100 mg / L.

25. The RNA in vitro transcription composition according to any one of the preceding claims, wherein after incubating the RNA in vitro transcription composition for at least 1 hour, less than 5% of the DNA beads contained in the composition aggregate.

26. Use of the RNA in vitro transcription composition according to any one of the preceding claims in a method for manufacturing RNA.

27. A method for producing RNA, the method comprising the steps of: (A) Providing an RNA in vitro transcription composition according to any one of claims 1 to 25, said RNA in vitro transcription composition comprising DNA beads in an IVT buffer contained in a reaction vessel; (B) IVT cycling is performed by mixing the DNA beads with the IVT buffer to produce RNA batches.

28. The method of claim 27, wherein the method further comprises the following steps: (C) The DNA beads are retained in the reaction vessel and the RNA batch is collected.

29. The method of claim 27 or 28, wherein the method further comprises at least one additional IVT cycle, the at least one additional IVT cycle comprising the steps of: (E) Add fresh IVT buffer to the reaction vessel containing the retained DNA beads; (F) Perform additional IVT cycles by mixing the DNA beads with newly added IVT buffer to produce additional RNA batches; (G) The DNA beads are retained in the reaction vessel and the additional RNA batches are collected.

30. The method of claim 29, wherein the at least one additional IVT cycle is repeated 1 to 20 times.

31. The method according to claims 27 to 30, further comprising the following steps: (D) Wash the retained DNA beads in washing buffer; Step D is performed after step C and / or after step G.

32. The method of claim 31, wherein the washing buffer comprises at least one chelating agent.

33. The method of claim 32, wherein the chelating agent is EDTA or a derivative of EDTA.

34. The method according to claims 31 to 33, wherein the washing buffer comprises urea.

35. The method according to claims 31 to 34, wherein step D reduces or prevents the aggregation of the DNA beads and / or reduces or prevents the attachment of RNA to the DNA beads.

36. The method according to claims 27 to 35, further comprising at least one NTP feeding step performed during the first IVT cycle (step B) and / or during a further IVT cycle (step F).

37. The method according to claims 27 to 36, wherein the collected RNA is substantially free of DNA beads or fragments thereof.

38. The method according to claims 27 to 37, wherein the collected RNA comprises RNA at a concentration of more than 500 mg / L, preferably more than 1 g / L.

39. The method according to claims 27 to 38, wherein the mixing of the DNA beads in steps B and / or F is performed by a magnetic unit, and / or wherein the retention of the DNA beads in steps C and / or G is performed by a magnetic unit.

40. The method according to claims 27 to 39, wherein the providing step (A) comprises a DNA production step (A1), a DNA fixation step (A2), and an optional DNA trimming step on the beads (A3).

41. The method according to claims 27 to 40, further comprising the step of purifying the collected RNA (H).

42. The method of claim 41, wherein the RNA purification step (H) comprises at least one step selected from the group consisting of: RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, nuclear bead flow chromatography, oligomer (dT) purification, cellulose-based purification, or any combination thereof.

43. The method according to claims 27 to 42, wherein the method does not include a DNA enzyme treatment step.

44. The method according to claims 27 to 43, wherein the method is used to generate RNA of at least 1,000 nucleotides in length.

45. The method according to claims 27 to 44, wherein the method is used to generate mRNA.

46. ​​An RNA manufacturing apparatus comprising an RNA in vitro transcription composition according to claims 1 to 25 and / or configured to perform the method according to any one of claims 27 to 45.

47. IVT buffer for use in RNA manufacturing processes to reduce or prevent DNA bead aggregation, wherein the IVT buffer contains less than 2 mM spermidine and Mg in a molar ratio not exceeding 1.

4. 2+ With NTP, and optionally less than 25 mM Mg 2+ .

48. The use according to claim 47, wherein the IVT buffer is further characterized by any of the features described in claims 2 to 14, and optionally, wherein the DNA beads are characterized by any of the features described in claims 18 to 23.

49. The use of a wash buffer containing a chelating agent for preventing or reducing DNA bead aggregation in an RNA manufacturing process, wherein the wash buffer contains at least one chelating agent. Preferably The concentration range is from 1 mM to 10 mM.

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